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Sample records for temperate phages tp901-1

  1. Construction of specific erythromycin resistance mutations in the temperate lactococcal bacteriophage TP901-1 and their use in studies of phage biology

    DEFF Research Database (Denmark)

    Koch, Birgit; Christiansen, Bettina; Evison, Tim

    1997-01-01

    A method for the construction and isolation of specifically designed mutations of the temperate lactococcal phage TP901-1 has been developed. Two different erm-labeled mutants were isolated. One was shown to be defective in lysogenization and excision. The other, showing normal lysogenization, wa...

  2. Temporal transcription of the lactococcal temperate phage TP901-1 and DNA sequence of the early promoter region

    DEFF Research Database (Denmark)

    Madsen, Hans Peter Lynge; Hammer, Karin

    1998-01-01

    , of which at least two (the integrase gene and putative repressor) are needed for lysogeny, and the divergent and longer transcriptional unit from PL, presumably encoding functions required for the lytic life cycle. ORFs with homology to proteins involved in DNA replication were identified on the latter......Transcriptional analysis by Northern blotting identified clusters of early, middle and late transcribed regions of the temperate lactococcal bacteriophage TP901-1 during one-step growth experiments. The latent period was found to be 65 min and the burst size 40 +/- 10. The eight early transcripts...

  3. Clear Plaque Mutants of Lactococcal Phage TP901-1

    DEFF Research Database (Denmark)

    Kot, Witold; Kilstrup, Mogens; Vogensen, Finn K.

    2016-01-01

    We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome...... protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1....

  4. Identification of DNA-binding sites for the activator involved in late transcription of the temperate lactococcal phage TP901-1

    DEFF Research Database (Denmark)

    Pedersen, Margit; Kilstrup, Mogens; Hammer, Karin

    2006-01-01

    Alt, encoded by the lactococcal phage TP901-1, is needed for late transcription. We identify Alt as a DNA-binding protein, and footprint analysis shows that Alt binds to a region containing four imperfect direct repeats (ALT boxes) located -76 to -32 relative to the P-late transcriptional start...... site. The importance of the ALT boxes was confirmed by deletion of one or two ALT boxes and by introducing mutations in ALT boxes 1 and 4. Alt is proposed to act as a tetramer or higher multimer activating transcription of TP901-1 late genes by binding to the four ALT boxes, and bending of the DNA may...... be important for transcriptional activation of P-late. Furthermore, our results suggest that DNA replication may be required for late transcription in TP901-1. Additionally, we identify gp28 of the related lactococcal phage Tuc2009 as an activator and show that the activators required for late transcription...

  5. Identification of operator sites of the CI repressor of phage TP901-1: evolutionary link to other phages

    DEFF Research Database (Denmark)

    Johansen, A.H.; Brøndsted, L.; Hammer, Karin

    2003-01-01

    The repressor encoded by the cI gene of the temperate Lactococcus lactis subsp. cremoris bacteriophage TP901-1 has been purified. Gel-retardation and footprinting analyses identified three palindromic operator sites (O-R, O-L, and O-D). The operator site O-R is located between the two divergent e...... of these repeats on the phage genomes correspond to those found in TP901-1, indicating that the same system of cooperative repression of early phage promoters has been inherited by modular evolution.......The repressor encoded by the cI gene of the temperate Lactococcus lactis subsp. cremoris bacteriophage TP901-1 has been purified. Gel-retardation and footprinting analyses identified three palindromic operator sites (O-R, O-L, and O-D). The operator site O-R is located between the two divergent...... forms of the repressor were observed, thus indicating that the repressor may bind simultaneously to all three operator sites. Inverted repeats with homology to the operator sites of TP901-1 were identified in phage genomes encoding repressors homologous to CI of TP901-1. Interestingly, the locations...

  6. Use of the integration elements encoded by the temperate lactococcal bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Brøndsted, Lone; Hammer, Karin

    1999-01-01

    . cremoris.In this work, a further analysis of the phage-encoded elements involved in integration was performed. Here we demonstrate that even when the orf1 gene is separated from the attP region, the Orf1 protein is able to promote site-specific integration of an attP-carrying plasmid into the attB site......P region seem to be necessary for site-specific integration of the temperate bacteriophage TP901-1. By use of the integrative elements (attP and orf1) expressed by the temperate lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions in L. lactis...

  7. An activator of transcription regulates phage TP901-1 late gene expression

    DEFF Research Database (Denmark)

    Brøndsted, Lone; Pedersen, Margit; Hammer, Karin

    2001-01-01

    A promoter active in the late phase of the lytic cycle of lactococcal bacteriophage TP901-1 has been identified. The promoter is tightly regulated and requires the product of the phage TP901-1 orf29 for activity. A deletion analysis of the late promoter region showed that a fragment as small as 99...

  8. Temperate phages TP901-1 and phi LC3, belonging to the P335 species, apparently use different pathways for DNA injection in Lactococcus lactis subsp cremoris 3107

    DEFF Research Database (Denmark)

    Breum, Solvej Østergaard; Neve, Horst; Heller, Knut J.

    2007-01-01

    was 10(3)-10(4) fold higher for 3107 than for the mutants. Mitomycin C induction of lysogenized mutants 3107 indicated that phage propagation was not affected in these four mutants. Electron microscopy and analysis of total DNA of infected cells showed that DNA was liberated from the phage particle...... during infection of strain 3107 with TP901-1 and that intracellular phage DNA replication occurred. This was not the case for mutants E121 and E126. This strongly suggests that some step starting with triggering DNA release and ending with DNA injection is impaired during infection with TP901......-1. As such impairment was not seen when infecting E119, E121 and E126 with phi LC3, we conclude that TP901-1 and phi LC3 either are differently triggered by their receptor or utilize different pathways of injection....

  9. Temperate phages TP901-1 and fLC3, belonging to the P335 species, apparently use different pathways for DNA injection in Lactococcus lactis subsp. cremoris 3107

    DEFF Research Database (Denmark)

    Breum, Solvej Østergaard; Neve, Horst; Heller, Knut J.

    2007-01-01

    was 103-104 fold higher for 3107 than for the mutants. Mitomycin C induction of lysogenized mutants 3107 indicated that phage propagation was not affected in these four mutant. Electron microscopy and analysis of total DNA of infected cells showed that DNA was liberated from the phage particle during...... infection of strain 3107 with TP901-1 and that intracellular phage DNA replication occurred. This was not the case for mutants E121 and E126. This strongly suggests that some step starting with triggering DNA release and ending with DNA injection is impaired during infection with TP901-1. As such impairment...... was not seen when infection E-119, E121 and E126 with fLC3, we conclude that TP901-1 and fLC3 either are differently triggered by their receptor or utilize differenct pathways of injection....

  10. Structural Biology of CI from Phage TP901-1

    DEFF Research Database (Denmark)

    Rasmussen, Kim Krighaar

    Viruses constitute the most abundant genetic pool on earth and are divided into several orders. One orders is the bacteriophages. A common characteristic of this order is the ability to infect bacteria. The order is further divided into families, subfamilies, genuses and species. Some species...... (CTD), separated by a linker of various lengths. The NTD is responsible for DNA binding and the CTD is responsible for oligomerization of the CI protein. A Helix-turn-Helix motif is usually responsible for DNA binding. This thesis presents a structural characterization of the CI repressor from TP901......-1 obtained by employing complementary techniques: X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy and Small Angle X-ray Scattering (SAXS). Employing crystallography, NMR and SAXS we show that NTD in solution and when crystallizes exists as a monomer. Furthermore, we here show...

  11. The genetic switch regulating activity of early promoters of the temperate lactococcal bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Madsen, P.L.; Johansen, Annette Helle; Hammer, Karin

    1999-01-01

    A functional analysis of open reading frame 4 (ORF4) and ORF5 from the temperate lactococcal phage TP901-1 was performed by mutant and deletion analysis combined with transcriptional studies of the early phage promoters p(R) and p(L). ORF4 (180 amino acids) was identified as a phage repressor...... or lysogenic life cycle after phage infection and that a protein complex consisting of ORF4 and ORF5 may constitute a new type of genetic switch in bacteriophages....

  12. Structural Biology of CI from Phage TP901-1

    DEFF Research Database (Denmark)

    Rasmussen, Kim Krighaar

    are virulent such as the T4 bacteriophage and some are temperate as the well studied λ bacteriophage. Temperate bacteriophages inherently have the ability to be bistable and thus can be in a lytic state or in a lysogenic state. A genetic switch determines the decision for lytic or lysogenic cycle. Temperate......), three Operator sites, Right, Left and Distance (OR, OL and OD) and two Promoters, Right and Left (PR and PL). The CI repressor is generally the major regulator of temperate bacteriophages genetic switches. In general CI repressors consist of two domains, the N-Terminal Domain (NTD) and C-Terminal Domain......Viruses constitute the most abundant genetic pool on earth and are divided into several orders. One orders is the bacteriophages. A common characteristic of this order is the ability to infect bacteria. The order is further divided into families, subfamilies, genuses and species. Some species...

  13. Analysis of the complete DNA sequence of the temperate bacteriophage TP901-1: Evolution, structure, and genome organization of lactococcal bacteriophages

    DEFF Research Database (Denmark)

    Brøndsted, Lone; Østergaard, Solvej; Pedersen, Margit

    2001-01-01

    A complete analysis of the entire genome of the temperate lactococcal bacteriophage TP901-1 has been performed and the function of 21 of 56 TP901-1-encoded ORFs has been assigned. This knowledge has been used to propose 10 functional modules each responsible for specific functions during bacterio......A complete analysis of the entire genome of the temperate lactococcal bacteriophage TP901-1 has been performed and the function of 21 of 56 TP901-1-encoded ORFs has been assigned. This knowledge has been used to propose 10 functional modules each responsible for specific functions during...... bacteriophage TP901-1 proliferation. Short regions of microhomology in intergenic regions present in several lactococcal bacteriophages and chromosomal fragments of Lactococcus lactis are suggested to be points of exchange of genetic material through homologous recombination. Our results indicate that TP901...... region of the TP901-1 genome were more homologous to proteins encoded by phages infecting gram-positive hosts other than L. lactis. This protein homology argues for the occurrence of horizontal genetic exchange among these bacteriophages and indicates that they have access to a common gene pool....

  14. A resolvase-like protein is requered for the site-specific integration of the temperate lactococcal bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Christiansen, Bettina; Brøndsted, Lone; Vogensen, Finn K.

    1996-01-01

    The integration system of the temperate lactococcal phage TP901-1 was characterized in Lactococcus lactis subsp. cremoris LM0230 and MG1363 with the use of deletion derivatives of the integration vector pBC143 (B. Christiansen, M.G. Johnsen, E. Stenby, F.K. Vogensen, and K. Hammer, J. Bacteriol. ...

  15. Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Breuner, Anne; Brøndsted, Lone; Hammer, Karin

    1999-01-01

    In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase...... of extended resolvases. Orf7 is a basic protein of 64 amino acids, and the corresponding gene (orf7) is the third gene in the early lytic operon. This location of an excisionase gene of a temperate bacteriophage has never been described before. The experiments are based on in vivo excision of specifically...... of genetic material based upon the upp gene (encoding uracil phosphoribosyltransferase) was designed, since upp mutants are resistant to fluorouracil. By using this system, frequencies of excision on the order of 10(-5) per cell could easily be measured. The described selection principle may be of general...

  16. Visualizing a Complete Siphoviridae Member by Single-Particle Electron Microscopy: the Structure of Lactococcal Phage TP901-1

    Science.gov (United States)

    Bebeacua, Cecilia; Lai, Livia; Vegge, Christina Skovgaard; Brøndsted, Lone; van Heel, Marin

    2013-01-01

    Tailed phages are genome delivery machines exhibiting unequaled efficiency acquired over more than 3 billion years of evolution. Siphophages from the P335 and 936 families infect the Gram-positive bacterium Lactococcus lactis using receptor-binding proteins anchored to the host adsorption apparatus (baseplate). Crystallographic and electron microscopy (EM) studies have shed light on the distinct adsorption strategies used by phages of these two families, suggesting that they might also rely on different infection mechanisms. Here, we report electron microscopy reconstructions of the whole phage TP901-1 (P335 species) and propose a composite EM model of this gigantic molecular machine. Our results suggest conservation of structural proteins among tailed phages and add to the growing body of evidence pointing to a common evolutionary origin for these virions. Finally, we propose that host adsorption apparatus architectures have evolved in correlation with the nature of the receptors used during infection. PMID:23135714

  17. Structure and molecular assignment of lactococcal phage TP901-1 baseplate

    DEFF Research Database (Denmark)

    Bebeacua, Cecilia; Bron, Patrick; Lai, Livia

    2010-01-01

    P335 lactococcal phages infect the Gram(+) bacterium Lactococcus lactis using a large multiprotein complex located at the distal part of the tail and termed baseplate (BP). The BP harbors the receptor-binding proteins (RBPs), which allow the specific recognition of saccharidic receptors localized...

  18. The Role of MOR and the CI Operator Sites on the Genetic Switch of the Temperate Bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Pedersen, Margit; Hammer, Karin

    2008-01-01

    A genetic switch controls whether the temperate bacteriophage TP901-1 will enter a lytic or a lysogenic life cycle after infection of its host, Lactococcus lactis. We studied this bistable switch encoded in a small DNA fragment of 979 bp by fusing it to a reporter gene on a low-copy-number plasmid...

  19. A Resolvase-like Protein is required for the Site-Specific Integration of the Temperate Lactococcal Bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Christiansen, Bettina; Brøndsted, Lone; Vogensen, Finn K.

    1996-01-01

    showed 38-44 % similarity to the resolvase group of site-specific integrases, while no similarity to known proteins was found in the C-terminal end. Bacteriophage TP901-1 therefore contains a unique integration system, not resembling the Int-class of site-specific integrases usually found in temperate...

  20. Key Players in the Genetic Switch of Bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Alsing, Anne; Pedersen, Margit; Sneppen, Kim

    2011-01-01

    After infection of a sensitive host temperate phages may enter either a lytic or a lysogenic pathway leading to new phage assembly or silencing as a prophage, respectively. The decision about which pathway to enter is centered in the genetic switch of the phage. In this work, we explore...... the bistable genetic switch of bacteriophage TP901-1 through experiments and statistical mechanical modeling. We examine the activity of the lysogenic promoter Pr at different concentrations of the phage repressor, CI, and compare the effect of CI on Pr in the presence or absence of the phage-encoded MOR...

  1. Identification of Quaternary Structure and Functional Domains of the CI Repressor from Bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Pedersen, Margit; Lo Leggio, Leila; Grossmann, J. Günter

    2008-01-01

    The bacteriophage-encoded repressor protein plays a key role in determining the life cycle of a temperate phage following infection of a sensitive host. The repressor protein Cl, which is encoded by the temperate lactococcal phage TP901-1, represses transcription from both the lytic promoter P......-L and the lysogenic promoter PR by binding to multiple operator sites on the DNA. In this study, we used a small bistable genetic switch element from phage TP901-1 to study the effect of cI deletions in vivo and showed that 43 amino acids could be removed from the C-terminal end of Cl without destroying the ability...

  2. Mutational analysis of the activator of late transcription, Alt , in the lactococcal bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Pedersen, Margit; Hammer, Karin

    2007-01-01

    An activator protein, Alt, synthesized during the early state of lytic infection is required for transcription of the late operon in the lactococcal phage TP901-1. In order to identify amino acid residues in the Alt protein required for activation of the TP901-1 late promoter, Plate, hydroxylamin...

  3. Mutational analysis of two structural genes of the remperate lactococcal bacteriophage TP901-1 involved in tail length determination and baseplate assembly

    DEFF Research Database (Denmark)

    Pedersen, Margit; Østergaard, Solvej; Bresciani, José

    2000-01-01

    phages to the indicator strain L. lactic ssp. cremoris 3107 were determined and electron micrographs were obtained. Specific mutations in orf tmp resulted in the production of mostly phage head structures without tails and a few wild-type looking phages. Furthermore, construction of an inframe deletion...... of the TP901-1 tail and baseplate structure is presented. Author Keywords: Lactococcus bacteriophage; TP901-1; mutagenesis; structural genes; adsorption; tail protein; tape measure protein; baseplate protein...

  4. Resolvase-like recombination performed by the TP901-1 integrase

    DEFF Research Database (Denmark)

    Breüner, A.; Brøndsted, Lone; Hammer, Karin

    2001-01-01

    are consistent with the hypothesis that the TC dinucleotide constitutes the TP901-1 overlap region. A 2 bp overlap region has been observed in recombination reactions catalysed by all other members of the resolvase/invertase family tested so far. By selecting for attB sites with a decreased ability...

  5. Stumbling across the Same Phage: Comparative Genomics of Widespread Temperate Phages Infecting the Fish Pathogen Vibrio anguillarum

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    Panos G. Kalatzis

    2017-05-01

    Full Text Available Nineteen Vibrio anguillarum-specific temperate bacteriophages isolated across Europe and Chile from aquaculture and environmental sites were genome sequenced and analyzed for host range, morphology and life cycle characteristics. The phages were classified as Siphoviridae with genome sizes between 46,006 and 54,201 bp. All 19 phages showed high genetic similarity, and 13 phages were genetically identical. Apart from sporadically distributed single nucleotide polymorphisms (SNPs, genetic diversifications were located in three variable regions (VR1, VR2 and VR3 in six of the phage genomes. Identification of specific genes, such as N6-adenine methyltransferase and lambda like repressor, as well as the presence of a tRNAArg, suggested a both mutualistic and parasitic interaction between phages and hosts. During short term phage exposure experiments, 28% of a V. anguillarum host population was lysogenized by the temperate phages and a genomic analysis of a collection of 31 virulent V. anguillarum showed that the isolated phages were present as prophages in >50% of the strains covering large geographical distances. Further, phage sequences were widely distributed among CRISPR-Cas arrays of publicly available sequenced Vibrios. The observed distribution of these specific temperate Vibriophages across large geographical scales may be explained by efficient dispersal of phages and bacteria in the marine environment combined with a mutualistic interaction between temperate phages and their hosts which selects for co-existence rather than arms race dynamics.

  6. Characterization of temperate phages infecting Clostridium difficile isolates of human and animal origins.

    Science.gov (United States)

    Sekulovic, Ognjen; Garneau, Julian R; Néron, Audrey; Fortier, Louis-Charles

    2014-04-01

    Clostridium difficile is a Gram-positive pathogen infecting humans and animals. Recent studies suggest that animals could represent potential reservoirs of C. difficile that could then transfer to humans. Temperate phages contribute to the evolution of most bacteria, for example, by promoting the transduction of virulence, fitness, and antibiotic resistance genes. In C. difficile, little is known about their role, mainly because suitable propagating hosts and conditions are lacking. Here we report the isolation, propagation, and preliminary characterization of nine temperate phages from animal and human C. difficile isolates. Prophages were induced by UV light from 58 C. difficile isolates of animal and human origins. Using soft agar overlays with 27 different C. difficile test strains, we isolated and further propagated nine temperate phages: two from horse isolates (ΦCD481-1 and ΦCD481-2), three from dog isolates (ΦCD505, ΦCD506, and ΦCD508), and four from human isolates (ΦCD24-2, ΦCD111, ΦCD146, and ΦCD526). Two phages are members of the Siphoviridae family (ΦCD111 and ΦCD146), while the others are Myoviridae phages. Pulsed-field gel electrophoresis and restriction enzyme analyses showed that all of the phages had unique double-stranded DNA genomes of 30 to 60 kb. Phages induced from human C. difficile isolates, especially the members of the Siphoviridae family, had a broader host range than phages from animal C. difficile isolates. Nevertheless, most of the phages could infect both human and animal strains. Phage transduction of antibiotic resistance was recently reported in C. difficile. Our findings therefore call for further investigation of the potential risk of transduction between animal and human C. difficile isolates.

  7. Prevalence, Host Range, and Comparative Genomic Analysis of Temperate Ochrobactrum Phages

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    Claudia Jäckel

    2017-06-01

    Full Text Available Ochrobactrum and Brucella are closely related bacteria that populate different habitats and differ in their pathogenic properties. Only little is known about mobile genetic elements in these genera which might be important for survival and virulence. Previous studies on Brucella lysogeny indicated that active phages are rare in this genus. To gain insight into the presence and nature of prophages in Ochrobactrum, temperate phages were isolated from various species and characterized in detail. In silico analyses disclosed numerous prophages in published Ochrobactrum genomes. Induction experiments showed that Ochrobactrum prophages can be induced by various stress factors and that some strains released phage particles even under non-induced conditions. Sixty percent of lysates prepared from 125 strains revealed lytic activity. The host range and DNA similarities of 19 phages belonging to the families Myoviridae, Siphoviridae, or Podoviridae were determined suggesting that they are highly diverse. Some phages showed relationship to the temperate Brucella inopinata phage BiPB01. The genomic sequences of the myovirus POA1180 (41,655 bp and podovirus POI1126 (60,065 bp were analyzed. Phage POA1180 is very similar to a prophage recently identified in a Brucella strain isolated from an exotic frog. The POA1180 genome contains genes which may confer resistance to chromate and the ability to take up sulfate. Phage POI1126 is related to podoviruses of Sinorhizobium meliloti (PCB5, Erwinia pyrifoliae (Pep14, and Burkholderia cenocepacia (BcepIL02 and almost identical to an unnamed plasmid of the Ochrobactrum intermedium strain LMG 3301. Further experiments revealed that the POI1126 prophage indeed replicates as an extrachromosomal element. The data demonstrate for the first time that active prophages are common in Ochrobactrum and suggest that atypical brucellae also may be a reservoir for temperate phages.

  8. Behaviour of temperate phage Mu in Salmonella typhi.

    Science.gov (United States)

    Soberon, M; Gama, M J; Richelle, J; Martuscelli, J

    1986-01-01

    We have developed a convenient system for genetic analysis of Salmonella typhi exploiting the properties of the mutator phage Mu. In spite of the fact that wild-type Salmonella typhi strains do not allow Mu to form plaques on them, we have shown that these strains are actually sensitive to the phage. It proved possible to use Mu to induce mutations and to promote intra- and interspecific genetic transfer, without having to introduce the phage into the bacteria by means other than infection. Furthermore, we isolated Salmonella typhi derivatives on which Mu formed plaques, and studied the behaviour of Mu in these and wild-type strains.

  9. Functional alignment of regulatory networks: a study of temperate phages.

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    Ala Trusina

    2005-12-01

    Full Text Available The relationship between the design and functionality of molecular networks is now a key issue in biology. Comparison of regulatory networks performing similar tasks can provide insights into how network architecture is constrained by the functions it directs. Here, we discuss methods of network comparison based on network architecture and signaling logic. Introducing local and global signaling scores for the difference between two networks, we quantify similarities between evolutionarily closely and distantly related bacteriophages. Despite the large evolutionary separation between phage lambda and 186, their networks are found to be similar when difference is measured in terms of global signaling. We finally discuss how network alignment can be used to pinpoint protein similarities viewed from the network perspective.

  10. Structure, Adsorption to Host, and Infection Mechanism of Virulent Lactococcal Phage p2

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    Bebeacua, Cecilia; Tremblay, Denise; Farenc, Carine; Chapot-Chartier, Marie-Pierre; Sadovskaya, Irina; van Heel, Marin; Veesler, David

    2013-01-01

    Lactococcal siphophages from the 936 and P335 groups infect the Gram-positive bacterium Lactococcus lactis using receptor binding proteins (RBPs) attached to their baseplate, a large multiprotein complex at the distal part of the tail. We have previously reported the crystal and electron microscopy (EM) structures of the baseplates of phages p2 (936 group) and TP901-1 (P335 group) as well as the full EM structure of the TP901-1 virion. Here, we report the complete EM structure of siphophage p2, including its capsid, connector complex, tail, and baseplate. Furthermore, we show that the p2 tail is characterized by the presence of protruding decorations, which are related to adhesins and are likely contributed by the major tail protein C-terminal domains. This feature is reminiscent of the tail of Escherichia coli phage λ and Bacillus subtilis phage SPP1 and might point to a common mechanism for establishing initial interactions with their bacterial hosts. Comparative analyses showed that the architecture of the phage p2 baseplate differs largely from that of lactococcal phage TP901-1. We quantified the interaction of its RBP with the saccharidic receptor and determined that specificity is due to lower koff values of the RBP/saccharidic dissociation. Taken together, these results suggest that the infection of L. lactis strains by phage p2 is a multistep process that involves reversible attachment, followed by baseplate activation, specific attachment of the RBPs to the saccharidic receptor, and DNA ejection. PMID:24027307

  11. Binding of the N-Terminal Domain of the Lactococcal Bacteriophage TP901-1 CI Repressor to Its Target DNA: A Crystallography, Small Angle Scattering, and Nuclear Magnetic Resonance Study

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner; Rasmussen, Kim K.; Jensen, Malene Ringkjøbing

    2013-01-01

    In most temperate bacteriophages, regulation of the choice of lysogenic or lytic life cycle is controlled by a CI repressor protein. Inhibition of transcription is dependent on a helix–turn–helix motif, often located in the N-terminal domain (NTD), which binds to specific DNA sequences (operator...

  12. Novel Temperate Phages of Salmonella enterica subsp. salamae and subsp. diarizonae and Their Activity against Pathogenic S. enterica subsp. enterica Isolates.

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    Lenka Mikalová

    Full Text Available Forty strains of Salmonella enterica (S. enterica subspecies salamae (II, arizonae (IIIa, diarizonae (IIIb, and houtenae (IV were isolated from human or environmental samples and tested for bacteriophage production. Production of bacteriophages was observed in 15 S. enterica strains (37.5% belonging to either the subspecies salamae (8 strains or diarizonae (7 strains. Activity of phages was tested against 52 pathogenic S. enterica subsp. enterica isolates and showed that phages produced by subsp. salamae had broader activity against pathogenic salmonellae compared to phages from the subsp. diarizonae. All 15 phages were analyzed using PCR amplification of phage-specific regions and 9 different amplification profiles were identified. Five phages (SEN1, SEN4, SEN5, SEN22, and SEN34 were completely sequenced and classified as temperate phages. Phages SEN4 and SEN5 were genetically identical, thus representing a single phage type (i.e. SEN4/5. SEN1 and SEN4/5 fit into the group of P2-like phages, while the SEN22 phage showed sequence relatedness to P22-like phages. Interestingly, while phage SEN34 was genetically distantly related to Lambda-like phages (Siphoviridae, it had the morphology of the Myoviridae family. Based on sequence analysis and electron microscopy, phages SEN1 and SEN4/5 were members of the Myoviridae family and phage SEN22 belonged to the Podoviridae family.

  13. A novel Pseudomonas aeruginosa bacteriophage, Ab31, a chimera formed from temperate phage PAJU2 and P. putida lytic phage AF: characteristics and mechanism of bacterial resistance.

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    Libera Latino

    Full Text Available A novel temperate bacteriophage of Pseudomonas aeruginosa, phage vB_PaeP_Tr60_Ab31 (alias Ab31 is described. Its genome is composed of structural genes related to those of lytic P. putida phage AF, and regulatory genes similar to those of temperate phage PAJU2. The virion structure resembles that of phage AF and other lytic Podoviridae (S. enterica Epsilon 15 and E. coli phiv10 with similar tail spikes. Ab31 was able to infect P. aeruginosa strain PA14 and two genetically related strains called Tr60 and Tr162, out of 35 diverse strains from cystic fibrosis patients. Analysis of resistant host variants revealed different phenotypes, including induction of pigment and alginate overproduction. Whole genome sequencing of resistant variants highlighted the existence of a large deletion of 234 kbp in two strains, encompassing a cluster of genes required for the production of CupA fimbriae. Stable lysogens formed by Ab31 in strain Tr60, permitted the identification of the insertion site. During colonization of the lung in cystic fibrosis patients, P. aeruginosa adapts by modifying its genome. We suggest that bacteriophages such as Ab31 may play an important role in this adaptation by selecting for bacterial characteristics that favor persistence of bacteria in the lung.

  14. Isolation of New Stenotrophomonas Bacteriophages and Genomic Characterization of Temperate Phage S1▿

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    García, Pilar; Monjardín, Cristina; Martín, Rebeca; Madera, Carmen; Soberón, Nora; Garcia, Eva; Meana, Álvaro; Suárez, Juan E.

    2008-01-01

    Twenty-two phages that infect Stenotrophomonas species were isolated through sewage enrichment and prophage induction. Of them, S1, S3, and S4 were selected due to their wide host ranges compared to those of the other phages. S1 and S4 are temperate siphoviruses, while S3 is a virulent myovirus. The genomes of S3 and S4, about 33 and 200 kb, were resistant to restriction digestion. The lytic cycles lasted 30 min for S3 and about 75 min for S1 and S4. The burst size for S3 was 100 virions/cell, while S1 and S4 produced about 75 virus particles/cell. The frequency of bacteriophage-insensitive host mutants, calculated by dividing the number of surviving colonies by the bacterial titer of a parallel, uninfected culture, ranged between 10−5 and 10−6 for S3 and 10−3 and 10−4 for S1 and S4. The 40,287-bp genome of S1 contains 48 open reading frames (ORFs) and 12-bp 5′ protruding cohesive ends. By using a combination of bioinformatics and experimental evidence, functions were ascribed to 21 ORFs. The morphogenetic and lysis modules are well-conserved, but no lysis-lysogeny switch or DNA replication gene clusters were recognized. Two major clusters of genes with respect to transcriptional orientation were observed. Interspersed among them were lysogenic conversion genes encoding phosphoadenosine phosphosulfate reductase and GspM, a protein involved in the general secretion system II. The attP site of S1 may be located within a gene that presents over 75% homology to a Stenotrophomonas chromosomal determinant. PMID:18952876

  15. Morphology, genome sequence, and structural proteome of type phage P335 from Lactococcus lactis

    DEFF Research Database (Denmark)

    Labrie, Simon J.; Josephsen, Jytte; Neve, Horst

    2008-01-01

    for a shorter tail and a different collar/whisker structure. Its 33,613-bp double-stranded DNA genome had 50 open reading frames. Putative functions were assigned to 29 of them. Unlike other sequenced genomes from lactococcal phages belonging to this species, P335 did not have a lysogeny module. However, it did...... carry a dUTPase gene, the most conserved gene among this phage species. Comparative genomic analyses revealed a high level of identity between the morphogenesis modules of the phages P335, ul36, TP901-1, and Tuc2009 and two putative prophages of L. lactis SK11. Differences were noted in genes coding...... for receptor-binding proteins, in agreement with their distinct host ranges. Sixteen structural proteins of phage P335 were identified by liquid chromatography-tandem mass spectrometry. A 2.8-kb insertion was recognized between the putative genes coding for the activator of late transcription (Alt...

  16. Escherichia coli and Staphylococcus phages: effect of translation initiation efficiency on differential codon adaptation mediated by virulent and temperate lifestyles.

    Science.gov (United States)

    Prabhakaran, Ramanandan; Chithambaram, Shivapriya; Xia, Xuhua

    2015-05-01

    Rapid biosynthesis is key to the success of bacteria and viruses. Highly expressed genes in bacteria exhibit a strong codon bias corresponding to the differential availability of tRNAs. However, a large clade of lambdoid coliphages exhibits relatively poor codon adaptation to the host translation machinery, in contrast to other coliphages that exhibit strong codon adaptation to the host. Three possible explanations were previously proposed but dismissed: (1) the phage-borne tRNA genes that reduce the dependence of phage translation on host tRNAs, (2) lack of time needed for evolving codon adaptation due to recent host switching, and (3) strong strand asymmetry with biased mutation disrupting codon adaptation. Here, we examined the possibility that phages with relatively poor codon adaptation have poor translation initiation which would weaken the selection on codon adaptation. We measured translation initiation by: (1) the strength and position of the Shine-Dalgarno (SD) sequence, and (2) the stability of the secondary structure of sequences flanking the SD and start codon known to affect accessibility of the SD sequence and start codon. Phage genes with strong codon adaptation had significantly stronger SD sequences than those with poor codon adaptation. The former also had significantly weaker secondary structure in sequences flanking the SD sequence and start codon than the latter. Thus, lambdoid phages do not exhibit strong codon adaptation because they have relatively inefficient translation initiation and would benefit little from increased elongation efficiency. We also provided evidence suggesting that phage lifestyle (virulent versus temperate) affected selection intensity on the efficiency of translation initiation and elongation. © 2015 The Authors.

  17. The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage.

    Science.gov (United States)

    Roszniowski, Bartosz; Latka, Agnieszka; Maciejewska, Barbara; Vandenheuvel, Dieter; Olszak, Tomasz; Briers, Yves; Holt, Giles S; Valvano, Miguel A; Lavigne, Rob; Smith, Darren L; Drulis-Kawa, Zuzanna

    2017-02-01

    Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7 %) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7-49.5 % identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29-98 % homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4-10 and are stable at 60 °C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest that the AP3 phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.

  18. Genetic engineering of a temperate phage-based delivery system for CRISPR/Cas9 antimicrobials against Staphylococcus aureus

    Science.gov (United States)

    Park, Joo Youn; Moon, Bo Youn; Park, Juw Won; Thornton, Justin A.; Park, Yong Ho; Seo, Keun Seok

    2017-01-01

    Discovery of clustered, regularly interspaced, short palindromic repeats and the Cas9 RNA-guided nuclease (CRISPR/Cas9) system provides a new opportunity to create programmable gene-specific antimicrobials that are far less likely to drive resistance than conventional antibiotics. However, the practical therapeutic use of CRISPR/Cas9 is still questionable due to current shortcomings in phage-based delivery systems such as inefficient delivery, narrow host range, and potential transfer of virulence genes by generalized transduction. In this study, we demonstrate genetic engineering strategies to overcome these shortcomings by integrating CRISPR/Cas9 system into a temperate phage genome, removing major virulence genes from the host chromosome, and expanding host specificity of the phage by complementing tail fiber protein. This significantly improved the efficacy and safety of CRISPR/Cas9 antimicrobials to therapeutic levels in both in vitro and in vivo assays. The genetic engineering tools and resources established in this study are expected to provide an efficacious and safe CRISPR/Cas9 antimicrobial, broadly applicable to Staphylococcus aureus. PMID:28322317

  19. Characterization of temperate Lactobacillus gasseri phage LgaI and its impact as prophage on autolysis of its lysogenic host strains.

    Science.gov (United States)

    Ismail, Elsayed A; Neve, Horst; Geis, Arnold; Heller, Knut J

    2009-06-01

    We show by electron microscopy that Lactobacillus gasseri phage LgaI, a temperate phage residing in the chromosome of Lactobacillus gasseri ATCC33323, belongs to the family of Myoviridae phages. The LgaI DNA is packed by the "head-full" mechanism, as demonstrated by analysis of restriction patterns of heated (74 degrees C) or non-heated DNA. By isolating prophage-cured cells, we were able to demonstrate phage LgaI to be responsible for the strong autolytic phenotype observed for Lactobacillus gasseri ATCC33323. In addition, we show that a copy of the LgaI prophage resides in the chromosome of Lactobacillus gasseri NCK102. The LgaI prophage was not inducible in L. gasseri NCK102-adh by mitomycin C, however, it apparently contributed to the autolytic phenotype of this strain.

  20. Stumbling across the same phage

    DEFF Research Database (Denmark)

    Kalatzis, Panagiotis; Rørbo, Nanna Iben; Castillo Bermúdez, Daniel Elías

    2017-01-01

    Nineteen Vibrio anguillarum-specific temperate bacteriophages isolated across Europe and Chile from aquaculture and environmental sites were genome sequenced and analyzed for host range, morphology and life cycle characteristics. The phages were classified as Siphoviridae with genome sizes between...... 46,006 and 54,201 bp. All 19 phages showed high genetic similarity, and 13 phages were genetically identical. Apart from sporadically distributed single nucleotide polymorphisms (SNPs), genetic diversifications were located in three variable regions (VR1, VR2 and VR3) in six of the phage genomes...... was lysogenized by the temperate phages and a genomic analysis of a collection of 31 virulent V. anguillarum showed that the isolated phages were present as prophages in >50% of the strains covering large geographical distances. Further, phage sequences were widely distributed among CRISPR-Cas arrays of publicly...

  1. In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae [Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736 (Korea, Republic of); Chang, Hyo-Ihl [College of Life Sciences & Biotechnology, Korea University, 5-1 Anam-dong, Seongbuk-gu, Seoul 136-701 (Korea, Republic of); Ha, Chang Hoon, E-mail: chhoonha@amc.seoul.kr [Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736 (Korea, Republic of)

    2016-04-22

    EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. - Highlights: • EFC-1 integrase-mediated recombination was site-specific and unidirectional system. • Serine 21 of EFC-1 integrase plays a major role in the catalytic domain. • The functional minimal sizes of attB and attP was defined 48 and 54 bp.

  2. The Significance of Mutualistic Phages for Bacterial Ecology and Evolution.

    Science.gov (United States)

    Obeng, Nancy; Pratama, Akbar Adjie; Elsas, Jan Dirk van

    2016-06-01

    Bacteria and phages have traditionally been viewed as 'antagonists'. However, temperate phages can transfer genes, which can broaden their bacterial hosts' metabolic repertoire, confer or enhance virulence, or eliminate competing organisms, and so enhance bacterial fitness. Recent evidence shows that phages can also promote biofilm formation leading to population-level benefits for their bacterial hosts. Here, we provide a perspective on the ecological and evolutionary consequences for the bacteria interacting with phages, when phage and host interests are aligned. Furthermore, we examine the question whether bacterial hosts can lower immune barriers to phage infection, thereby facilitating infection by beneficial phages. Taking recent evidence together, we suggest that in many cases temperate phages are to be considered as being mutualistic as well as parasitic, at the same time. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Rasmussen, Kim Krighaar; Frandsen, Kristian E. H.; Erba, Elisabetta Boeri

    2016-01-01

    repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CIΔ58 interacts with the O-L operator site as one dimer bound to both half-sites, and with much higher affinity than...

  4. Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

    Science.gov (United States)

    Rousseau, Geneviève M.; Capra, María L.; Quiberoni, Andrea; Tremblay, Denise M.; Labrie, Simon J.

    2015-01-01

    Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts. PMID:26475105

  5. Filamentous bacteriophage: biology, phage display and nanotechnology applications.

    Science.gov (United States)

    Rakonjac, Jasna; Bennett, Nicholas J; Spagnuolo, Julian; Gagic, Dragana; Russel, Marjorie

    2011-01-01

    Filamentous bacteriophage, long and thin filaments that are secreted from the host cells without killing them, have been an antithesis to the standard view of head-and-tail bacterial killing machines. Episomally replicating filamentous phage Ff of Escherichia coli provide the majority of information about the principles and mechanisms of filamentous phage infection, episomal replication and assembly. Chromosomally- integrated "temperate" filamentous phage have complex replication and integration, which are currently under active investigation. The latter are directly or indirectly implicated in diseases caused by bacterial pathogens Vibrio cholerae, Pseudomonas aeruginosa and Neisseria meningitidis. In the first half of the review, both the Ff and temperate phage are described and compared. A large section of the review is devoted to an overview of phage display technology and its applications in nanotechnology.

  6. The Magistral Phage.

    Science.gov (United States)

    Pirnay, Jean-Paul; Verbeken, Gilbert; Ceyssens, Pieter-Jan; Huys, Isabelle; De Vos, Daniel; Ameloot, Charlotte; Fauconnier, Alan

    2018-02-06

    Since time immemorial, phages-the viral parasites of bacteria-have been protecting Earth's biosphere against bacterial overgrowth. Today, phages could help address the antibiotic resistance crisis that affects all of society. The greatest hurdle to the introduction of phage therapy in Western medicine is the lack of an appropriate legal and regulatory framework. Belgium is now implementing a pragmatic phage therapy framework that centers on the magistral preparation (compounding pharmacy in the US) of tailor-made phage medicines.

  7. The Staphylococci Phages Family: An Overview

    Directory of Open Access Journals (Sweden)

    Laurence Van Melderen

    2012-11-01

    Full Text Available Due to their crucial role in pathogenesis and virulence, phages of Staphylococcus aureus have been extensively studied. Most of them encode and disseminate potent staphylococcal virulence factors. In addition, their movements contribute to the extraordinary versatility and adaptability of this prominent pathogen by improving genome plasticity. In addition to S. aureus, phages from coagulase-negative Staphylococci (CoNS are gaining increasing interest. Some of these species, such as S. epidermidis, cause nosocomial infections and are therefore problematic for public health. This review provides an overview of the staphylococcal phages family extended to CoNS phages. At the morphological level, all these phages characterized so far belong to the Caudovirales order and are mainly temperate Siphoviridae. At the molecular level, comparative genomics revealed an extensive mosaicism, with genes organized into functional modules that are frequently exchanged between phages. Evolutionary relationships within this family, as well as with other families, have been highlighted. All these aspects are of crucial importance for our understanding of evolution and emergence of pathogens among bacterial species such as Staphylococci.

  8. Engineered phages for electronics.

    Science.gov (United States)

    Cui, Yue

    2016-11-15

    Phages are traditionally widely studied in biology and chemistry. In recent years, engineered phages have attracted significant attentions for functionalization or construction of electronic devices, due to their specific binding, catalytic, nucleating or electronic properties. To apply the engineered phages in electronics, these are a number of interesting questions: how to engineer phages for electronics? How are the engineered phages characterized? How to assemble materials with engineered phages? How are the engineered phages micro or nanopatterned? What are the strategies to construct electronics devices with engineered phages? This review will highlight the early attempts to address these questions and explore the fundamental and practical aspects of engineered phages in electronics, including the approaches for selection or expression of specific peptides on phage coat proteins, characterization of engineered phages in electronics, assembly of electronic materials, patterning of engineered phages, and construction of electronic devices. It provides the methodologies and opens up ex-cit-ing op-por-tu-ni-ties for the development of a variety of new electronic materials and devices based on engineered phages for future applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria.

    Science.gov (United States)

    Proux, Caroline; van Sinderen, Douwe; Suarez, Juan; Garcia, Pilar; Ladero, Victor; Fitzgerald, Gerald F; Desiere, Frank; Brüssow, Harald

    2002-11-01

    The complete genome sequences of two dairy phages, Streptococcus thermophilus phage 7201 and Lactobacillus casei phage A2, are reported. Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus of Siphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria. Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared. Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness. This varying range of relatedness was found within Sfi21-like phages from a single species as demonstrated by the different prophages harbored by Lactococcus lactis strain IL1403. A systematic dot plot analysis with 11 complete L. lactis phage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages. The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster. With respect to structural genes, four DNA homology groups could be defined within temperate L. lactis phages. Closely related structural modules for all four DNA homology groups were detected in phages from Streptococcus or Listeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved in L. lactis. It seems reasonable to base phage taxonomy on data from comparative genomics. However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics. For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera. We propose to base phage taxonomy on comparative genomics of a single structural gene

  10. Bacteria, phages and septicemia.

    Directory of Open Access Journals (Sweden)

    Ausra Gaidelyte

    Full Text Available The use of phages is an attractive option to battle antibiotic resistant bacteria in certain bacterial infections, but the role of phage ecology in bacterial infections is obscure. Here we surveyed the phage ecology in septicemia, the most severe type of bacterial infection. We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease. Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis-causing bacterial clones play a role in clonal selection during bacterial invasion.

  11. Lysogenic Conversion and Phage Resistance Development in Phage Exposed Escherichia coli Biofilms

    Directory of Open Access Journals (Sweden)

    Abram Aertsen

    2013-01-01

    Full Text Available In this study, three-day old mature biofilms of Escherichia coli were exposed once to either a temperate Shiga-toxin encoding phage (H-19B or an obligatory lytic phage (T7, after which further dynamics in the biofilm were monitored. As such, it was found that a single dose of H-19B could rapidly lead to a near complete lysogenization of the biofilm, with a subsequent continuous release of infectious H-19B particles. On the other hand, a single dose of T7 rapidly led to resistance development in the biofilm population. Together, our data indicates a profound impact of phages on the dynamics within structured bacterial populations.

  12. Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection

    Science.gov (United States)

    2011-01-01

    Background Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy. Results We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S. aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (cat) gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80). Treatment with the endolysin-deficient phage rescued mice from the fatal S. aureus infection. Conclusions A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage

  13. Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection

    Directory of Open Access Journals (Sweden)

    Kempashanaiah Nanjundappa

    2011-08-01

    Full Text Available Abstract Background Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy. Results We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S. aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (cat gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80. Treatment with the endolysin-deficient phage rescued mice from the fatal S. aureus infection. Conclusions A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host

  14. The Magistral Phage

    Directory of Open Access Journals (Sweden)

    Jean-Paul Pirnay

    2018-02-01

    Full Text Available Since time immemorial, phages—the viral parasites of bacteria—have been protecting Earth’s biosphere against bacterial overgrowth. Today, phages could help address the antibiotic resistance crisis that affects all of society. The greatest hurdle to the introduction of phage therapy in Western medicine is the lack of an appropriate legal and regulatory framework. Belgium is now implementing a pragmatic phage therapy framework that centers on the magistral preparation (compounding pharmacy in the US of tailor-made phage medicines.

  15. Comparative genomics and transduction potential of Enterococcus faecalis temperate bacteriophages.

    Science.gov (United States)

    Yasmin, Azra; Kenny, John G; Shankar, Jayendra; Darby, Alistair C; Hall, Neil; Edwards, Clive; Horsburgh, Malcolm J

    2010-02-01

    To determine the relative importance of temperate bacteriophage in the horizontal gene transfer of fitness and virulence determinants of Enterococcus faecalis, a panel of 47 bacteremia isolates were treated with the inducing agents mitomycin C, norfloxacin, and UV radiation. Thirty-four phages were purified from culture supernatants and discriminated using pulsed-field gel electrophoresis (PFGE) and restriction mapping. From these analyses the genomes of eight representative phages were pyrosequenced, revealing four distinct groups of phages. Three groups of phages, PhiFL1 to 3, were found to be sequence related, with PhiFL1A to C and PhiFL2A and B sharing the greatest identity (87 to 88%), while PhiFL3A and B share 37 to 41% identity with PhiFL1 and 2. PhiFL4A shares 3 to 12% identity with the phages PhiFL1 to 3. The PhiFL3A and B phages possess a high DNA sequence identity with the morphogenesis and lysis modules of Lactococcus lactis subsp. cremoris prophages. Homologs of the Streptococcus mitis platelet binding phage tail proteins, PblA and PblB, are encoded on each sequenced E. faecalis phage. Few other phage genes encoding potential virulence functions were identified, and there was little evidence of carriage of lysogenic conversion genes distal to endolysin, as has been observed with genomes of many temperate phages from the opportunist pathogens Staphylococcus aureus and Streptococcus pyogenes. E. faecalis JH2-2 lysogens were generated using the eight phages, and these were examined for their relative fitness in Galleria mellonella. Several lysogens exhibited different effects upon survival of G. mellonella compared to their isogenic parent. The eight phages were tested for their ability to package host DNA, and three were shown to be very effective for generalized transduction of naive host cells of the laboratory strains OG1RF and JH2-2.

  16. Tales of diversity: Genomic and morphological characteristics of forty-six Arthrobacter phages.

    Directory of Open Access Journals (Sweden)

    Karen K Klyczek

    Full Text Available The vast bacteriophage population harbors an immense reservoir of genetic information. Almost 2000 phage genomes have been sequenced from phages infecting hosts in the phylum Actinobacteria, and analysis of these genomes reveals substantial diversity, pervasive mosaicism, and novel mechanisms for phage replication and lysogeny. Here, we describe the isolation and genomic characterization of 46 phages from environmental samples at various geographic locations in the U.S. infecting a single Arthrobacter sp. strain. These phages include representatives of all three virion morphologies, and Jasmine is the first sequenced podovirus of an actinobacterial host. The phages also span considerable sequence diversity, and can be grouped into 10 clusters according to their nucleotide diversity, and two singletons each with no close relatives. However, the clusters/singletons appear to be genomically well separated from each other, and relatively few genes are shared between clusters. Genome size varies from among the smallest of siphoviral phages (15,319 bp to over 70 kbp, and G+C contents range from 45-68%, compared to 63.4% for the host genome. Although temperate phages are common among other actinobacterial hosts, these Arthrobacter phages are primarily lytic, and only the singleton Galaxy is likely temperate.

  17. Phage therapy: present and future

    Science.gov (United States)

    Kolesnikova, S. G.; Tulyakova, E. N.; Moiseeva, I. Y.

    2017-01-01

    In recent years, bacteriophages are known to have become an effective alternative to antibiotic drugs. The article describes the current and potential applications of bacteriophages and phage endolysins. Also of interest is the devastating effect of phages on biofilms. The development of phage resistance is touched upon as well. Furthermore, the authors discuss the issue of laying down the rules of rational phage therapy.

  18. Estimating richness from phage metagenomes

    Science.gov (United States)

    Bacteriophages are important drivers of ecosystem functions, yet little is known about the vast majority of phages. Phage metagenomics, or the study of the collective genome of an assemblage of phages, enables the investigation of broad ecological questions in phage communities. One ecological cha...

  19. ORFeome Phage Display.

    Science.gov (United States)

    Zantow, Jonas; Moreira, Gustavo Marçal Schmidt Garcia; Dübel, Stefan; Hust, Michael

    2018-01-01

    ORFeome phage display allows the efficient functional screening of entire proteomes or even metaproteomes to identify immunogenic proteins. For this purpose, randomly fragmented, whole genomes or metagenomes are cloned into a phage-display vector allowing positive selection for open reading frames (ORF) to improve the library quality. These libraries display all possible proteins encoded by a pathogen or a microbiome on the phage surface. Consequently, immunogenic proteins can be selected from these libraries using disease-related immunoglobulins from patient serum. ORFeome phage display in particular allows the identification of immunogenic proteins that are only expressed in the host-pathogen interaction but not in cultivation, as well as the detection of very low expressed and very small immunogens and immunogenic proteins of non-cultivable organisms. The identified immunogenic proteins are potential biomarkers for the development of diagnostic assays or vaccines. These articles will give an introduction to ORFeome phage-display technology and give detailed protocols to identify immunogenic proteins by phage display.

  20. Temper Foam

    Science.gov (United States)

    1981-01-01

    Fabricated by Expanded Rubber & Plastics Corporation, Temper Foam provides better impact protection for airplane passengers and enhances passenger comfort on long flights because it distributes body weight and pressure evenly over the entire contact area. Called a "memory foam" it matches the contour of the body pressing against it and returns to its original shape once the pressure is removed. As a shock absorber, a three-inch foam pad has the ability to absorb the impact of a 10-foot fall by an adult. Applications include seat cushioning for transportation vehicles, padding for furniture and a variety of athletic equipment medical applications including wheelchair padding, artificial limb socket lining, finger splint and hand padding for burn patients, special mattresses for the bedridden and dental stools. Production and sales rights are owned by Temper Foam, Inc. Material is manufactured under license by the Dewey and Almy Division of Grace Chemical Corporation. Distributors of the product are Kees Goebel Medical Specialties, Inc. and Alimed, Inc. They sell Temper Foam in bulk to the fabricators who trim it to shapes required by their customers.

  1. Clostridium difficile phages: still difficult?

    Directory of Open Access Journals (Sweden)

    Katherine Rose Hargreaves

    2014-04-01

    Full Text Available Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however developing suitable phages is challenging. In this review we summarise the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics.Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage-host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution.No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using whole-phages are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen.

  2. TEMPERED FRACTIONAL CALCULUS

    Science.gov (United States)

    MEERSCHAERT, MARK M.; SABZIKAR, FARZAD; CHEN, JINGHUA

    2014-01-01

    Fractional derivatives and integrals are convolutions with a power law. Multiplying by an exponential factor leads to tempered fractional derivatives and integrals. Tempered fractional diffusion equations, where the usual second derivative in space is replaced by a tempered fractional derivative, govern the limits of random walk models with an exponentially tempered power law jump distribution. The limiting tempered stable probability densities exhibit semi-heavy tails, which are commonly observed in finance. Tempered power law waiting times lead to tempered fractional time derivatives, which have proven useful in geophysics. The tempered fractional derivative or integral of a Brownian motion, called a tempered fractional Brownian motion, can exhibit semi-long range dependence. The increments of this process, called tempered fractional Gaussian noise, provide a useful new stochastic model for wind speed data. A tempered difference forms the basis for numerical methods to solve tempered fractional diffusion equations, and it also provides a useful new correlation model in time series. PMID:26085690

  3. Tempered fractional calculus

    Energy Technology Data Exchange (ETDEWEB)

    Sabzikar, Farzad, E-mail: sabzika2@stt.msu.edu [Department of Statistics and Probability, Michigan State University, East Lansing, MI 48823 (United States); Meerschaert, Mark M., E-mail: mcubed@stt.msu.edu [Department of Statistics and Probability, Michigan State University, East Lansing, MI 48823 (United States); Chen, Jinghua, E-mail: cjhdzdz@163.com [School of Sciences, Jimei University, Xiamen, Fujian, 361021 (China)

    2015-07-15

    Fractional derivatives and integrals are convolutions with a power law. Multiplying by an exponential factor leads to tempered fractional derivatives and integrals. Tempered fractional diffusion equations, where the usual second derivative in space is replaced by a tempered fractional derivative, govern the limits of random walk models with an exponentially tempered power law jump distribution. The limiting tempered stable probability densities exhibit semi-heavy tails, which are commonly observed in finance. Tempered power law waiting times lead to tempered fractional time derivatives, which have proven useful in geophysics. The tempered fractional derivative or integral of a Brownian motion, called a tempered fractional Brownian motion, can exhibit semi-long range dependence. The increments of this process, called tempered fractional Gaussian noise, provide a useful new stochastic model for wind speed data. A tempered fractional difference forms the basis for numerical methods to solve tempered fractional diffusion equations, and it also provides a useful new correlation model in time series.

  4. TEMPERED FRACTIONAL CALCULUS.

    Science.gov (United States)

    Meerschaert, Mark M; Sabzikar, Farzad; Chen, Jinghua

    2015-07-15

    Fractional derivatives and integrals are convolutions with a power law. Multiplying by an exponential factor leads to tempered fractional derivatives and integrals. Tempered fractional diffusion equations, where the usual second derivative in space is replaced by a tempered fractional derivative, govern the limits of random walk models with an exponentially tempered power law jump distribution. The limiting tempered stable probability densities exhibit semi-heavy tails, which are commonly observed in finance. Tempered power law waiting times lead to tempered fractional time derivatives, which have proven useful in geophysics. The tempered fractional derivative or integral of a Brownian motion, called a tempered fractional Brownian motion, can exhibit semi-long range dependence. The increments of this process, called tempered fractional Gaussian noise, provide a useful new stochastic model for wind speed data. A tempered difference forms the basis for numerical methods to solve tempered fractional diffusion equations, and it also provides a useful new correlation model in time series.

  5. A Genetic Approach to the Development of New Therapeutic Phages to Fight Pseudomonas Aeruginosa in Wound Infections

    Science.gov (United States)

    Krylov, Victor; Shaburova, Olga; Krylov, Sergey; Pleteneva, Elena

    2012-01-01

    Pseudomonas aeruginosa is a frequent participant in wound infections. Emergence of multiple antibiotic resistant strains has created significant problems in the treatment of infected wounds. Phage therapy (PT) has been proposed as a possible alternative approach. Infected wounds are the perfect place for PT applications, since the basic condition for PT is ensured; namely, the direct contact of bacteria and their viruses. Plenty of virulent (“lytic”) and temperate (“lysogenic”) bacteriophages are known in P. aeruginosa. However, the number of virulent phage species acceptable for PT and their mutability are limited. Besides, there are different deviations in the behavior of virulent (and temperate) phages from their expected canonical models of development. We consider some examples of non-canonical phage-bacterium interactions and the possibility of their use in PT. In addition, some optimal approaches to the development of phage therapy will be discussed from the point of view of a biologist, considering the danger of phage-assisted horizontal gene transfer (HGT), and from the point of view of a surgeon who has accepted the Hippocrates Oath to cure patients by all possible means. It is also time now to discuss the possible approaches in international cooperation for the development of PT. We think it would be advantageous to make phage therapy a kind of personalized medicine. PMID:23344559

  6. A Genetic Approach to the Development of New Therapeutic Phages to Fight Pseudomonas Aeruginosa in Wound Infections

    Directory of Open Access Journals (Sweden)

    Elena Pleteneva

    2012-12-01

    Full Text Available Pseudomonas aeruginosa is a frequent participant in wound infections. Emergence of multiple antibiotic resistant strains has created significant problems in the treatment of infected wounds. Phage therapy (PT has been proposed as a possible alternative approach. Infected wounds are the perfect place for PT applications, since the basic condition for PT is ensured; namely, the direct contact of bacteria and their viruses. Plenty of virulent (“lytic” and temperate (“lysogenic” bacteriophages are known in P. aeruginosa. However, the number of virulent phage species acceptable for PT and their mutability are limited. Besides, there are different deviations in the behavior of virulent (and temperate phages from their expected canonical models of development. We consider some examples of non-canonical phage-bacterium interactions and the possibility of their use in PT. In addition, some optimal approaches to the development of phage therapy will be discussed from the point of view of a biologist, considering the danger of phage-assisted horizontal gene transfer (HGT, and from the point of view of a surgeon who has accepted the Hippocrates Oath to cure patients by all possible means. It is also time now to discuss the possible approaches in international cooperation for the development of PT. We think it would be advantageous to make phage therapy a kind of personalized medicine.

  7. TEMPERED FRACTIONAL CALCULUS

    OpenAIRE

    MEERSCHAERT, MARK M.; SABZIKAR, FARZAD; CHEN, JINGHUA

    2015-01-01

    Fractional derivatives and integrals are convolutions with a power law. Multiplying by an exponential factor leads to tempered fractional derivatives and integrals. Tempered fractional diffusion equations, where the usual second derivative in space is replaced by a tempered fractional derivative, govern the limits of random walk models with an exponentially tempered power law jump distribution. The limiting tempered stable probability densities exhibit semi-heavy tails, which are commonly obs...

  8. The use of genomic signature distance between bacteriophages and their hosts displays evolutionary relationships and phage growth cycle determination

    Directory of Open Access Journals (Sweden)

    Regeard Christophe

    2010-07-01

    Full Text Available Abstract Background Bacteriophage classification is mainly based on morphological traits and genome characteristics combined with host information and in some cases on phage growth lifestyle. A lack of molecular tools can impede more precise studies on phylogenetic relationships or even a taxonomic classification. The use of methods to analyze genome sequences without the requirement for homology has allowed advances in classification. Results Here, we proposed to use genome sequence signature to characterize bacteriophages and to compare them to their host genome signature in order to obtain host-phage relationships and information on their lifestyle. We analyze the host-phage relationships in the four most representative groups of Caudoviridae, the dsDNA group of phages. We demonstrate that the use of phage genomic signature and its comparison with that of the host allows a grouping of phages and is also able to predict the host-phage relationships (lytic vs. temperate. Conclusions We can thus condense, in relatively simple figures, this phage information dispersed over many publications.

  9. Phages in the Human Body.

    Science.gov (United States)

    Navarro, Ferran; Muniesa, Maite

    2017-01-01

    Bacteriophages, viruses that infect bacteria, have re-emerged as powerful regulators of bacterial populations in natural ecosystems. Phages invade the human body, just as they do other natural environments, to such an extent that they are the most numerous group in the human virome. This was only revealed in recent metagenomic studies, despite the fact that the presence of phages in the human body was reported decades ago. The influence of the presence of phages in humans has yet to be evaluated; but as in marine environments, a clear role in the regulation of bacterial populations could be envisaged, that might have an impact on human health. Moreover, phages are excellent vehicles of genetic transfer, and they contribute to the evolution of bacterial cells in the human body by spreading and acquiring DNA horizontally. The abundance of phages in the human body does not pass unnoticed and the immune system reacts to them, although it is not clear to what extent. Finally, the presence of phages in human samples, which most of the time is not considered, can influence and bias microbiological and molecular results; and, in view of the evidences, some studies suggest that more attention needs to be paid to their interference.

  10. Advance in phage display technology for bioanalysis.

    Science.gov (United States)

    Tan, Yuyu; Tian, Tian; Liu, Wenli; Zhu, Zhi; J Yang, Chaoyong

    2016-06-01

    Phage display technology has emerged as a powerful tool for target gene expression and target-specific ligand selection. It is widely used to screen peptides, proteins and antibodies with the advantages of simplicity, high efficiency and low cost. A variety of targets, including ions, small molecules, inorganic materials, natural and biological polymers, nanostructures, cells, bacteria, and even tissues, have been demonstrated to generate specific binding ligands by phage display. Phages and target-specific ligands screened by phage display have been widely used as affinity reagents in therapeutics, diagnostics and biosensors. In this review, comparisons of different types of phage display systems are first presented. Particularly, microfluidic-based phage display, which enables screening with high throughput, high efficiency and integration, is highlighted. More importantly, we emphasize the advances in biosensors based on phages or phage-derived probes, including nonlytic phages, lytic phages, peptides or proteins screened by phage display, phage assemblies and phage-nanomaterial complexes. However, more efficient and higher throughput phage display methods are still needed to meet an explosion in demand for bioanalysis. Furthermore, screening of cyclic peptides and functional peptides will be the hotspot in bioanalysis. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Serial tempering without exchange.

    Science.gov (United States)

    Nymeyer, Hugh

    2010-09-21

    Serial tempering is a computational method that turns the temperature T (or more generally any independent λ parameter) into a dynamical variable. It is shown that, under conditions for which this variable is fast, serial tempering is equivalent to the umbrella sampling method with a single effective potential. This equivalence is demonstrated using both a small one-dimensional system and a small solvated peptide. The suggestion is then made to replace the serial tempering protocol with the equivalent umbrella sampling calculation. This approach, serial tempering without exchange (STeWiE), has the same performance as serial tempering in the limit that exchanges are frequent, is simpler to implement, and has fewer adjustable parameters than conventional serial tempering. The equivalence of serial tempering and STeWiE also provides a convenient route for estimating and optimizing the performance of serial tempering simulations and other generalized-ensemble methods.

  12. Synthetic Phage for Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    So Young Yoo

    2014-01-01

    Full Text Available Controlling structural organization and signaling motif display is of great importance to design the functional tissue regenerating materials. Synthetic phage, genetically engineered M13 bacteriophage has been recently introduced as novel tissue regeneration materials to display a high density of cell-signaling peptides on their major coat proteins for tissue regeneration purposes. Structural advantages of their long-rod shape and monodispersity can be taken together to construct nanofibrous scaffolds which support cell proliferation and differentiation as well as direct orientation of their growth in two or three dimensions. This review demonstrated how functional synthetic phage is designed and subsequently utilized for tissue regeneration that offers potential cell therapy.

  13. Phage Genome Annotation Using the RAST Pipeline.

    Science.gov (United States)

    McNair, Katelyn; Aziz, Ramy Karam; Pusch, Gordon D; Overbeek, Ross; Dutilh, Bas E; Edwards, Robert

    2018-01-01

    Phages are complex biomolecular machineries that have to survive in a bacterial world. Phage genomes show many adaptations to their lifestyle such as shorter genes, reduced capacity for redundant DNA sequences, and the inclusion of tRNAs in their genomes. In addition, phages are not free-living, they require a host for replication and survival. These unique adaptations provide challenges for the bioinformatics analysis of phage genomes. In particular, ORF calling, genome annotation, noncoding RNA (ncRNA) identification, and the identification of transposons and insertions are all complicated in phage genome analysis. We provide a road map through the phage genome annotation pipeline, and discuss the challenges and solutions for phage genome annotation as we have implemented in the rapid annotation using subsystems (RAST) pipeline.

  14. The Need for Temperance

    Directory of Open Access Journals (Sweden)

    Karl Inge Tangen

    2015-11-01

    Full Text Available This article explores how temperance as a virtue relates to organizational leadership. The study begins with a short survey of classical Greek and Christian notions of temperance before proceeding to ex-plore temperance in relation to self-leadership, visionary and strategic leadership, and relational lead-ership. The final part of the article offers reflections on how temperance might be cultivated from a theological perspective. Temperance is understood not only as sound thinking but also as embodied self-control and active patience. On the level of self-leadership, it is argued that temperance enables the leader to establish forms of integrity that protect the leader’s self from chaos and destruction. Moreover, temperance may also nurture focused visionary leadership that accepts ethical limits and has an eye to the common good. The study also suggests that organizations should cultivate a culture of strategic discipline that is capable of realizing such visions. On the interpersonal level, temperance is viewed as critical in terms of enabling leaders to treat co-workers with respect and wisdom and han-dle conflict with consideration. Finally, is argued that that the cultivation of temperance is not a one-way street from the inside to the outside or a subordination of feelings to reason but rather a very complex process that includes interpersonal humility, finds vision in an encounter with the good, and yet remains a personal responsibility.

  15. Recombinant phage probes for Listeria monocytogenes

    Science.gov (United States)

    Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

    2007-10-01

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

  16. Recombinant phage probes for Listeria monocytogenes

    Energy Technology Data Exchange (ETDEWEB)

    Carnazza, S; Gioffre, G; Felici, F; Guglielmino, S [Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina (Italy)

    2007-10-03

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 10{sup 4} cells ml{sup -1}. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

  17. Control of Pierce's Disease by Phage.

    Directory of Open Access Journals (Sweden)

    Mayukh Das

    Full Text Available Pierce's Disease (PD of grapevines, caused by Xylella fastidiosa subsp. fastidiosa (Xf, is a limiting factor in the cultivation of grapevines in the US. There are presently no effective control methods to prevent or treat PD. The therapeutic and prophylactic efficacy of a phage cocktail composed of four virulent (lytic phages was evaluated for control of PD. Xf levels in grapevines were significantly reduced in therapeutically or prophylactically treated grapevines. PD symptoms ceased to progress one week post-therapeutic treatment and symptoms were not observed in prophylactically treated grapevines. Cocktail phage levels increased in grapevines in the presence of the host. No in planta phage-resistant Xf isolates were obtained. Moreover, Xf mutants selected for phage resistance in vitro did not cause PD symptoms. Our results indicate that phages have great potential for biocontrol of PD and other economically important diseases caused by Xylella.

  18. Genomics of phages with therapeutic potential

    DEFF Research Database (Denmark)

    Zschach, Henrike

    for alternatives, phage therapy once again takes center stage. Phage therapy holds the promise of substantial benefits both from the economic as well as the public health perspective but also holds distinct challenges. The aim of this PhD was to address how bioinformatics tools, specifically genomics......D. Chapters 1 - 3 deal with phages, their use in therapy and the nosocomial pathogen Staphylococcus aureus. Following that, Chapter 4 and 5 provide an overview of Next Generation Sequencing as well as commonly employed genomics tools, while Chapter 6 details basics of Machine Learning. The second part...... to each other as well as to databases of available phage genomes. It revealed a great diversity in the sequences, many of which were distant from all known phages. The phage content of the different sample locations exhibited a rather stable genomic distance that was not influenced by whether...

  19. Salmonella typhimurium phage typing for pathogens.

    Science.gov (United States)

    Rabsch, Wolfgang

    2007-01-01

    Phage typing provides a rapid, accurate, and cheap method of investigating Salmonella strains for epidemiological use. Salmonella strains within a particular serovar may be differentiated into a number of phage types by their pattern of susceptibility to lysis by a set of phages with different specificity. Characterization based on the pattern of phage lysis of wild strains isolated from different patients, carriers, or other sources is valuable in epidemiological study. The phages must have well-defined propagation strains that allow reproducible discrimination between different Salmonella Typhimurium strains. Different schemes have been developed for this serovar in different countries. The Felix/Callow (England) and Lilleengen typing systems (Sweden) used for laboratory-based epidemiological analysis were helpful for control of salmonellosis. More recently, the extended phage-typing system of Anderson (England) that distinguishes more than 300 definitive phage types (DTs) has been used worldwide in Europe, the United States, and Australia. The use of this method for decades show us that some phage types (DT204 in the 1970s and DT104 in the 1990s) have a broad host range and are distributed worldwide, other phage types such as DT2 or DT99 are frequently associated with disease in pigeons, indicative of a narrow host range.

  20. Influence of environmental variables in the efficiency of phage therapy in aquaculture.

    Science.gov (United States)

    Silva, Yolanda J; Costa, Liliana; Pereira, Carla; Cunha, Ângela; Calado, Ricardo; Gomes, Newton C M; Almeida, Adelaide

    2014-09-01

    Aquaculture facilities worldwide continue to experience significant economic losses because of disease caused by pathogenic bacteria, including multidrug-resistant strains. This scenario drives the search for alternative methods to inactivate pathogenic bacteria. Phage therapy is currently considered as a viable alternative to antibiotics for inactivation of bacterial pathogens in aquaculture systems. While phage therapy appears to represent a useful and flexible tool for microbiological decontamination of aquaculture effluents, the effect of physical and chemical properties of culture waters on the efficiency of this technology has never been reported. The present study aimed to evaluate the effect of physical and chemical properties of aquaculture waters (e.g. pH, temperature, salinity and organic matter content) on the efficiency of phage therapy under controlled experimental conditions in order to provide a basis for the selection of the most suitable protocol for subsequent experiments. A bioluminescent genetically transformed Escherichia coli was selected as a model microorganism to monitor real-time phage therapy kinetics through the measurement of bioluminescence, thus avoiding the laborious and time-consuming conventional method of counting colony-forming units (CFU). For all experiments, a bacterial concentration of ≈ 10(5) CFU ml(-1) and a phage concentration of ≈ 10(6-8) plaque forming unit ml(-1) were used. Phage survival was not significantly affected by the natural variability of pH (6.5-7.4), temperature (10-25 °C), salinity (0-30 g NaCl l(-1) ) and organic matter concentration of aquaculture waters in a temperate climate. Nonetheless, the efficiency of phage therapy was mostly affected by the variation of salinity and organic matter content. As the effectiveness of phage therapy increases with water salt content, this approach appears to be a suitable choice for marine aquaculture systems. The success of phage therapy may also be enhanced in

  1. Development of a phage typing system for Staphylococcus hyicus

    DEFF Research Database (Denmark)

    Wegener, Henrik Caspar

    1993-01-01

    originating from other countries. Although phages were isolated from porcine skin strains exclusively, the system produced phage types in S. hyicus strains of bovine origin. Ten strains of S. aureus and S. chromogenes were not typable by these phages. Strains belonging to one phage type (A/B/C/W) were...... strains isolated from Danish pig herds, the phages were able to type 92% of the strains, producing 16 different phage types. Reproducibility of the phage typing system after subculture of the strains and using fresh phage stock was 96%. Typability ranged from 52 to 80% when typing porcine strains...

  2. Coastal Temperate Rainforest Symposium

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — The North Pacific LCC is helping sponsor the April 2012 science symposium - Coastal Temperate Rainforests: Integrating Communities, Climate Science, and Resource...

  3. Differences in Shiga toxin and phage production among stx2g-positive STEC strains

    Directory of Open Access Journals (Sweden)

    Claudia Viviana Granobles Velandia

    2012-06-01

    Full Text Available Shigatoxigenic E. coli (STEC are characterized by the production of Shiga toxins (Stx encoded by temperate bacteriophages. Stx production is linked to the induction of the phage lytic cycle. Several stx variants have been described and differentially associated with the risk of developing severe illness.The variant named stx2g was first identified in a STEC strain isolated from the faeces of healthy cattle. Analysis of stx2g-positive strains isolated from humans, animals and environmental sources have shown that they have a close relationship. In this study, stx2g-positive STEC isolated from cattle were analyzed for phage and Stx production, with the aim to relate the results to differences observed in cytotoxicity.The presence of inducible phages was assessed by analyzing the bacterial growth/lysis curves and also by plaque assay. Bacterial growth curves in the absence of induction were similar for all isolates, however, notably differed among induced cultures. The two strains that clearly evidenced bacteriolysis under this condition also showed higher phage titers in plaque assays. However, only the phage plaques produced by one of these strains (FB 62 hybridized with a stx2-probe. Furthermore, the production of Stx was evaluated by EIA and Western immunoblotting in overnight supernatants. By EIA, we detected Stx only in supernatants of FB 62, with a higher signal with induced than in uninduced cultures. By immunoblotting, Stx2 could be detected after induction in all stx2g-positive isolates, but with lower amounts of Stx2B subunit in those supernatants where phages could not be detected.Taking into account all the results, several differences could be found among stx2g-positive strains. The strain with the highest cytotoxic titer showed higher levels of stx2-phages and toxin production by EIA, and the opposite was observed for strains that previously showed low cytotoxic titers, confirming that in stx2g-positive strains Stx production is

  4. HostPhinder: A Phage Host Prediction Tool

    DEFF Research Database (Denmark)

    Villarroel, Julia; Kleinheinz, Kortine Annina; Jurtz, Vanessa Isabell

    2016-01-01

    and significantly outperforming BLAST on phages for which both had predictions. HostPhinder predictions on phage draft genomes from the INTESTI phage cocktail corresponded well with the advertised targets of the cocktail. Our study indicates that for most phages genomic similarity correlates well with related...

  5. Therapeutic use of chimeric bacteriophage (phage) lysins in staphylococcal endophthalmitis

    Science.gov (United States)

    Purpose: Phage endolysins are peptidoglycan hydrolases that are produced at the end of the phage lytic cycle to digest the host bacterial cell wall, facilitating the release of mature phage progeny. The aim of this study is to determine the antimicrobial activity of chimeric phage lysins against cli...

  6. Simulated Solute Tempering.

    Science.gov (United States)

    Denschlag, Robert; Lingenheil, Martin; Tavan, Paul; Mathias, Gerald

    2009-10-13

    For the enhanced conformational sampling in molecular dynamics (MD) simulations, we present "simulated solute tempering" (SST) which is an easy to implement variant of simulated tempering. SST extends conventional simulated tempering (CST) by key concepts of "replica exchange with solute tempering" (REST, Liu et al. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 13749). We have applied SST, CST, and REST to molecular dynamics (MD) simulations of an alanine octapeptide in explicit water. The weight parameters required for CST and SST are determined by two different formulas whose performance is compared. For SST only one of them yields a uniform sampling of the temperature space. Compared to CST and REST, SST provides the highest exchange probabilities between neighboring rungs in the temperature ladder. Concomitantly, SST leads to the fastest diffusion of the simulation system through the temperature space, in particular, if the "even-odd" exchange scheme is employed in SST. As a result, SST exhibits the highest sampling speed of the investigated tempering methods.

  7. Phage Therapy - Everything Old is New Again.

    Science.gov (United States)

    Kropinski, Andrew M

    2006-09-01

    The study of bacterial viruses (bacteriophages or phages) proved pivotal in the nascence of the disciplines of molecular biology and microbial genetics, providing important information on the central processes of the bacterial cell (DNA replication, transcription and translation) and on how DNA can be transferred from one cell to another. As a result of the pioneering genetics studies and modern genomics, it is now known that phages have contributed to the evolution of the microbial cell and to its pathogenic potential. Because of their ability to transmit genes, phages have been exploited to develop cloning vector systems. They also provide a plethora of enzymes for the modern molecular biologist. Until the introduction of antibiotics, phages were used to treat bacterial infections (with variable success). Western science is now having to re-evaluate the application of phage therapy - a therapeutic modality that never went out of vogue in Eastern Europe - because of the emergence of an alarming number of antibiotic-resistant bacteria. The present article introduces the reader to phage biology, and the benefits and pitfalls of phage therapy in humans and animals.

  8. Phage Therapy -- Everything Old Is New again

    Directory of Open Access Journals (Sweden)

    Andrew M Kropinski

    2006-01-01

    Full Text Available The study of bacterial viruses (bacteriophages or phages proved pivotal in the nascence of the disciplines of molecular biology and microbial genetics, providing important information on the central processes of the bacterial cell (DNA replication, transcription and translation and on how DNA can be transferred from one cell to another. As a result of the pioneering genetics studies and modern genomics, it is now known that phages have contributed to the evolution of the microbial cell and to its pathogenic potential. Because of their ability to transmit genes, phages have been exploited to develop cloning vector systems. They also provide a plethora of enzymes for the modern molecular biologist. Until the introduction of antibiotics, phages were used to treat bacterial infections (with variable success. Western science is now having to re-evaluate the application of phage therapy -- a therapeutic modality that never went out of vogue in Eastern Europe -- because of the emergence of an alarming number of antibiotic-resistant bacteria. The present article introduces the reader to phage biology, and the benefits and pitfalls of phage therapy in humans and animals.

  9. Characterization of a Vibrio cholerae phage isolated from the coastal water of Peru.

    Science.gov (United States)

    Talledo, Miguel; Rivera, Irma N G; Lipp, Erin K; Neale, Angela; Karaolis, David; Huq, Anwar; Colwell, Rita R

    2003-05-01

    A Vibrio cholerae bacteriophage, family Myoviridae, was isolated from seawater collected from the coastal water of Lima, Peru. Genome size was estimated to be 29 kbp. The temperate phage was specific to V. cholerae and infected 12/13 V. cholerae O1 strains and half of the four non-O1/non-O139 strains tested in this study. Vibrio cholerae O139 strains were resistant to infection and highest infection rates were obtained in low nutrient media amended with NaCl or prepared using seawater as diluent.

  10. Phage-host interplay: examples from tailed phages and Gram-negative bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Soraya eChaturongakul

    2014-08-01

    Full Text Available Complex interactions between bacteriophages and their bacterial hosts play significant roles in shaping the structure of environmental microbial communities, not only by genetic transduction but also by modification of bacterial gene expression patterns. Survival of phages solely depends on their ability to infect their bacterial hosts, most importantly during phage entry. Successful dynamic adaptation of bacteriophages when facing selective pressures, such as host adaptation and resistance, dictates their abundance and diversification. Co-evolution of the phage tail fibers and bacterial receptors determine bacterial host ranges, mechanisms of phage entry and other infection parameters. This review summarizes the current knowledge about the physical interactions between tailed bacteriophages and bacterial pathogens (e.g., Salmonella enterica and Pseudomonas aeruginosa and the influences of the phage on host gene expression. Understanding these interactions can offer insights into phage-host dynamics and suggest novel strategies for the design of bacterial pathogen biological controls.

  11. Three New Escherichia coli Phages from the Human Gut Show Promising Potential for Phage Therapy.

    Directory of Open Access Journals (Sweden)

    Marion Dalmasso

    Full Text Available With the emergence of multi-drug resistant bacteria the use of bacteriophages (phages is gaining renewed interest as promising anti-microbial agents. The aim of this study was to isolate and characterize phages from human fecal samples. Three new coliphages, ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03, were isolated. Their phenotypic and genomic characteristics, and lytic activity against biofilm, and in combination with ciprofloxacin, were investigated. All three phages reduced the growth of E. coli strain DPC6051 at multiplicity of infection (MOI between 10-3 and 105. A cocktail of all three phages completely inhibited the growth of E. coli. The phage cocktail also reduced biofilm formation and prevented the emergence of phage-resistant mutants which occurred with single phage. When combined with ciprofloxacin, phage alone or in cocktail inhibited the growth of E. coli and prevented the emergence of resistant mutants. These three new phages are promising biocontrol agents for E. coli infections.

  12. Supersize me: Cronobacter sakazakii phage GAP32

    Energy Technology Data Exchange (ETDEWEB)

    Abbasifar, Reza; Griffiths, Mansel W. [Canadian Research Institute for Food Safety, University of Guelph, Guelph, ON, Canada N1G 2W1 (Canada); Sabour, Parviz M. [Agriculture and Agri-Food Canada, Guelph Food Research Centre, Guelph, ON, Canada N1G 5C9 (Canada); Ackermann, Hans-Wolfgang [Department of Microbiology-Infectiology and Immunology, Faculty of Medicine, Université Laval, Quebec, QC (Canada); Vandersteegen, Katrien; Lavigne, Rob [Laboratory of Gene Technology, Katholieke Universiteit Leuven, Leuven (Belgium); Noben, Jean-Paul [Biomedical Research Institute and Transnational University Limburg, School of Life Sciences, Hasselt University, Diepenbeek (Belgium); Alanis Villa, Argentina; Abbasifar, Arash [Canadian Research Institute for Food Safety, University of Guelph, Guelph, ON, Canada N1G 2W1 (Canada); Nash, John H.E. [Public Health Agency of Canada, Laboratory for Foodborne Zoonoses, Guelph, ON, Canada N1G 3W4 (Canada); Kropinski, Andrew M., E-mail: akropins@uoguelph.ca [Public Health Agency of Canada, Laboratory for Foodborne Zoonoses, Guelph, ON, Canada N1G 3W4 (Canada); Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1 (Canada)

    2014-07-15

    Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB{sub C}saM{sub G}AP32 (GAP32), which possesses the second largest sequenced phage genome (358,663 bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily. - Highlights: • Cronobacter sakazakii phage vB{sub C}saM{sub G}AP32 has a genome of 358,663 bp. • It encodes 545 proteins which is more than Mycoplasma genitalium G37. • It is a member of the Myoviridae. • It is peripherally related to coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2. • GAP32 encodes a chromosome condensation protein.

  13. Phage Life Cycles Behind Bacterial Biodiversity.

    Science.gov (United States)

    Olszak, Tomasz; Latka, Agnieszka; Roszniowski, Bartosz; Valvano, Miguel A; Drulis-Kawa, Zuzanna

    2017-11-24

    Bacteriophages (phages or bacterial viruses) are the most abundant biological entities in our planet; their influence reaches far beyond the microorganisms they parasitize. Phages are present in every environment and shape up every bacterial population in both active and passive ways. They participate in the circulation of organic matter and drive the evolution of microorganisms by horizontal gene transfer at unprecedented scales. The mass flow of genetic information in the microbial world influences the biosphere and poses challenges for science and medicine. The genetic flow, however, depends on the fate of the viral DNA injected into the bacterial cell. The archetypal notion of phages only engaging in predatorprey relationships is slowly fading. Because of their varied development cycles, environmental conditions, and the diversity of microorganisms they parasitize, phages form a dense and highly complex web of dependencies, which has important consequences for life on Earth. The sophisticated phage-bacteria interplay includes both aggressive action (bacterial lysis) and "diplomatic negotiations" (prophage domestication). Here, we review the most important mechanisms of interactions between phages and bacteria and their evolutionary consequences influencing their biodiversity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. Infective and inactivated filamentous phage as carriers for immunogenic peptides.

    Science.gov (United States)

    Samoylova, Tatiana I; Norris, Mandy D; Samoylov, Alexandre M; Cochran, Anna M; Wolfe, Karen G; Petrenko, Valery A; Cox, Nancy R

    2012-07-01

    The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Colonisation of a phage susceptible Campylobacter jejuni population in two phage positive broiler flocks.

    Directory of Open Access Journals (Sweden)

    Sophie Kittler

    Full Text Available The pathogens Campylobacter jejuni and Campylobacter coli are commensals in the poultry intestine and campylobacteriosis is one of the most frequent foodborne diseases in developed and developing countries. Phages were identified to be effective in reducing intestinal Campylobacter load and this was evaluated, in the first field trials which were recently carried out. The aim of this study was to further investigate Campylobacter population dynamics during phage application on a commercial broiler farm. This study determines the superiority in colonisation of a Campylobacter type found in a field trial that was susceptible to phages in in vitro tests. The colonisation factors, i.e. motility and gamma glutamyl transferase activity, were increased in this type. The clustering in phylogenetic comparisons of MALDI-TOF spectra did not match the ST, biochemical phenotype and phage susceptibility. Occurrence of Campylobacter jejuni strains and phage susceptibility types with different colonisation potential seem to play a very important role in the success of phage therapy in commercial broiler houses. Thus, mechanisms of both, phage susceptibility and Campylobacter colonisation should be further investigated and considered when composing phage cocktails.

  16. Toward Understanding Phage:Host Interactions in the Rumen; Complete Genome Sequences of Lytic Phages Infecting Rumen Bacteria

    Directory of Open Access Journals (Sweden)

    Rosalind A. Gilbert

    2017-12-01

    Full Text Available The rumen is known to harbor dense populations of bacteriophages (phages predicted to be capable of infecting a diverse range of rumen bacteria. While bacterial genome sequencing projects are revealing the presence of phages which can integrate their DNA into the genome of their host to form stable, lysogenic associations, little is known of the genetics of phages which utilize lytic replication. These phages infect and replicate within the host, culminating in host lysis, and the release of progeny phage particles. While lytic phages for rumen bacteria have been previously isolated, their genomes have remained largely uncharacterized. Here we report the first complete genome sequences of lytic phage isolates specifically infecting three genera of rumen bacteria: Bacteroides, Ruminococcus, and Streptococcus. All phages were classified within the viral order Caudovirales and include two phage morphotypes, representative of the Siphoviridae and Podoviridae families. The phage genomes displayed modular organization and conserved viral genes were identified which enabled further classification and determination of closest phage relatives. Co-examination of bacterial host genomes led to the identification of several genes responsible for modulating phage:host interactions, including CRISPR/Cas elements and restriction-modification phage defense systems. These findings provide new genetic information and insights into how lytic phages may interact with bacteria of the rumen microbiome.

  17. Morphological evidence for phages in Xylella fastidiosa

    Directory of Open Access Journals (Sweden)

    Civerolo Edwin L

    2008-06-01

    Full Text Available Abstract Presumptive phage particles associated with Xylella fastidiosa strain Temecula-1 grown in PW broth were observed by transmission electron microscopy (TEM in ultrathin sections of bacterial cell-containing low speed centrifugation pellets and in partially purified preparations from CsCl equilibrium centrifugation density gradients. Ultrathin-sectioned cell pellets contained icosahedral particles of about 45 nm in diameter. Samples collected from CsCl density gradients revealed mostly non-tailed icosahedral but also tailed particles. The icosahedral particles could be divided into two types: a large type (about 45 nm and a small type (about 30 nm. Filamentous phage-like particles (17 × 120 to 6,300 nm were also observed. The presence of different types of phage-like particles resembling to those in several bacteriophage families provides new physical evidence, in addition to X. fastidiosa genomic information, that X. fastidiosa possesses active phages. This is the first report of phage particles released in X. fastidiosa cultures.

  18. Evidence for Metaviromic Islands in Marine phages

    Directory of Open Access Journals (Sweden)

    Carolina Megumi Mizuno

    2014-02-01

    Full Text Available Metagenomic islands have been defined as genomic regions in prokaryotic genomes that under-recruit from metagenomes where most of the same genome recruits at close to 100% identity over most of its length. The presence of metagenomic islands in prokaryotes has been associated to the diversity of concurrent lineages that vary at this level to disperse the predatory pressure of phages that, reciprocally, maintain high clonal diversity in the population and improve ecosystem performance. This was proposed as a Constant-Diversity (C-D model. Here we have investigated the regions of phage genomes under-recruiting in a metavirome constructed with a sample from the same habitat where they were retrieved. Some of the genes found to under-recruit are involved in host recognition as would be expected from the C-D model. Furthermore, the recruitment of intragenic regions known to be involved in molecular recognition also had a significant under-recruitment compared to the rest of the gene. However, other genes apparently disconnected from the recognition process under-recruited often, specifically the terminases involved in packaging of the phage genome in the capsid and a few others. In addition, some highly related phage genomes (at nucleotide sequence level had no metaviromic islands. We speculate that the latter might be generalist phages with broad infection range that do not require clone specific lineages.

  19. Current taxonomy of phages infecting lactic acid bacteria

    Directory of Open Access Journals (Sweden)

    Jennifer eMahony

    2014-01-01

    Full Text Available Phages infecting lactic acid bacteria have been the focus of significant research attention over the past three decades. Through the isolation and characterization of hundreds of phage isolates, it has been possible to classify phages of the dairy starter and adjunct bacteria Lactococus lactis, Streptococcus thermophilus, Leuconostoc spp. and Lactobacillus spp. Among these, phages of L. lactis have been most thoroughly scrutinized and serve as an excellent model system to address issues that arise when attempting taxonomic classification of phages infecting other LAB species. Here, we present an overview of the current taxonomy of phages infecting LAB genera of industrial significance, the methods employed in these taxonomic efforts and how these may be employed for the taxonomy of phages of currently underrepresented and emerging phage species.

  20. Sequencing and characterization of Pseudomonas aeruginosa phage JG004

    National Research Council Canada - National Science Library

    Garbe, Julia; Bunk, Boyke; Rohde, Manfred; Schobert, Max

    2011-01-01

    .... Pseudomonas aeruginosa. For an effective use of bacteriophages as antimicrobial agents, it is important to understand phage biology but also genes of the bacterial host essential for phage infection...

  1. Phages of Listeria offer novel tools for diagnostics and biocontrol

    Directory of Open Access Journals (Sweden)

    Martin J Loessner

    2014-04-01

    Full Text Available Historically, bacteriophages infecting their hosts have perhaps been best known and even notorious for being a nuisance in dairy-fermentation processes. However, with the rapid progress in molecular microbiology and microbial ecology, a new dawn has risen for phages. This review will provide an overview on possible uses and applications of Listeria phages, including phage-typing, reporter phage for bacterial diagnostics, and use of phage as biocontrol agents for food safety. The use of phage-encoded enzymes such as endolysins for the detection and as antimicrobial will also be addressed. Desirable properties of candidate phages for biocontrol will be discussed. While emphasizing the enormous future potential for applications, we will also consider some of the intrinsic limitations dictated by both phage and bacterial ecology.

  2. Phage display—A powerful technique for immunotherapy

    Science.gov (United States)

    Bazan, Justyna; Całkosiński, Ireneusz; Gamian, Andrzej

    2012-01-01

    One of the most effective molecular diversity techniques is phage display. This technology is based on a direct linkage between phage phenotype and its encapsulated genotype, which leads to presentation of molecule libraries on the phage surface. Phage display is utilized in studying protein-ligand interactions, receptor binding sites and in improving or modifying the affinity of proteins for their binding partners. Generating monoclonal antibodies and improving their affinity, cloning antibodies from unstable hybridoma cells and identifying epitopes, mimotopes and functional or accessible sites from antigens are also important advantages of this technology. Techniques originating from phage display have been applied to transfusion medicine, neurological disorders, mapping vascular addresses and tissue homing of peptides. Phages have been applicable to immunization therapies, which may lead to development of new tools used for treating autoimmune and cancer diseases. This review describes the phage display technology and presents the recent advancements in therapeutic applications of phage display. PMID:22906939

  3. Kajian Pembuatan Cokelat Batang dengan Metode Tempering dan Tanpa Tempering

    OpenAIRE

    Eti Indarti; Normalina Arpi; Slamet Budijanto*

    2013-01-01

    This research is aimed to improve stability of milk chocolate bars by tempering process. The making of chocolate bars consisted of two formulations, namely a higher fat bar (40%) and low fat bar (21.5%).The study includes the chocolate bar preparation with and without tempering results. The melting point of milk chocolate bars that use cocoa butter tempering (L1) is higher than the milk chocolate bars that use fat without tempering (L2) for all treatments. Solid fat content (SFC) of F1 has hi...

  4. Analysis of novel mycobacteriophages indicates the existence of different strategies for phage inheritance in mycobacteria.

    Directory of Open Access Journals (Sweden)

    Emma J Stella

    Full Text Available Mycobacteriophages have been essential in the development of mycobacterial genetics through their use in the construction of tools for genetic manipulation. Due to the simplicity of their isolation and variety of exploitable molecular features, we searched for and isolated 18 novel mycobacteriophages from environmental samples collected from several geographic locations. Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100-400 nm, genome size in the 50-70 kbp, morphological features compatible with those corresponding to the Siphoviridae family, however novel characteristics for propagation were noticed. Although all the mycobacteriophages propagated at 30°C, eight of them failed to propagate at 37°C. Since some of our phages yielded pinpoint plaques, we improved plaque detection by including sub-inhibitory concentrations of isoniazid or ampicillin-sulbactam in the culture medium. Thus, searches for novel mycobacteriophages at low temperature and in the presence of these drugs would allow for the isolation of novel members that would otherwise not be detected. Importantly, while eight phages lysogenized Mycobacterium smegmatis, four of them were also capable of lysogenizing Mycobacterium tuberculosis. Analysis of the complete genome sequence obtained for twelve mycobacteriophages (the remaining six rendered partial genomic sequences allowed for the identification of a new singleton. Surprisingly, sequence analysis revealed the presence of parA or parA/parB genes in 7/18 phages including four that behaved as temperate in M. tuberculosis. In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.

  5. Phage therapy in the food industry.

    Science.gov (United States)

    Endersen, Lorraine; O'Mahony, Jim; Hill, Colin; Ross, R Paul; McAuliffe, Olivia; Coffey, Aidan

    2014-01-01

    Despite advances in modern technologies, the food industry is continuously challenged with the threat of microbial contamination. The overuse of antibiotics has further escalated this problem, resulting in the increasing emergence of antibiotic-resistant foodborne pathogens. Efforts to develop new methods for controlling microbial contamination in food and the food processing environment are extremely important. Accordingly, bacteriophages (phages) and their derivatives have emerged as novel, viable, and safe options for the prevention, treatment, and/or eradication of these contaminants in a range of foods and food processing environments. Whole phages, modified phages, and their derivatives are discussed in terms of current uses and future potential as antimicrobials in the traditional farm-to-fork context, encompassing areas such as primary production, postharvest processing, biosanitation, and biodetection. The review also presents some safety concerns to ensure safe and effective exploitation of bacteriophages in the future.

  6. Pitfalls to avoid when using phage display for snake toxins

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard; Lauridsen, Line Præst; Lomonte, Bruno

    2017-01-01

    when animal toxins are used as targets for phage display selection. Here, we report selected critical challenges from our own phage display experiments associated with biotinylation of antigens, clone picking, and the presence of amber codons within antibody fragment structures in some phage display...

  7. A method for generation phage cocktail with great therapeutic potential.

    Directory of Open Access Journals (Sweden)

    Jingmin Gu

    Full Text Available BACKGROUND: Bacteriophage could be an alternative to conventional antibiotic therapy against multidrug-resistant bacteria. However, the emergence of resistant variants after phage treatment limited its therapeutic application. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an approach, named "Step-by-Step" (SBS, has been established. This method takes advantage of the occurrence of phage-resistant bacteria variants and ensures that phages lytic for wild-type strain and its phage-resistant variants are selected. A phage cocktail lytic for Klebsiella pneumoniae was established by the SBS method. This phage cocktail consisted of three phages (GH-K1, GH-K2 and GH-K3 which have different but overlapping host strains. Several phage-resistant variants of Klebsiella pneumoniae were isolated after different phages treatments. The virulence of these variants was much weaker [minimal lethal doses (MLD>1.3×10(9 cfu/mouse] than that of wild-type K7 countpart (MLD = 2.5×10(3 cfu/mouse. Compared with any single phage, the phage cocktail significantly reduced the mutation frequency of Klebsiella pneumoniae and effectively rescued Klebsiella pneumoniae bacteremia in a murine K7 strain challenge model. The minimal protective dose (MPD of the phage cocktail which was sufficient to protect bacteremic mice from lethal K7 infection was only 3.0×10(4 pfu, significantly smaller (p<0.01 than that of single monophage. Moreover, a delayed administration of this phage cocktail was still effective in protection against K7 challenge. CONCLUSIONS/SIGNIFICANCE: Our data showed that the phage cocktail was more effective in reducing bacterial mutation frequency and in the rescue of murine bacteremia than monophage suggesting that phage cocktail established by SBS method has great therapeutic potential for multidrug-resistant bacteria infection.

  8. Recognition of epoxy with phage displayed peptides.

    Science.gov (United States)

    Swaminathan, Swathi; Cui, Yue

    2013-07-01

    The development of a general approach for non-destructive chemical and biological functionalization of epoxy could expand opportunities for both fundamental studies and creating various device platforms. Epoxy shows unique electrical, mechanical, chemical and biological compatibility and has been widely used for fabricating a variety of devices. Phage display has emerged as a powerful method for selecting peptides that possess enhanced selectivity and binding affinity toward a variety of targets. In this letter, we demonstrate for the first time a powerful yet benign approach for identifying binding motifs to epoxy via comprehensively screened phage displayed peptides. Our results show that the epoxy can be selectively recognized with peptide-displaying phages. Further, along with the development of epoxy-based microstructures; recognition of the epoxy with phage displayed peptides can be specifically localized in these microstructures. We anticipate that these results could open up exciting opportunities in the use of peptide-recognized epoxy in fundamental biochemical recognition studies, as well as in applications ranging from analytical devices, hybrid materials, surface and interface, to cell biology. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. PHAGE AMPLIFICATION TECHNOLOGY IN THE DIAGNOSIS OF ...

    African Journals Online (AJOL)

    The World Health Organization recognizes the need for new, affordable, rapid and highly sensitive diagnostics for use in developing countries. Phage amplification technology employs a specific mycobacteriophage which infects a live TB bacillus if represent in a sample. These replicate and lyse the cells to release progeny ...

  10. Interaction Analysis through Proteomic Phage Display

    Directory of Open Access Journals (Sweden)

    Gustav N. Sundell

    2014-01-01

    Full Text Available Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs, or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance.

  11. Interaction Analysis through Proteomic Phage Display

    Science.gov (United States)

    2014-01-01

    Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. PMID:25295249

  12. Anatomy of a lactococcal phage tail

    NARCIS (Netherlands)

    Mc Grath, S; Neve, H; Seegers, JFML; Eijlander, R; Vegge, CS; Brondsted, L; Heller, KJ; Fitzgerald, GF; Vogensen, FK; van Sinderen, D

    Bacteriophages of the Siphoviridae family utilize a long noncontractile tail to recognize, adsorb to, and inject DNA into their bacterial host. The tail anatomy of the archetypal Siphoviridae X has been well studied, in contrast to phages infecting gram-positive bacteria. This report outlines a

  13. Optimization of lytic phage manufacturing in bioreactor using monolithic supports.

    Science.gov (United States)

    Smrekar, Franc; Ciringer, Mateja; Jančar, Janez; Raspor, Peter; Štrancar, Aleš; Podgornik, Aleš

    2011-08-01

    A process for manufacturing large quantities of lytic bacteriophages was developed. Determination of cultivation termination was found to be essential to achieve high phage quantity and purity. When optimal cultivation termination is missed, phage fraction was found to be highly contaminated with deoxyribonucleic acid released from Escherichia coli cells. Besides, an already established method for monitoring of phage cultivation based on optical density, where its peak indicates point when maximal phage titer is achieved, a new indirect chromatographic method using methacrylate monoliths is proposed for on-line estimation of phage titer. It is based on the measurement of released E. coli deoxyribonucleic acid and shows high correlation with phage titer obtained from plaque assay. Its main advantage is that the information is obtained within few minutes. In addition, the same method can also be used to determine purity of a final phage fraction. Two strategies to obtain highly pure phage fractions are proposed: an immediate purification of phage lysate using monolithic columns or an addition of EDTA before chromatographic purification. The developed protocol was shown to give phage purity above 90% and it is completed within one working day including cultivation and phage titer in the final formulation using developed chromatographic method. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Phage-bacteria interaction network in human oral microbiome.

    Science.gov (United States)

    Wang, Jinfeng; Gao, Yuan; Zhao, Fangqing

    2016-07-01

    Although increasing knowledge suggests that bacteriophages play important roles in regulating microbial ecosystems, phage-bacteria interaction in human oral cavities remains less understood. Here we performed a metagenomic analysis to explore the composition and variation of oral dsDNA phage populations and potential phage-bacteria interaction. A total of 1,711 contigs assembled with more than 100 Gb shotgun sequencing data were annotated to 104 phages based on their best BLAST matches against the NR database. Bray-Curtis dissimilarities demonstrated that both phage and bacterial composition are highly diverse between periodontally healthy samples but show a trend towards homogenization in diseased gingivae samples. Significantly, according to the CRISPR arrays that record infection relationship between bacteria and phage, we found certain oral phages were able to invade other bacteria besides their putative bacterial hosts. These cross-infective phages were positively correlated with commensal bacteria while were negatively correlated with major periodontal pathogens, suggesting possible connection between these phages and microbial community structure in oral cavities. By characterizing phage-bacteria interaction as networks rather than exclusively pairwise predator-prey relationships, our study provides the first insight into the participation of cross-infective phages in forming human oral microbiota. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  15. Twelve previously unknown phage genera are ubiquitous in global oceans.

    Science.gov (United States)

    Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh; Corrier, Kristen; Riemann, Lasse; Verberkmoes, Nathan C; Sullivan, Matthew B

    2013-07-30

    Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in "unknowns" dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage-host systems for experimental hypothesis testing.

  16. Formulation, stabilisation and encapsulation of bacteriophage for phage therapy.

    Science.gov (United States)

    Malik, Danish J; Sokolov, Ilya J; Vinner, Gurinder K; Mancuso, Francesco; Cinquerrui, Salvatore; Vladisavljevic, Goran T; Clokie, Martha R J; Garton, Natalie J; Stapley, Andrew G F; Kirpichnikova, Anna

    2017-05-14

    Against a backdrop of global antibiotic resistance and increasing awareness of the importance of the human microbiota, there has been resurgent interest in the potential use of bacteriophages for therapeutic purposes, known as phage therapy. A number of phage therapy phase I and II clinical trials have concluded, and shown phages don't present significant adverse safety concerns. These clinical trials used simple phage suspensions without any formulation and phage stability was of secondary concern. Phages have a limited stability in solution, and undergo a significant drop in phage titre during processing and storage which is unacceptable if phages are to become regulated pharmaceuticals, where stable dosage and well defined pharmacokinetics and pharmacodynamics are de rigueur. Animal studies have shown that the efficacy of phage therapy outcomes depend on the phage concentration (i.e. the dose) delivered at the site of infection, and their ability to target and kill bacteria, arresting bacterial growth and clearing the infection. In addition, in vitro and animal studies have shown the importance of using phage cocktails rather than single phage preparations to achieve better therapy outcomes. The in vivo reduction of phage concentration due to interactions with host antibodies or other clearance mechanisms may necessitate repeated dosing of phages, or sustained release approaches. Modelling of phage-bacterium population dynamics reinforces these points. Surprisingly little attention has been devoted to the effect of formulation on phage therapy outcomes, given the need for phage cocktails, where each phage within a cocktail may require significantly different formulation to retain a high enough infective dose. This review firstly looks at the clinical needs and challenges (informed through a review of key animal studies evaluating phage therapy) associated with treatment of acute and chronic infections and the drivers for phage encapsulation. An important driver

  17. Exploration of Phage-Host Interactions in Fish Pathogen Vibrio anguillarum and Anti-Phage Defense Strategies

    DEFF Research Database (Denmark)

    Tan, Demeng

    T to repress ompK expression. It was demonstrated that QS controls the choice of anti-phage defense strategies in the V. anguillarum strain PF430-3, suggesting the presence of dynamic, temporary adaptations to phage infection pressure, while still securing the ability to produce a functional OmpK receptor...... of bacterial pathogenicity development. Therefore, successful application of phage therapy in the treatment of vibriosis requires a detailed understanding of phage-host interactions, especially with regards to anti-phage defense mechanisms in the host. Part I. As a first approach, 24 V. anguillarum and 13....... In conclusion, this thesis provides a first insight into the dynamic vibriophage-host interactions, indicating the complexity of phage therapy in the treatment of vibriosis, regarding the evolution of anti-phage defense mechanisms, gene regulation, quorum sensing, biofilm formation, as well as pathogenesis...

  18. Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity

    Directory of Open Access Journals (Sweden)

    Mandal Nitai C

    2007-06-01

    Full Text Available Abstract Background Lysogenic mode of life cycle of a temperate bacteriophage is generally maintained by a protein called 'repressor'. Repressor proteins of temperate lambdoid phages bind to a few symmetric operator DNAs in order to regulate their gene expression. In contrast, repressor molecules of temperate mycobacteriophages and some other phages bind to multiple asymmetric operator DNAs. Very little is known at present about the structure-function relationship of any mycobacteriophage repressor. Results Using highly purified repressor (CI of temperate mycobacteriophage L1, we have demonstrated here that L1 CI harbors an N-terminal domain (NTD and a C-terminal domain (CTD which are separated by a small hinge region. Interestingly, CTD is more compact than NTD at 25°C. Both CTD and CI contain significant amount of α-helix at 30°C but unfold partly at 42°C. At nearly 200 nM concentration, both proteins form appreciable amount of dimers in solution. Additional studies reveal that CI binds to O64 and OL types of asymmetric operators of L1 with variable affinity at 25°C. Interestingly, repressor – operator interaction is affected drastically at 42°C. The conformational change of CI is most possibly responsible for its reduced operator binding affinity at 42°C. Conclusion Repressors encoded by mycobacteriophages differ significantly from the repressor proteins of λ and related phages at functional level but at structural level they are nearly similar.

  19. Humoral immune responses against gonadotropin releasing hormone elicited by immunization with phage-peptide constructs obtained via phage display.

    Science.gov (United States)

    Samoylov, Alexandre; Cochran, Anna; Schemera, Bettina; Kutzler, Michelle; Donovan, Caitlin; Petrenko, Valery; Bartol, Frank; Samoylova, Tatiana

    2015-12-20

    Phage display is based on genetic engineering of phage coat proteins resulting in fusion peptides displayed on the surface of phage particles. The technology is widely used for generation of phages with novel characteristics for numerous applications in biomedicine and far beyond. The focus of this study was on development of phage-peptide constructs that stimulate production of antibodies against gonadotropin releasing hormone (GnRH). Phage-peptide constructs that elicit production of neutralizing GnRH antibodies can be used for anti-fertility and anti-cancer applications. Phage-GnRH constructs were generated via selection from a phage display library using several types of GnRH antibodies as selection targets. Such phage constructs were characterized for sequence similarities to GnRH peptide and frequency of their occurrence in the selection rounds. Five of the constructs with suitable characteristics were tested in mice as a single dose 5×10(11) virions (vir) vaccine and were found to be able to stimulate production of GnRH-specific antibodies, but not to suppress testosterone (indirect indicator of GnRH antibody neutralizing properties). Next, one of the constructs was tested at a higher dose of 2×10(12) vir per mouse in combination with a poly(lactide-co-glycolide) (PLGA)-based adjuvant. This resulted in multifold increase in GnRH antibody production and significant reduction of serum testosterone, indicating that antibodies produced in response to the phage-GnRH immunization possess neutralizing properties. To achieve optimal immune responses for desired applications, phage-GnRH constructs can be modified with respect to flanking sequences of GnRH-like peptides displayed on phage. Anticipated therapeutic effects also might be attained using optimized phage doses, a combination of several constructs in a single treatment, or application of adjuvants and advanced phage delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Bioinformatic analysis of the Acinetobacter baumannii phage AB1 genome.

    Science.gov (United States)

    Li, Peizhen; Chen, Biaobang; Song, Zhijian; Song, Yulong; Yang, Yanmei; Ma, Ping; Wang, Huifeng; Ying, Jun; Ren, Ping; Yang, Lei; Gao, Guohui; Jin, Shouguang; Bao, Qiyu; Yang, Hongjiang

    2012-10-10

    As one of the pathogens of hospital-acquired infections, Acinetobacter baumannii poses great challenges to the public health. A. baumannii phage could be an effective way to fight multi-resistant A. baumannii. Here, we completed the whole genome sequencing of the complete genome of A. baumannii phage AB1, which consists of 45,159 bp and is a double-stranded DNA molecule with an average GC content of 37.7%. The genome encodes one tRNA gene and 85 open reading frames (ORFs) and the average size of the ORF is 531 bp in length. Among 85 ORFs, only 14 have been identified to share significant sequence similarities to the genes with known functions, while 28 are similar in sequence to the genes with function-unknown genes in the database and 43 ORFs are uniquely present in the phage AB1 genome. Fourteen function-assigned genes with putative functions include five phage structure proteins, an RNA polymerase, a big sub-unit and a small sub-unit of a terminase, a methylase and a recombinase and the proteins involved in DNA replication and so on. Multiple sequence alignment was conducted among those homologous proteins and the phylogenetic trees were reconstructed to analyze the evolutionary courses of these essential genes. From comparative genomics analysis, it turned out clearly that the frame of the phage genome mainly consisted of genes from Xanthomonas phages, Burkholderia ambifaria phages and Enterobacteria phages and while it comprises genes of its host A. baumannii only sporadically. The mosaic feature of the phage genome suggested that the horizontal gene transfer occurred among the phage genomes and between the phages and the host bacterium genomes. Analyzing the genome sequences of the phages should lay sound foundation to investigate how phages adapt to the environment and infect their hosts, and even help to facilitate the development of biological agents to deal with pathogenic bacteria. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Keratinocyte specific markers isolated using phage display

    DEFF Research Database (Denmark)

    Jensen, K.B.; Jensen, Ole Nørregaard; Ravn, P.

    2003-01-01

    Specific molecular markers for various normal and pathogenic cell states and cell types provide knowledge of basic biological systems and have a direct application in targeted therapy. We describe a proteomic method based on the combination of new and improved phage display antibody technologies...... display method was applied to analysis of human skin keratinocytes resulting in the isolation of a panel of antibodies. Fourteen of these antibodies were further characterized, half of which predominantly recognized keratinocytes in a screen of a range of different cell types. Three cognate keratinocyte...... antigens were subsequently identified by mass spectrometry as laminin-5, plectin, and fibronectin. The combination of phage display technology with mass spectrometry methods for protein identification is a general and promising approach for proteomic analysis of cell surface complexity....

  2. An Unusual Phage Repressor Encoded by Mycobacteriophage BPs.

    Science.gov (United States)

    Villanueva, Valerie M; Oldfield, Lauren M; Hatfull, Graham F

    2015-01-01

    Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection-lysogenic or lytic growth-as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33-34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.

  3. Delivering phage therapy per os: benefits and barriers.

    Science.gov (United States)

    Zelasko, Susan; Gorski, Andrzej; Dabrowska, Krystyna

    2017-02-01

    Multidrug-resistant bacterial infections of the gastrointestinal tract pose a serious public health concern. High levels of antibiotic drug resistance, along with the potential for antibiotics to precipitate disease or alter the gut microbiome has prompted research into alternative treatment methods. Evidence suggests that bacteriophage therapy delivered per os may be well-suited to target such infections. Areas covered: Herein, we discuss the specific advantages and challenges of using orally administered phage therapy. Our literature review encompasses recent works using phages to target various clinically-relevant bacteria in vivo. We also provide insights into methods that aim to overcome the barriers to effective phage transit through the harsh gastrointestinal environment. Expert commentary: Evidence from a number of in vivo animal studies suggests that targeting bacterial infections using phages delivered orally holds potential. Efficacious oral phage therapy depends on the delivery of sufficient phage titers to the infection site, which may be hindered by the host's gastrointestinal tract and immune response.

  4. Trivalent Cation Induced Bundle Formation of Filamentous fd Phages.

    Science.gov (United States)

    Korkmaz Zirpel, Nuriye; Park, Eun Jin

    2015-09-01

    Bacteriophages are filamentous polyelectrolyte viral rods infecting only bacteria. In this study, we investigate the bundle formation of fd phages with trivalent cations having different ionic radii (Al(3+) , La(3+) and Y(3+) ) at various phage and counterion concentrations, and at varying bundling times. Aggregated phage bundles were detected at relatively low trivalent counterion concentrations (1 mM). Although 10 mM and 100 mM Y(3+) and La(3+) treatments formed larger and more intertwined phage bundles, Al(3+) and Fe(3+) treatments lead to the formation of networking filaments. Energy dispersive X-ray spectroscopy (EDX) analyses confirmed the presence of C, N and O peaks on densely packed phage bundles. Immunofluorescence labelling and ELISA analyses with anti-p8 antibodies showed the presence of phage filaments after bundling. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Complete Genome Sequence of Pseudomonas aeruginosa Phage AAT-1.

    Science.gov (United States)

    Andrade-Domínguez, Andrés; Kolter, Roberto

    2016-08-25

    Aspects of the interaction between phages and animals are of interest and importance for medical applications. Here, we report the genome sequence of the lytic Pseudomonas phage AAT-1, isolated from mammalian serum. AAT-1 is a double-stranded DNA phage, with a genome of 57,599 bp, containing 76 predicted open reading frames. Copyright © 2016 Andrade-Domínguez and Kolter.

  6. Probing Tumor Microenvironment With In Vivo Phage Display

    Science.gov (United States)

    2014-10-01

    technology. A, Peptides found in the “Phage pool alone” group are listed in a descending order of frequency. Note that CISQERGESC (CIS) and CIFSGEGESC ( CIF ...expressing CISQERGESC (CIS: 0.6% of the recovered phage clones) and 202 phages expressing a relevant peptide CIFSGEGESC ( CIF : 0.2%) (Table 1A). The...term ended showed that CIS and CIF , which have very similar amino acid sequences, bind to cultured hb6011 CAFs especially to filopodia and fibrous

  7. Satellite phage TLCφ enables toxigenic conversion by CTX phage through dif site alteration.

    Science.gov (United States)

    Hassan, Faizule; Kamruzzaman, M; Mekalanos, John J; Faruque, Shah M

    2010-10-21

    Bacterial chromosomes often carry integrated genetic elements (for example plasmids, transposons, prophages and islands) whose precise function and contribution to the evolutionary fitness of the host bacterium are unknown. The CTXφ prophage, which encodes cholera toxin in Vibrio cholerae, is known to be adjacent to a chromosomally integrated element of unknown function termed the toxin-linked cryptic (TLC). Here we report the characterization of a TLC-related element that corresponds to the genome of a satellite filamentous phage (TLC-Knφ1), which uses the morphogenesis genes of another filamentous phage (fs2φ) to form infectious TLC-Knφ1 phage particles. The TLC-Knφ1 phage genome carries a sequence similar to the dif recombination sequence, which functions in chromosome dimer resolution using XerC and XerD recombinases. The dif sequence is also exploited by lysogenic filamentous phages (for example CTXφ) for chromosomal integration of their genomes. Bacterial cells defective in the dimer resolution often show an aberrant filamentous cell morphology. We found that acquisition and chromosomal integration of the TLC-Knφ1 genome restored a perfect dif site and normal morphology to V. cholerae wild-type and mutant strains with dif(-) filamentation phenotypes. Furthermore, lysogeny of a dif(-) non-toxigenic V. cholerae with TLC-Knφ1 promoted its subsequent toxigenic conversion through integration of CTXφ into the restored dif site. These results reveal a remarkable level of cooperative interactions between multiple filamentous phages in the emergence of the bacterial pathogen that causes cholera.

  8. Isolation and Characterization of Phages Infecting Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Anna Krasowska

    2015-01-01

    Full Text Available Bacteriophages have been suggested as an alternative approach to reduce the amount of pathogens in various applications. Bacteriophages of various specificity and virulence were isolated as a means of controlling food-borne pathogens. We studied the interaction of bacteriophages with Bacillus species, which are very often persistent in industrial applications such as food production due to their antibiotic resistance and spore formation. A comparative study using electron microscopy, PFGE, and SDS-PAGE as well as determination of host range, pH and temperature resistance, adsorption rate, latent time, and phage burst size was performed on three phages of the Myoviridae family and one phage of the Siphoviridae family which infected Bacillus subtilis strains. The phages are morphologically different and characterized by icosahedral heads and contractile (SIOΦ, SUBω, and SPOσ phages or noncontractile (ARπ phage tails. The genomes of SIOΦ and SUBω are composed of 154 kb. The capsid of SIOΦ is composed of four proteins. Bacteriophages SPOσ and ARπ have genome sizes of 25 kbp and 40 kbp, respectively. Both phages as well as SUBω phage have 14 proteins in their capsids. Phages SIOΦ and SPOσ are resistant to high temperatures and to the acid (4.0 and alkaline (9.0 and 10.0 pH.

  9. Evolution of phage display technology: from discovery to application.

    Science.gov (United States)

    Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Ahmadzadeh, Vahideh; Akbari, Bahman

    2017-03-01

    Phage display technology as a selection-based system is an attractive method for evolution of new biological drugs. Unique ability of phage libraries for displaying proteins on bacteriophage surfaces enable them to make a major contribution in diverse fields of researches related to the diagnosis and therapy of diseases. One of the great challenges facing researchers is the modification of phage display technology and the development of new applications. This article reviews the molecular basis of phage display library, and summarizes the novel and specific applications of this technique in the field of biological drugs development including therapeutic antibodies, peptides, vaccines, and catalytic antibodies.

  10. [lambda] Phage Nanobioparticle Expressing Apoptin Efficiently Suppress Human Breast Carcinoma Tumor Growth In Vivo

    National Research Council Canada - National Science Library

    Shoae-Hassani, Alireza; Keyhanvar, Peyman; Seifalian, Alexander; Mortazavi-Tabatabaei, Seyed; Ghaderi, Narmin; Issazadeh, Khosro; Amirmozafari, Nour; Verdi, Javad

    2013-01-01

      Using phages is a novel field of cancer therapy and phage nanobioparticles (NBPs) such as λ phage could be modified to deliver and express genetic cassettes into eukaryotic cells safely in contrast with animal viruses...

  11. λ Phage nanobioparticle expressing apoptin efficiently suppress human breast carcinoma tumor growth in vivo

    National Research Council Canada - National Science Library

    Shoae-Hassani, Alireza; Keyhanvar, Peyman; Seifalian, Alexander Marcus; Mortazavi-Tabatabaei, Seyed Abdolreza; Ghaderi, Narmin; Issazadeh, Khosro; Amirmozafari, Nour; Verdi, Javad

    2013-01-01

    Using phages is a novel field of cancer therapy and phage nanobioparticles (NBPs) such as λ phage could be modified to deliver and express genetic cassettes into eukaryotic cells safely in contrast with animal viruses...

  12. Three novel Pseudomonas phages isolated from composting provide insights into the evolution and diversity of tailed phages.

    Science.gov (United States)

    Amgarten, Deyvid; Martins, Layla Farage; Lombardi, Karen Cristina; Antunes, Luciana Principal; de Souza, Ana Paula Silva; Nicastro, Gianlucca Gonçalves; Kitajima, Elliott Watanabe; Quaggio, Ronaldo Bento; Upton, Chris; Setubal, João Carlos; da Silva, Aline Maria

    2017-05-04

    Among viruses, bacteriophages are a group of special interest due to their capacity of infecting bacteria that are important for biotechnology and human health. Composting is a microbial-driven process in which complex organic matter is converted into humus-like substances. In thermophilic composting, the degradation activity is carried out primarily by bacteria and little is known about the presence and role of bacteriophages in this process. Using Pseudomonas aeruginosa as host, we isolated three new phages from a composting operation at the Sao Paulo Zoo Park (Brazil). One of the isolated phages is similar to Pseudomonas phage Ab18 and belongs to the Siphoviridae YuA-like viral genus. The other two isolated phages are similar to each other and present genomes sharing low similarity with phage genomes in public databases; we therefore hypothesize that they belong to a new genus in the Podoviridae family. Detailed genomic descriptions and comparisons of the three phages are presented, as well as two new clusters of phage genomes in the Viral Orthologous Clusters database of large DNA viruses. We found sequences encoding homing endonucleases that disrupt a putative ribonucleotide reductase gene and an RNA polymerase subunit 2 gene in two of the phages. These findings provide insights about the evolution of two-subunits RNA polymerases and the possible role of homing endonucleases in this process. Infection tests on 30 different strains of bacteria reveal a narrow host range for the three phages, restricted to P. aeruginosa PA14 and three other P. aeruginosa clinical isolates. Biofilm dissolution assays suggest that these phages could be promising antimicrobial agents against P. aeruginosa PA14 infections. Analyses on composting metagenomic and metatranscriptomic data indicate association between abundance variations in both phage and host populations in the environment. The results about the newly discovered and described phages contribute to the understanding of

  13. Genome characteristics of a novel phage from Bacillus thuringiensis showing high similarity with phage from Bacillus cereus.

    Directory of Open Access Journals (Sweden)

    Yihui Yuan

    Full Text Available Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs. It had a typical genome structure consisting of three modules: the "late" region, the "lysogeny-lysis" region and the "early" region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor.

  14. Genome characteristics of a novel phage from Bacillus thuringiensis showing high similarity with phage from Bacillus cereus.

    Science.gov (United States)

    Yuan, Yihui; Gao, Meiying; Wu, Dandan; Liu, Pengming; Wu, Yan

    2012-01-01

    Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the "late" region, the "lysogeny-lysis" region and the "early" region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor.

  15. Identification of multiple integration sites for Stx-phage Phi24B in the Escherichia coli genome, description of a novel integrase and evidence for a functional anti-repressor.

    Science.gov (United States)

    Fogg, Paul C M; Gossage, Sharon M; Smith, Darren L; Saunders, Jon R; McCarthy, Alan J; Allison, Heather E

    2007-12-01

    The key virulence factor in Shiga-toxigenic Escherichia coli is the expression of Shiga toxin (Stx), which is conferred by Stx-encoding temperate lambdoid phages (Stx-phages). It had been assumed that Stx-phages would behave similarly to lambda phage. However, contrary to the lambda superinfection immunity model, it has been demonstrated that double lysogens can be produced with the Stx-phage Phi24(B). Here, the Phi24(B) integrase gene is identified, and the preferred site of integration defined. Although an E. coli int gene was identified close to the Phi24(B) integration site, it was shown not to be involved in the phage integration event. An additional six potential integration sites were identified in the E. coli genome, and three of these were confirmed experimentally. Two of the other potential sites lie within genes predicted to be essential to E. coli and are therefore unlikely to support phage integration. A Phi24(B) gene, possessing similarity to the well-characterized P22 ant gene, was identified. RT-PCR was used to demonstrate that ant is transcribed in a Phi24(B) E. coli lysogen, and expression of an anti-repressor is the likely explanation for the absence of immunity to superinfection. Demonstration of the ability of Phi24(B) to form multiple lysogens has two potentially serious impacts. First, multiple integrated prophages will drive the evolution of bacterial pathogens as novel Stx-phages emerge following intracellular mutation/recombination events. Second, multiple copies of the stx gene may lead to an increase in toxin production and consequently increased virulence.

  16. Kajian Pembuatan Cokelat Batang dengan Metode Tempering dan Tanpa Tempering

    Directory of Open Access Journals (Sweden)

    Eti Indarti

    2013-02-01

    Full Text Available This research is aimed to improve stability of milk chocolate bars by tempering process. The making of chocolate bars consisted of two formulations, namely a higher fat bar (40% and low fat bar (21.5%.The study includes the chocolate bar preparation with and without tempering results. The melting point of milk chocolate bars that use cocoa butter tempering (L1 is higher than the milk chocolate bars that use fat without tempering (L2 for all treatments. Solid fat content (SFC of F1 has higher solid phase at room temperature (55-60% in all treatments compared with milk chocolate bar F2 (40-43% and chocolate produced by UKM (Malaysia 40-48 % and soccolatte 35-38% at the same temperature (350C. Blooming was not formed on the milk chocolate bars containing cocoa butter L1, while the milk chocolate bars showed blooming with L2 treatment. Keywords: chocolate bar, tempering, moulding, melting point, solid fat content, blooming

  17. Identification of bacterial adhesins through phage display.

    OpenAIRE

    Ana Tung Ching Ching

    2012-01-01

    A leptospirose é uma zoonose de importância mundial causada por bactérias do gênero Leptospira. No Brasil, a maioria dos casos é causada por L. interrogans sorovar Copenhageni. O objetivo destre trabalho foi identificar adesinas de leptospira pela técnica de Phage display. Bibliotecas com fragmentos genômicos resultaram na idendificação de ligantes de leptospira com afinidade por tecidos de hamster. Uma varredura dessas bibliotecas contra heparan sulfato proteoglicano (HSPG) identificou como ...

  18. Inhibition of phage infection in capsule-producing Streptococcus ...

    African Journals Online (AJOL)

    SERVER

    2007-10-04

    Oct 4, 2007 ... Lactic cultures that produce capsular polysaccharides are widely used in the dairy industry. However, little information is available on their phage-cell interactions. Concanavalin A (Con A), lysozyme, and saccharides were investigated for their ability to modify phage-cell interactions in such a manner as to.

  19. Heat tolerance of dairy lactococcal c2 phages

    DEFF Research Database (Denmark)

    Nielsen, Cecilie Lykke Marvig; Basheer, Aideh; Neve, H.

    2011-01-01

    Nine Lactococcus lactis c2 phages propagated on different hosts were screened for thermal resistance in skimmed milk. Pronounced variations in thermal resistance were found. Three phages displayed high sensitivity towards heat resulting in >8 log reductions after 70 °C for 5 min, whereas the most...

  20. Production of a phage-displayed single chain variable fragment ...

    African Journals Online (AJOL)

    Purpose: To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology. Methods: Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the ...

  1. Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

    Science.gov (United States)

    Gillis, Annika; Mahillon, Jacques

    2014-01-01

    Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here. PMID:25010767

  2. Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

    Directory of Open Access Journals (Sweden)

    Annika Gillis

    2014-07-01

    Full Text Available Many bacteriophages (phages have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here.

  3. Attaching quantum dots to HER2 specific phage antibodies

    Science.gov (United States)

    Chu, Viet Ha; Nghiem, Thi Ha Lien; Huyen La, Thi; Dieu Thuy Ung, Thi; Huan Le, Quang; Thuan Tong, Kim; Liem Nguyen, Quang; Nhung Tran, Hong

    2010-06-01

    This work presents the results of the attachment of Qdot 655 ITKTM amino (PEG) quantum dots (QDs) (Invitrogen) and CdTe QDs (provided by Institute of Materials Science, VAST) to HER2 (Human Epidermal growth factor Receptor 2) specific phage antibodies (Abs) (provided by Institute of Biotechnology, VAST) in solution. The QDs were attached to the phage display specific HER2 Abs to form a complex QD-Ab. The QDs and complex QD-Ab were characterized by UV-VIS spectroscopy, transmission electron microscopy (TEM) and fluorescence microscopy. The fluorescence images show the QDs conjugated to the phage. Due to the QDs attaching to the surface, the phage dimensions were amplified, so its shape could be observed by optical microscopy. The complex QD-Ab was stable and lasted for a month. The results illustrate the value of the HER2 phage-QD complex as a cancer detection platform.

  4. Challenges in predicting the evolutionary maintenance of a phage transgene.

    Science.gov (United States)

    Schmerer, Matthew; Molineux, Ian J; Ally, Dilara; Tyerman, Jabus; Cecchini, Nicole; Bull, James J

    2014-01-01

    In prior work, a phage engineered with a biofilm-degrading enzyme (dispersin B) cleared artificial, short-term biofilms more fully than the phage lacking the enzyme. An unresolved question is whether the transgene will be lost or maintained during phage growth - its loss would limit the utility of the engineering. Broadly supported evolutionary theory suggests that transgenes will be lost through a 'tragedy of the commons' mechanism unless the ecology of growth in biofilms meets specific requirements. We test that theory here. Functional properties of the transgenic phage were identified. Consistent with the previous study, the dispersin phage was superior to unmodified phage at clearing short term biofilms grown in broth, shown here to be an effect attributable to free enzyme. However, the dispersin phage was only marginally better than control phages on short term biofilms in minimal media and was no better than control phages in clearing long term biofilms. There was little empirical support for the tragedy of the commons framework despite a strong theoretical foundation for its supposed relevance. The framework requires that the transgene imposes an intrinsic cost, yet the transgene was intrinsically neutral or beneficial when expressed from one part of the phage genome. Expressed from a different part of the genome, the transgene did behave as if intrinsically costly, but its maintenance did not benefit from spatially structured growth per se - violating the tragedy framework. Overall, the transgene was beneficial under many conditions, but no insight to its maintenance was attributable to the established evolutionary framework. The failure likely resides in system details that would be used to parameterize the models. Our study cautions against naive applications of evolutionary theory to synthetic biology, even qualitatively.

  5. Staphylococcus aureus phage types and their correlation to antibiotic resistance

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    Mehndiratta P

    2010-10-01

    Full Text Available Context: Staphylococcus aureus is one of the most devastating human pathogen. The organism has a differential ability to spread and cause outbreak of infections. Characterization of these strains is important to control the spread of infection in the hospitals as well as in the community. Aim: To identify the currently existing phage groups of Staphylococcus aureus, their prevalence and resistance to antibiotics. Materials and Methods: Study was undertaken on 252 Staphylococcus aureus strains isolated from clinical samples. Strains were phage typed and their resistance to antibiotics was determined following standard microbiological procedures. Statistical Analysis: Chi square test was used to compare the antibiotic susceptibility between methicillin resistant Staph. aureus (MRSA and methicillin sensitive S. aureus (MSSA strains. Results: Prevalence of MRSA and MSSA strains was found to be 29.36% and 70.65% respectively. Of these 17.56% of MRSA and 40.44% of MSSA strains were community acquired. All the MSSA strains belonging to phage type 81 from the community were sensitive to all the antibiotics tested including clindamycin and were resistant to penicillin. Forty five percent strains of phage group III and 39% of non-typable MRSA strains from the hospital were resistant to multiple antibiotics. Conclusion: The study revealed that predominant phage group amongst MRSA strains was phage group III and amongst MSSA from the community was phage group NA (phage type 81. MSSA strains isolated from the community differed significantly from hospital strains in their phage type and antibiotic susceptibility. A good correlation was observed between community acquired strains of phage type 81 and sensitivity to gentamycin and clindamycin.

  6. The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

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    Jens Andre Hammerl

    2015-06-01

    Full Text Available Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O\\(_{\\rm{R}}\\3 in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O\\(_{\\rm{R}}\\3 upstream of the prophage repressor gene. The O\\(_{\\rm{R}}\\3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages.

  7. Phage-Host Interactions in Flavobacterium psychrophilum and the Potential for Phage Therapy in Aquaculture

    DEFF Research Database (Denmark)

    Christiansen, Rói Hammershaimb

    , the increasing problem with antibiotic resistance has led to increased attention to the use of phages for controlling F. psychrophilum infections in aquaculture. In a synopsis and four scientific papers, this PhD project studies the potential and optimizes the use of phage therapy for treatment and prevention...... temperatures below 15°C and typically with fry mortality rates of 50-60%. Several attempts of vaccine development against RTFS have been made, but according to my knowledge no commercial vaccine is yet available. Bacterial chemotherapy is still the most effective and used treatment of RTFS today. However......, the dispersal and survival of a F. psychrophilum phage in vivo in juvenile rainbow trout after administration by three different methods was examined. Following phage administration by bath and oral intubation into the stomach, phages could be detected in the intestine, spleen, brain and kidney for 24 h before...

  8. Safety and efficacy of phage therapy via the intravenous route.

    Science.gov (United States)

    Speck, Peter; Smithyman, Anthony

    2016-02-01

    Increasing development of antimicrobial resistance is driving a resurgence in interest in phage therapy: the use of bacteriophages to treat bacterial infections. As the lytic action of bacteriophages is unaffected by the antibiotic resistance status of their bacterial target, it is thought that phage therapy may have considerable potential in the treatment of a wide range of topical and localized infections. As yet this interest has not extended to intravenous (IV) use, which is surprising given that the historical record shows that phages are likely to be safe and effective when delivered by this route. Starting almost 100 years ago, phages were administered intravenously in treatment of systemic infections including typhoid, and Staphylococcal bacteremia. There was extensive IV use of phages in the 1940s to treat typhoid, reportedly with outstanding efficacy and safety. The safety of IV phage administration is also underpinned by the detailed work of Ochs and colleagues in Seattle who have over four decades' experience with IV injection into human subjects of large doses of highly purified coliphage PhiX174. Though these subjects included a large number of immune-deficient children, no serious side effects were observed over this extended time period. The large and continuing global health problems of typhoid and Staphylococcus aureus are exacerbated by the increasing antibiotic resistance of these pathogens. We contend that these infections are excellent candidates for use of IV phage therapy. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Coevolution of CRISPR bacteria and phage in 2 dimensions

    Science.gov (United States)

    Han, Pu; Deem, Michael

    2014-03-01

    CRISPR (cluster regularly interspaced short palindromic repeats) is a newly discovered adaptive, heritable immune system of prokaryotes. It can prevent infection of prokaryotes by phage. Most bacteria and almost all archae have CRISPR. The CRISPR system incorporates short nucleotide sequences from viruses. These incorporated sequences provide a historical record of the host and predator coevolution. We simulate the coevolution of bacteria and phage in 2 dimensions. Each phage has multiple proto-spacers that the bacteria can incorporate. Each bacterium can store multiple spacers in its CRISPR. Phages can escape recognition by the CRISPR system via point mutation or recombination. We will discuss the different evolutionary consequences of point mutation or recombination on the coevolution of bacteria and phage. We will also discuss an intriguing ``dynamic phase transition'' in the number of phage as a function of time and mutation rate. We will show that due to the arm race between phages and bacteria, the frequency of spacers and proto-spacers in a population can oscillate quite rapidly.

  10. Oligopeptide M13 Phage Display in Pathogen Research

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    Michael Hust

    2013-10-01

    Full Text Available Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.

  11. Identification and genomic comparison of temperate bacteriophages derived from emetic Bacillus cereus.

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    Peiling Geng

    Full Text Available Cereulide-producing Bacillus cereus isolates can cause serious emetic (vomiting syndrome and even acute lethality. As mobile genetic elements, the exploration of prophages derived from emetic B. cereus isolates will help in our understanding of the genetic diversity and evolution of these pathogens. In this study, five temperate phages derived from cereulide-producing B. cereus strains were induced, with four of them undergoing genomic sequencing. Sequencing revealed that they all belong to the Siphoviridae family, but presented in different forms in their hosts. PfNC7401 and PfIS075 have typical icosahedral heads, probably existing alone as phagemids in the host with self-replicating capability in the lysogenic state. PfEFR-4, PfEFR-5, and PfATCC7953 have elongated heads, with the genomes of the former two identified as linear dsDNA, which could be integrated into the host genome during the lysogenic state. Genomic comparison of the four phages with others also derived from emetic B. cereus isolates showed similar genome structures and core genes, thus displaying host spectrum specificity. In addition, phylogenic analysis based on the complete genome and conserved tail fiber proteins of 36 Bacillus species-derived phages confirmed that the phages derived from emetic B. cereus strains were highly similar. Furthermore, one endolysin LysPfEFR-4 was cloned and showed lytic activity against all tested emetic B. cereus strains and cross-lytic activity against some other pathogenic bacteria, implying a potential to control bacterial contamination in the food supply.

  12. Identification and genomic comparison of temperate bacteriophages derived from emetic Bacillus cereus.

    Science.gov (United States)

    Geng, Peiling; Tian, Shen; Yuan, Zhiming; Hu, Xiaomin

    2017-01-01

    Cereulide-producing Bacillus cereus isolates can cause serious emetic (vomiting) syndrome and even acute lethality. As mobile genetic elements, the exploration of prophages derived from emetic B. cereus isolates will help in our understanding of the genetic diversity and evolution of these pathogens. In this study, five temperate phages derived from cereulide-producing B. cereus strains were induced, with four of them undergoing genomic sequencing. Sequencing revealed that they all belong to the Siphoviridae family, but presented in different forms in their hosts. PfNC7401 and PfIS075 have typical icosahedral heads, probably existing alone as phagemids in the host with self-replicating capability in the lysogenic state. PfEFR-4, PfEFR-5, and PfATCC7953 have elongated heads, with the genomes of the former two identified as linear dsDNA, which could be integrated into the host genome during the lysogenic state. Genomic comparison of the four phages with others also derived from emetic B. cereus isolates showed similar genome structures and core genes, thus displaying host spectrum specificity. In addition, phylogenic analysis based on the complete genome and conserved tail fiber proteins of 36 Bacillus species-derived phages confirmed that the phages derived from emetic B. cereus strains were highly similar. Furthermore, one endolysin LysPfEFR-4 was cloned and showed lytic activity against all tested emetic B. cereus strains and cross-lytic activity against some other pathogenic bacteria, implying a potential to control bacterial contamination in the food supply.

  13. Induction of temperate cyanophage AS-1 by heavy metal – copper

    Directory of Open Access Journals (Sweden)

    Chu Tin-Chun

    2006-02-01

    Full Text Available Abstract Background It has been reported that some marine cyanophage are temperate and can be induced from a lysogenic phase to a lytic phase by different agents such as heavy metals. However, to date no significant reports have focused on the temperate nature of freshwater cyanophage/cyanobacteria. Previous experiments with cyanophage AS-1 and cyanobacteria Anacystis nidulans have provided some evidence that AS-1 may have a lysogenic life cycle in addition to the characterized lytic cycle. Results In this study, the possible temperate A. nidulans was treated with different concentrations of heavy metal-copper. CuSO4 with concentrations of 3.1 × 10-3 M, 3.1 × 10-4 M, 3.1 × 10-5 M and 3.1 × 10-6 M were used to detect the induction of AS-1 from A. nidulans. The population of the host, unicellular cyanobacteria Anacystis nidulans, was monitored by direct count and turbidity while the amount of virus produced was derived from plaque forming units (PFU by a direct plating method. The ratio of AS-1 release from A. nidulans was also determined. From these results it appears that AS-1 lysogenic phage can be induced by copper at concentrations from 3.1 × 10-6 M to 3.1 × 10-4 M. Maximal phage induction occurred at 6 hours after addition of copper, with an optimal concentration of 3.1 × 10-6 M. Conclusion Cu2+ is a significant inducer for lysogenic cyanobacterial cells and consequently would be a potential control agent in the cyanobacteria population in fresh water ecosystems.

  14. Phage display of peptide / major histocompatibility class I complexes

    DEFF Research Database (Denmark)

    Vest Hansen, N; Ostergaard Pedersen, L; Stryhn, A

    2001-01-01

    and subsequently that ot the T cell receptor for peptide-MHC-I complex), we have fused a single chain peptide-MHC-I complex to the phage minor coat protein, gpIII, and displayed it on filamentous phage. Expression of peptide-MHC-I complexes was shown with relevant conformation-specific monoclonal antibodies and......, more importantly, with a unique "T cell receptor-like" (i. e. peptide-specific, MHC-I-restricted) antibody. Thus, properly assembled and folded peptide-MHC-I complexes can be displayed on filamentous phage. Despite the successful display, interaction with T cells could not be demonstrated....

  15. CRISPR/Cas9-mediated phage resistance is not impeded by the DNA modifications of phage T4.

    Science.gov (United States)

    Yaung, Stephanie J; Esvelt, Kevin M; Church, George M

    2014-01-01

    Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems. Certain phages have evolved countermeasures that are known to block endonucleases. For example, phage T4 not only adds hydroxymethyl groups to all of its cytosines, but also glucosylates them, a strategy that defeats almost all restriction enzymes. We sought to determine whether these DNA modifications can similarly impede CRISPR-based defenses. In a bioinformatics search, we found naturally occurring CRISPR spacers that potentially target phages known to modify their DNA. Experimentally, we show that the Cas9 nuclease from the Type II CRISPR system of Streptococcus pyogenes can overcome a variety of DNA modifications in Escherichia coli. The levels of Cas9-mediated phage resistance to bacteriophage T4 and the mutant phage T4 gt, which contains hydroxymethylated but not glucosylated cytosines, were comparable to phages with unmodified cytosines, T7 and the T4-like phage RB49. Our results demonstrate that Cas9 is not impeded by N6-methyladenine, 5-methylcytosine, 5-hydroxymethylated cytosine, or glucosylated 5-hydroxymethylated cytosine.

  16. Plasmids and packaging cell lines for use in phage display

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  17. Phage display creates innovative applications to combat hepatitis B virus

    Science.gov (United States)

    Tan, Wen Siang; Ho, Kok Lian

    2014-01-01

    Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20th century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases. PMID:25206271

  18. [Biology of two lysogenic phages from Bacillus thuringiensis MZ1].

    Science.gov (United States)

    Liao, Wei; Sun, Fan; Song, Shao-yun; Shi, Wei; Pang, Yi

    2007-02-01

    A Bacillus thuringiensis (Bt) fermentative strain MZ1 (subsp. kurstaki) , from a company in Meixian County of Guangdong Province, produce toxins during sporulation and are extensively used in the field to control pest insects (Lepidoptera) in China. But some unknown or random factors that inhibited or stopped B. t growth in the fermentation can be regarded as reflecting the exist of lysogenic phage. Therefore, strain MZI and its lysogenic phages were studied in this paper. With indicator strain ZK1, two kind of phage plaques, one with about 3mm diameter and the other with about 1mm diameter, can be observed after strain MZ1 cultured in plates or flasks was induced by mitomycin C. Then, two lysogenic phages, namely MZTP01 and MZTP02, were isolated and characterized in biology. They belonged to family Siphoviridae, which had icosahedral heads (MZTP01 :82nm x 85nm; MZTP02: 75nm x 55nm) and long tails (MZTP01: 220nm x 18nm; MZTP02: 183nm x 12nm) without flexibility. Host range examination showed that six and seven (including indicator strain ZKl) out of 113 B. t strains saved in our laboratory were sensitive to MZTP01 and MZTP02, respectively. MZTP01 was more stable than MZTP02 against pH value, ultraviolet and heat treatment, but contrary against organic solvents. For MZTP01 and MZTP02, K values in the neutralization reactions were 45 and 326, respectively. Both phages had no relationship in their antigenicity. Burst size of phage MZTP02 was 175, two times more than that of MZTP01. Latent time of MZTP02 was 40min, one times shorter than that of MZTP01. It was suggested that both phages DNA be linear dsDNA through the typical absorption curves, reaction with diphenylamine, DNase sensitivity and acridine orange staining. And this was in good accord with the previous findings that all tailed phages being dsDNA moleculars. The genomic DNA and their restriction maps showed that both molecular weights should be between 9.4 - 23kb. Both phage genomic DNAs were digested by

  19. Primary isolation strain determines both phage type and receptors recognised by Campylobacter jejuni bacteriophages.

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    Martine C Holst Sørensen

    Full Text Available In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb, host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220 as well as receptors (CPS or flagella recognised by the isolated phages.

  20. “French Phage Network”—Second Meeting Report

    Science.gov (United States)

    Torres-Barceló, Clara; Kaltz, Oliver; Froissart, Rémy; Gandon, Sylvain; Ginet, Nicolas; Ansaldi, Mireille

    2017-01-01

    The study of bacteriophages (viruses of bacteria) includes a variety of approaches, such as structural biology, genetics, ecology, and evolution, with increasingly important implications for therapeutic and industrial uses. Researchers working with phages in France have recently established a network to facilitate the exchange on complementary approaches, but also to engage new collaborations. Here, we provide a summary of the topics presented during the second meeting of the French Phage Network that took place in Marseille in November 2016. PMID:28430166

  1. Killing cancer cells by targeted drug-carrying phage nanomedicines

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    Yacoby Iftach

    2008-04-01

    Full Text Available Abstract Background Systemic administration of chemotherapeutic agents, in addition to its anti-tumor benefits, results in indiscriminate drug distribution and severe toxicity. This shortcoming may be overcome by targeted drug-carrying platforms that ferry the drug to the tumor site while limiting exposure to non-target tissues and organs. Results We present a new form of targeted anti-cancer therapy in the form of targeted drug-carrying phage nanoparticles. Our approach is based on genetically-modified and chemically manipulated filamentous bacteriophages. The genetic manipulation endows the phages with the ability to display a host-specificity-conferring ligand. The phages are loaded with a large payload of a cytotoxic drug by chemical conjugation. In the presented examples we used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. We show that targeting of phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. Conclusion The results of the proof-of concept study presented here reveal important features regarding the potential of filamentous phages to serve as drug-delivery platform, on the affect of drug solubility or hydrophobicity on the target specificity of the platform and on the effect of drug release mechanism on the potency of the platform. These results define targeted drug-carrying filamentous phage nanoparticles as a unique type of antibody-drug conjugates.

  2. Stochastic cellular fate decision making by multiple infecting lambda phage.

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    Matthew L Robb

    Full Text Available Bacteriophage lambda is a classic system for the study of cellular decision making. Both experiments and mathematical models have demonstrated the importance of viral concentration in the lysis-lysogeny decision outcome in lambda phage. However, a recent experimental study using single cell and single phage resolution reported that cells with the same viral concentrations but different numbers of infecting phage (multiplicity of infection can have markedly different rates of lysogeny. Thus the decision depends on not only viral concentration, but also directly on the number of infecting phage. Here, we attempt to provide a mechanistic explanation of these results using a simple stochastic model of the lambda phage genetic network. Several potential factors including intrinsic gene expression noise, spatial dynamics and cell-cycle effects are investigated. We find that interplay between the level of intrinsic noise and viral protein decision threshold is a major factor that produces dependence on multiplicity of infection. However, simulations suggest spatial segregation of phage particles does not play a significant role. Cellular image processing is used to re-analyse the original time-lapse movies from the recent study and it is found that higher numbers of infecting phage reduce the cell elongation rate. This could also contribute to the observed phenomena as cellular growth rate can affect transcription rates. Our model further predicts that rate of lysogeny is dependent on bacterial growth rate, which can be experimentally tested. Our study provides new insight on the mechanisms of individual phage decision making. More generally, our results are relevant for the understanding of gene-dosage compensation in cellular systems.

  3. Phage therapy against Enterococcus faecalis in dental root canals

    Directory of Open Access Journals (Sweden)

    Leron Khalifa

    2016-09-01

    Full Text Available Antibiotic resistance is an ever-growing problem faced by all major sectors of health care, including dentistry. Recurrent infections related to multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant enterococci (VRE in hospitals are untreatable and question the effectiveness of notable drugs. Two major reasons for these recurrent infections are acquired antibiotic resistance genes and biofilm formation. None of the traditionally known effective techniques have been able to efficiently resolve these issues. Hence, development of a highly effective antibacterial practice has become inevitable. One example of a hard-to-eradicate pathogen in dentistry is Enterococcus faecalis, which is one of the most common threats observed in recurrent root canal treatment failures, of which the most problematic to treat are its biofilm-forming VRE strains. An effective response against such infections could be the use of bacteriophages (phages. Phage therapy was found to be highly effective against biofilm and multidrug-resistant bacteria and has other advantages like ease of isolation and possibilities for genetic manipulations. The potential of phage therapy in dentistry, in particular against E. faecalis biofilms in root canals, is almost unexplored. Here we review the efforts to develop phage therapy against biofilms. We also focus on the phages isolated against E. faecalis and discuss the possibility of using phages against E. faecalis biofilm in root canals.

  4. The Legacy of 20th Century Phage Research.

    Science.gov (United States)

    Campbell, Allan M

    2010-09-01

    The Golden Age of Phage Research, where phage was the favored material for attacking many basic questions in molecular biology, lasted from about 1940 to 1970. The era was initiated by Ellis and Delbrück, whose analysis defined the relevant parameters to measure in studying phage growth, and depended on the fact that the contents of a plaque can comprise descendants of a single infecting particle. It ended around 1970 because definitive methods had then become available for answering the same questions in other systems. Some of the accomplishments of phage research were the demonstration by Hershey and Chase that the genetic material of phage T2 is largely composed of DNA, the construction of linkage maps of T2 and T4 by Hershey and Rotman and their extension to very short molecular distances by Benzer, and the isolation of conditionally lethal mutants in T4 by Epstein et al. and in λ by Campbell. The dissection of the phage life cycle into causal chains was explored by Edgar and Wood for T4 assembly and later in the regulation of lysogeny by Kaiser, extended to the molecular level by Ptashne and others. Restriction/modification was discovered in λ by Bertani and Weigle, and the biochemical mechanism was elucidated by Arber and by Smith.

  5. Biodiversity and biogeography of phages in modern stromatolites and thrombolites.

    Science.gov (United States)

    Desnues, Christelle; Rodriguez-Brito, Beltran; Rayhawk, Steve; Kelley, Scott; Tran, Tuong; Haynes, Matthew; Liu, Hong; Furlan, Mike; Wegley, Linda; Chau, Betty; Ruan, Yijun; Hall, Dana; Angly, Florent E; Edwards, Robert A; Li, Linlin; Thurber, Rebecca Vega; Reid, R Pamela; Siefert, Janet; Souza, Valeria; Valentine, David L; Swan, Brandon K; Breitbart, Mya; Rohwer, Forest

    2008-03-20

    Viruses, and more particularly phages (viruses that infect bacteria), represent one of the most abundant living entities in aquatic and terrestrial environments. The biogeography of phages has only recently been investigated and so far reveals a cosmopolitan distribution of phage genetic material (or genotypes). Here we address this cosmopolitan distribution through the analysis of phage communities in modern microbialites, the living representatives of one of the most ancient life forms on Earth. On the basis of a comparative metagenomic analysis of viral communities associated with marine (Highborne Cay, Bahamas) and freshwater (Pozas Azules II and Rio Mesquites, Mexico) microbialites, we show that some phage genotypes are geographically restricted. The high percentage of unknown sequences recovered from the three metagenomes (>97%), the low percentage similarities with sequences from other environmental viral (n = 42) and microbial (n = 36) metagenomes, and the absence of viral genotypes shared among microbialites indicate that viruses are genetically unique in these environments. Identifiable sequences in the Highborne Cay metagenome were dominated by single-stranded DNA microphages that were not detected in any other samples examined, including sea water, fresh water, sediment, terrestrial, extreme, metazoan-associated and marine microbial mats. Finally, a marine signature was present in the phage community of the Pozas Azules II microbialites, even though this environment has not been in contact with the ocean for tens of millions of years. Taken together, these results prove that viruses in modern microbialites display biogeographical variability and suggest that they may be derived from an ancient community.

  6. Pitfalls to avoid when using phage display for snake toxins.

    Science.gov (United States)

    Laustsen, Andreas Hougaard; Lauridsen, Line Præst; Lomonte, Bruno; Andersen, Mikael Rørdam; Lohse, Brian

    2017-02-01

    Antivenoms against bites and stings from snakes, spiders, and scorpions are associated with immunological side effects and high cost of production, since these therapies are still derived from the serum of hyper-immunized production animals. Biotechnological innovations within envenoming therapies are thus warranted, and phage display technology may be a promising avenue for bringing antivenoms into the modern era of biologics. Although phage display technology represents a robust and high-throughput approach for the discovery of antibody-based antitoxins, several pitfalls may present themselves when animal toxins are used as targets for phage display selection. Here, we report selected critical challenges from our own phage display experiments associated with biotinylation of antigens, clone picking, and the presence of amber codons within antibody fragment structures in some phage display libraries. These challenges may be detrimental to the outcome of phage display experiments, and we aim to help other researchers avoiding these pitfalls by presenting their solutions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Phage therapy against Enterococcus faecalis in dental root canals

    Science.gov (United States)

    Khalifa, Leron; Shlezinger, Mor; Beyth, Shaul; Houri-Haddad, Yael; Coppenhagen-Glazer, Shunit; Beyth, Nurit; Hazan, Ronen

    2016-01-01

    Antibiotic resistance is an ever-growing problem faced by all major sectors of health care, including dentistry. Recurrent infections related to multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant enterococci (VRE) in hospitals are untreatable and question the effectiveness of notable drugs. Two major reasons for these recurrent infections are acquired antibiotic resistance genes and biofilm formation. None of the traditionally known effective techniques have been able to efficiently resolve these issues. Hence, development of a highly effective antibacterial practice has become inevitable. One example of a hard-to-eradicate pathogen in dentistry is Enterococcus faecalis, which is one of the most common threats observed in recurrent root canal treatment failures, of which the most problematic to treat are its biofilm-forming VRE strains. An effective response against such infections could be the use of bacteriophages (phages). Phage therapy was found to be highly effective against biofilm and multidrug-resistant bacteria and has other advantages like ease of isolation and possibilities for genetic manipulations. The potential of phage therapy in dentistry, in particular against E. faecalis biofilms in root canals, is almost unexplored. Here we review the efforts to develop phage therapy against biofilms. We also focus on the phages isolated against E. faecalis and discuss the possibility of using phages against E. faecalis biofilm in root canals. PMID:27640530

  8. A novel helper phage for HaloTag-mediated co-display of enzyme and substrate on phage.

    Science.gov (United States)

    Delespaul, Wouter; Peeters, Yves; Herdewijn, Piet; Robben, Johan

    2015-05-01

    Phage display is an established technique for the molecular evolution of peptides and proteins. For the selection of enzymes based on catalytic activity however, simultaneous coupling of an enzyme and its substrate to the phage surface is required. To facilitate this process of co-display, we developed a new helper phage displaying HaloTag, a modified haloalkane dehalogenase that binds specifically and covalently to functionalized haloalkane ligands. The display of functional HaloTag was demonstrated by capture on streptavidin-coated magnetic beads, after coupling a biotinylated haloalkane ligand, or after on-phage extension of a DNA oligonucleotide primer with a biotinylated nucleotide by phi29 DNA polymerase. We also achieved co-display of HaloTag and phi29 DNA polymerase, thereby opening perspectives for the molecular evolution of this enzyme (and others) towards new substrate specificities. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Genome organisation of the Acinetobacter lytic phage ZZ1 and comparison with other T4-like Acinetobacter phages.

    Science.gov (United States)

    Jin, Jing; Li, Zhen-Jiang; Wang, Shu-Wei; Wang, Shan-Mei; Chen, Song-Jian; Huang, De-Hai; Zhang, Gai; Li, Ya-Hui; Wang, Xiao-Ting; Wang, Jin; Zhao, Guo-Qiang

    2014-09-14

    Phage ZZ1, which efficiently infects pathogenic Acinetobacter baumannii strains, is the fifth completely sequenced T4-like Acinetobacter phage to date. To gain a better understanding of the genetic characteristics of ZZ1, bioinformatics and comparative genomic analyses of the T4 phages were performed. The 166,687-bp double-stranded DNA genome of ZZ1 has the lowest GC content (34.4%) of the sequenced T4-like Acinetobacter phages. A total of 256 protein-coding genes and 8 tRNA genes were predicted. Forty-three percent of the predicted ZZ1 proteins share up to 73% amino acid identity with T4 proteins, and the homologous genes generally retained the same order and transcriptional direction. Beyond the conserved structural and DNA replication modules, T4 and ZZ1 have diverged substantially by the acquisition and deletion of large blocks of unrelated genes, especially in the first halves of their genomes. In addition, ZZ1 and the four other T4-like Acinetobacter phage genomes (Acj9, Acj61, 133, and Ac42) share a well-organised and highly conserved core genome, particularly in the regions encoding DNA replication and virion structural proteins. Of the ZZ1 proteins, 70, 64, 61, and 56% share up to 86, 85, 81, and 83% amino acid identity with Acj9, Acj61, 133, and Ac42 proteins, respectively. ZZ1 has a different number and types of tRNAs than the other 4 Acinetobacter phages, although some of the ZZ1-encoded tRNAs share high sequence similarity with the tRNAs from these phages. Over half of ZZ1-encoded tRNAs (5 out of 8) are related to optimal codon usage for ZZ1 proteins. However, this correlation was not present in any of the other 4 Acinetobacter phages. The comparative genomic analysis of these phages provided some new insights into the evolution and diversity of Acinetobacter phages, which might elucidate the evolutionary origin and host-specific adaptation of these phages.

  10. Effect of Bacteriophages on the Growth of Flavobacterium psychrophilum and Development of Phage-Resistant Strains

    DEFF Research Database (Denmark)

    Christiansen, Rói Hammershaimb; Madsen, Lone; Dalsgaard, Inger

    2016-01-01

    concentration (MOI = 0.3–4) was crucial for efficient viral lysis, resulting in a 104–105-fold reduction of phage-sensitive cells (both single phages and phage cocktails), which was maintained throughout the incubation (>10 days). Following cell lysis, regrowth of phage-resistant strains was examined...... and resistant strains were isolated for further characterization. The application of a mathematical model allowed simulation of phage-host interactions and resistance development, confirming indications from strain isolations that phage-sensitive strains dominated the regrowing population (>99.8 %) at low MOI...... and phage-resistant strains (>87.8 %) dominated at high MOI. A cross-infectivity test covering 68 isolated strains and 22 phages resulted in 23 different host susceptibility patterns, with 20 of the isolates being resistant to all the applied phages. Eleven isolated strains with different susceptibility...

  11. Comparative Omics and Trait Analyses of Marine Pseudoalteromonas Phages Advance the Phage OTU Concept

    Directory of Open Access Journals (Sweden)

    Melissa B. Duhaime

    2017-07-01

    Full Text Available Viruses influence the ecology and evolutionary trajectory of microbial communities. Yet our understanding of their roles in ecosystems is limited by the paucity of model systems available for hypothesis generation and testing. Further, virology is limited by the lack of a broadly accepted conceptual framework to classify viral diversity into evolutionary and ecologically cohesive units. Here, we introduce genomes, structural proteomes, and quantitative host range data for eight Pseudoalteromonas phages isolated from Helgoland (North Sea, Germany and use these data to advance a genome-based viral operational taxonomic unit (OTU definition. These viruses represent five new genera and inform 498 unaffiliated or unannotated protein clusters (PCs from global virus metagenomes. In a comparison of previously sequenced Pseudoalteromonas phage isolates (n = 7 and predicted prophages (n = 31, the eight phages are unique. They share a genus with only one other isolate, Pseudoalteromonas podophage RIO-1 (East Sea, South Korea and two Pseudoalteromonas prophages. Mass-spectrometry of purified viral particles identified 12–20 structural proteins per phage. When combined with 3-D structural predictions, these data led to the functional characterization of five previously unidentified major capsid proteins. Protein functional predictions revealed mechanisms for hijacking host metabolism and resources. Further, they uncovered a hybrid sipho-myovirus that encodes genes for Mu-like infection rarely described in ocean systems. Finally, we used these data to evaluate a recently introduced definition for virus populations that requires members of the same population to have >95% average nucleotide identity across at least 80% of their genes. Using physiological traits and genomics, we proposed a conceptual model for a viral OTU definition that captures evolutionarily cohesive and ecologically distinct units. In this trait-based framework, sensitive hosts are

  12. Four Escherichia coli O157:H7 phages: a new bacteriophage genus and taxonomic classification of T1-like phages.

    Directory of Open Access Journals (Sweden)

    Yan D Niu

    Full Text Available The T1-like bacteriophages vB_EcoS_AHP24, AHS24, AHP42 and AKS96 of the family Siphoviridae were shown to lyse common phage types of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157:H7, but not non-O157 E. coli. All contained circularly permuted genomes of 45.7-46.8 kb (43.8-44 mol% G+C encoding 74-81 open reading frames and 1 arginyl-tRNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the structural proteins were identical among the four phages. Further proteomic analysis identified seven structural proteins responsible for tail fiber, tail tape measure protein, major capsid, portal protein as well as major and minor tail proteins. Bioinformatic analyses on the proteins revealed that genomes of AHP24, AHS24, AHP42 and AKS96 did not encode for bacterial virulence factors, integration-related proteins or antibiotic resistance determinants. All four phages were highly lytic to STEC O157:H7 with considerable potential as biocontrol agents. Comparative genomic, proteomic and phylogenetic analysis suggested that the four phages along with 17 T1-like phage genomes from database of National Center for Biotechnology Information (NCBI can be assigned into a proposed subfamily "Tunavirinae" with further classification into five genera, namely "Tlslikevirus" (TLS, FSL SP-126, "Kp36likevirus" (KP36, F20, Tunalikevirus (T1, ADB-2 and Shf1, "Rtplikevirus" (RTP, vB_EcoS_ACG-M12 and "Jk06likevirus" (JK06, vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96, phiJLA23, phiKP26, phiEB49. The fact that the viruses related to JK06 have been isolated independently in Israel (JK06 (GenBank Assession #, NC_007291, Canada (vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96 and Mexico (phiKP26, phiJLA23 (between 2005 and 2011 indicates that these similar phages are widely distributed, and that horizontal gene transfer does not always prevent the characterization of bacteriophage evolution. With this new scheme, any new discovered phages with same type

  13. The long-term effects of phage concentration on the inhibition of planktonic bacterial cultures.

    Science.gov (United States)

    Worley-Morse, Thomas O; Zhang, Lucy; Gunsch, Claudia K

    2014-01-01

    Since the early 1920s there has been an interest in using bacteriophages (phages) for the control of bacterial pathogens. While there are many factors that have limited the success of phage bio-control, one particular problem is the variability of outcomes between phages and bacteria. Specifically, there is a significant need for a better understanding of how initial phage concentrations affect long-term bacterial inhibition. In work reported herein three phages were isolated for Escherichia coli K12, Pseudomonas aeruginosa PAO1, as well as Bacillus cereus and bio-control experiments were performed with phage concentrations ranging from 10(5) to 10(8) plaque forming units per mL over the course of 72 h. For four of the nine phages isolated there was a linear relationship between inhibition and phage concentration, suggesting the effect of phage concentration is important at longer time scales. For three of the isolated phages, phage concentrations had no effect on bacterial inhibition suggesting that even at the lowest concentration the method of action was saturated and lower concentrations might still be effective. Additionally, a cocktail was created and was compared to the previously isolated phages. There was no statistical difference between the cocktail and the best performing phage highlighting the importance of selecting the appropriate phages for treatment. These results suggest that, for certain phages, there is a strong relationship between phage concentration and long-term bacterial growth inhibition and the initial phage concentration is an important indicator of the long-term outcome.

  14. Lactococcal 936-type phages and dairy fermentation problems: from detection to evolution and prevention

    Directory of Open Access Journals (Sweden)

    Jennifer eMahony

    2012-09-01

    Full Text Available The so-called 936-type phages are the most frequently encountered lactococcal phage species in dairy fermentations, where they cause slow or even failed fermentations with concomitant economic losses. Several dairy phage population studies, performed in different geographical locations, have detailed their dominance in dairy phage populations, while various phage-resistance mechanisms have been assessed in a bid to protect against this virulent phage group. The impact of thermal and chemical treatments on 936 phages is an important aspect for dairy technologists and has been assessed in several studies, and has indicated that these phages have adapted to better resist such treatments. The abundance of 936 phage genome sequences has permitted a focused view on genomic content and regions of variation, and the role of such variable regions in the evolution of these phages. Here, we present an overview on detection and global prevalence of the 936 phages, together with their tolerance to industrial treatments and anti-phage strategies. Furthermore, we present a comprehensive review on the comparative genomic analyses of members of this fascinating phage species.

  15. Lactococcal 936-type phages and dairy fermentation problems: from detection to evolution and prevention.

    Science.gov (United States)

    Mahony, Jennifer; Murphy, James; van Sinderen, Douwe

    2012-01-01

    The so-called 936-type phages are the most frequently encountered lactococcal phage species in dairy fermentations, where they cause slow or even failed fermentations with concomitant economic losses. Several dairy phage population studies, performed in different geographical locations, have detailed their dominance in dairy phage populations, while various phage-resistance mechanisms have been assessed in a bid to protect against this virulent phage group. The impact of thermal and chemical treatments on 936 phages is an important aspect for dairy technologists and has been assessed in several studies, and has indicated that these phages have adapted to better resist such treatments. The abundance of 936 phage genome sequences has permitted a focused view on genomic content and regions of variation, and the role of such variable regions in the evolution of these phages. Here, we present an overview on detection and global prevalence of the 936 phages, together with their tolerance to industrial treatments and anti-phage strategies. Furthermore, we present a comprehensive review on the comparative genomic analyses of members of this fascinating phage species.

  16. Impact of a Single Phage and a Phage Cocktail Application in Broilers on Reduction of Campylobacter jejuni and Development of Resistance

    Science.gov (United States)

    Fischer, Samuel; Kittler, Sophie; Klein, Günter; Glünder, Gerhard

    2013-01-01

    Campylobacteriosis is currently the most frequent foodborne zoonosis in many countries. One main source is poultry. The aim of this study was to enhance the knowledge about the potential of bacteriophages in reducing colonization of broilers with Campylobacter , as there are only a few in vivo studies published. Commercial broilers were inoculated with 104 CFU/bird of a Campylobacter jejuni field strain. Groups of 88 birds each were subsequently treated with a single phage or a four-phage cocktail (107 PFU/bird in CaCO3 buffered SM-Buffer). Control birds received the solvent only. Afterwards, subgroups of eleven birds each were examined for their loads with phages and Campylobacter on day 1, 3, 7, 14, 21, 28, 35 and 42 after phage application. The susceptibility of the Campylobacter population to phage infection was determined using ten isolates per bird. In total 4180 re-isolates were examined. The study demonstrated that the deployed phages persisted over the whole investigation period. The Campylobacter load was permanently reduced by the phage-cocktail as well as by the single phage. The reduction was significant between one and four weeks after treatment and reached a maximum of log10 2.8 CFU/g cecal contents. Phage resistance rates of initially up to 43% in the single phage treated group and 24% in the cocktail treated group later stabilized at low levels. The occurrence of phage resistance influenced but did not override the Campylobacter reducing effect. Regarding the reduction potential, the cocktail treatment had only a small advantage over the singe phage treatment directly after phage administration. However, the cocktail moderated and delayed the emergence of phage resistance. PMID:24205254

  17. Diversity and Geographical Distribution of Flavobacterium psychrophilum Isolates and Their Phages: Patterns of Susceptibility to Phage Infection and Phage Host Range

    DEFF Research Database (Denmark)

    Castillo, Daniel; Christiansen, Rói Hammershaimb; Espejo, Romilio

    2014-01-01

    in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme...

  18. Genome sequence of temperate bacteriophage Psymv2 from Antarctic Dry Valley soil isolate Psychrobacter sp. MV2.

    Science.gov (United States)

    Meiring, Tracy L; Tuffin, I Marla; Cary, Craig; Cowan, Don A

    2012-09-01

    A temperate phage, Psymv2, was isolated from an Antarctic soil bacterium, Psychrobacter sp. MV2. The morphology of Psymv2 was typical of the Siphoviridae, with an isometric head and non-contractile tail. The Psymv2 genome was found to be 35,725 bp in length, had a G + C content of 44.5 %, with 49 protein-coding genes and one tRNA gene predicted. Integration of Psymv2 occurred at an ssrA gene, with the last 27 bases of this gene directly repeated at the prophage ends. The genome was organised in a modular fashion: integration, regulation, packaging, head assembly, tail assembly, host specificity and lysis. While the genome sequence had little similarity on a nucleotide level to previously reported phage sequences, the genome architecture resembled that of Siphoviridae of low G + C Gram-positive bacteria. The closest relatives to Psymv2 were uncharacterized putative prophages within the P. arcticus 273-4 and Acinetobacter baumannii 6013113 genomes. Global alignment of the Psymv2 genome and these prophages revealed significant conservation of the structural modules despite the large spatial divergence of their hosts. A number of unique ORFs were identified in the Psymv2 genome that may contribute to phage and lysogen fitness.

  19. A selective integrated tempering method.

    Science.gov (United States)

    Yang, Lijiang; Qin Gao, Yi

    2009-12-07

    In this paper, based on the integrated tempering sampling we introduce a selective integrated tempering sampling (SITS) method for the efficient conformation sampling and thermodynamics calculations for a subsystem in a large one, such as biomolecules solvated in aqueous solutions. By introducing a potential surface scaled with temperature, the sampling over the configuration space of interest (e.g., the solvated biomolecule) is selectively enhanced but the rest of the system (e.g., the solvent) stays largely unperturbed. The applications of this method to biomolecular systems allow highly efficient sampling over both energy and configuration spaces of interest. Comparing to the popular and powerful replica exchange molecular dynamics (REMD), the method presented in this paper is significantly more efficient in yielding relevant thermodynamics quantities (such as the potential of mean force for biomolecular conformational changes in aqueous solutions). It is more important that SITS but not REMD yielded results that are consistent with the traditional umbrella sampling free energy calculations when explicit solvent model is used since SITS avoids the sampling of the irrelevant phase space (such as the boiling water at high temperatures).

  20. Selection of phage antibodies to surface epitopes of Phytophthora infestans.

    Science.gov (United States)

    Gough, K C; Li, Y; Vaughan, T J; Williams, A J; Cockburn, W; Whitelam, G C

    1999-08-31

    Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen, Phytophthora infestans, were isolated from a diverse phage library displaying single-chain Fv (scFv) antibody fragments. The library was subpanned against external soluble components released from mycelia, sporangia and germlings and a discrete population of phage antibodies isolated. Binding of monoclonal phage antibodies was demonstrated by enzyme-linked immunosorbent assay (ELISA) and diversity was established by BstNI restriction enzyme digest patterns. Antibodies were subcloned as fusions at the C-terminus of maltose binding protein (MBP) and expressed as soluble proteins in Escherichia coli. These antibody fusion proteins bound to P. infestans germlings and to mycelial homogenates from various Phytophthora species. The binding activities to mycelial homogenates of fungal species not belonging to the order Peronosporales were substantially lower. Several phage-displayed scFvs were used in conjunction with fluorescently labelled antiphage antibody to visualise the distribution of their cognate epitopes on the surface of the germlings. The combination of procedures developed here with Phytophthora demonstrates the potential of phage antibody technology in isolating antibodies to cell surface and external soluble components of pathogens, some of which may play a role in host/pathogen interactions.

  1. Nongenetic individuality in the host-phage interaction.

    Directory of Open Access Journals (Sweden)

    Sivan Pearl

    2008-05-01

    Full Text Available Isogenic bacteria can exhibit a range of phenotypes, even in homogeneous environmental conditions. Such nongenetic individuality has been observed in a wide range of biological processes, including differentiation and stress response. A striking example is the heterogeneous response of bacteria to antibiotics, whereby a small fraction of drug-sensitive bacteria can persist under extensive antibiotic treatments. We have previously shown that persistent bacteria enter a phenotypic state, identified by slow growth or dormancy, which protects them from the lethal action of antibiotics. Here, we studied the effect of persistence on the interaction between Escherichia coli and phage lambda. We used long-term time-lapse microscopy to follow the expression of green fluorescent protein (GFP under the phage lytic promoter, as well as cellular fate, in single infected bacteria. Intriguingly, we found that, whereas persistent bacteria are protected from prophage induction, they are not protected from lytic infection. Quantitative analysis of gene expression reveals that the expression of lytic genes is suppressed in persistent bacteria. However, when persistent bacteria switch to normal growth, the infecting phage resumes the process of gene expression, ultimately causing cell lysis. Using mathematical models for these two host-phage interactions, we found that the bacteria's nongenetic individuality can significantly affect the population dynamics, and might be relevant for understanding the coevolution of bacterial hosts and phages.

  2. Temper Fragileness Study for RUL 2 Steel

    Directory of Open Access Journals (Sweden)

    Rodica Rădulea

    2007-10-01

    Full Text Available This paper presents an experimental study about the tenacity variation depending on the tempering temperature of the steel RUL2 grade, within q wide range of temperatures. By this analysis it is possible to study the cooling of the above mentioned heat treatment on the temper fragileness.

  3. Phenotypic resistance and the dynamics of bacterial escape from phage control

    DEFF Research Database (Denmark)

    Bull, James J.; Vegge, Christina Skovgaard; Schmerer, Matthew

    2014-01-01

    The canonical view of phage - bacterial interactions in dense, liquid cultures is that the phage will eliminate most of the sensitive cells; genetic resistance will then ascend to restore high bacterial densities. Yet there are various mechanisms by which bacteria may remain sensitive to phages...... be specific to the receptors used by phages, awareness of its mechanisms may identify ways of improving the choice of phages for therapy. Phenotypic resistance can also explain several enigmas in the ecology of phage-bacterial dynamics. Phenotypic resistance does not preclude the evolution of genetic...... but still attain high densities in their presence - because bacteria enter a transient state of reduced adsorption. Importantly, these mechanisms may be cryptic and inapparent prior to the addition of phage yet result in a rapid rebound of bacterial density after phage are introduced. We describe...

  4. Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform

    National Research Council Canada - National Science Library

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Borgogni, Erica; Castellino, Flora; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    ...), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts...

  5. Characterization of a phiBP endolysin encoded by the Paenibacillus polymyxa CCM 7400 phage.

    Science.gov (United States)

    Ugorcakova, Jana; Medzova, Livia; Solteszova, Barbora; Bukovska, Gabriela

    2015-07-01

    Endolysin (gp1.2) from the Paenibacillus polymyxa CCM 7400 temperate phage phiBP has a modular structure consisting of an N-terminal region with a catalytic glycosyl hydrolase 25 domain and a C-terminal cell wall-binding domain. The entire gene of this endolysin and fragments containing its catalytic and binding domains separately were cloned into expression vectors and the corresponding recombinant proteins were expressed in Escherichia coli and purified by affinity chromatography. The lytic activities of endolysin and its catalytic domain were tested on cell wall substrates from paenibacilli, bacilli, corynebacteria and E. coli. The presence of a cell wall-binding domain was found to be essential, as the phiBP endolysin was fully active only as a full-length protein. The binding ability of the cell wall-binding domain alone and in fusion with green fluorescent protein was demonstrated by specific binding assays to the cell surface of P. polymyxa CCM 7400 and to those of other Paenibacillus strains. Thus the ability of phiBP endolysin to hydrolyze the paenibacilli cell wall was confirmed. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. A multiplex real-time PCR for the detection and differentiation of Campylobacter phages.

    Science.gov (United States)

    Jäckel, Claudia; Hammerl, Jens A; Rau, Jörg; Hertwig, Stefan

    2017-01-01

    Campylobacter jejuni and C. coli are important food-borne pathogens that are widespread in animal husbandry. To combat Campylobacter along the food chain, the application of lytic phages has been shown to be a promising tool. Campylobacter phages are currently classified into three groups, of which group II and group III phages are the most common. Members of each group are closely related, whereas the two groups share only little DNA similarity. Moreover, while group III phages are specific for C. jejuni, group II phages additionally infect C. coli. Phage cocktails intended to be used for applications should be composed of various phages that differ in their host range and growth kinetics. The isolation of phages is generally performed by plaque assays. This approach has the limitation that phages are merely identified by their lytic activity on certain indicator strains and that relatively high numbers of phages must be present in a tested sample. Therefore, a more sensitive molecular detection system would be beneficial, which allows a pre-screening of samples and the quick detection and discrimination of group II and group III phages. New phages can then be isolated by use of indicator strains that may be different from those typically applied. On the basis of available Campylobacter phage genome sequences, we developed a multiplex PCR system for group II and group III phages selecting the tail tube gene and the gene for the base plate wedge, respectively, as target. Phages of both groups could be identified with primers deduced from the putative tail fiber gene. Efficient release of phage DNA from capsids was achieved by an extended heat treatment or digestion of phage particles with proteinase K/SDS yielding a detection limit of 1 pfu/ml. Individual detection of group II phages, group III phages and of both groups was studied with artificially contaminated chicken skin. To recover phages that had strongly adhered to the skin, stomaching was the most efficient

  7. Parallel tempering for the traveling salesman problem

    Energy Technology Data Exchange (ETDEWEB)

    Percus, Allon [Los Alamos National Laboratory; Wang, Richard [UCLA MATH DEPT; Hyman, Jeffrey [UCLA MATH DEPT; Caflisch, Russel [UCLA MATH DEPT

    2008-01-01

    We explore the potential of parallel tempering as a combinatorial optimization method, applying it to the traveling salesman problem. We compare simulation results of parallel tempering with a benchmark implementation of simulated annealing, and study how different choices of parameters affect the relative performance of the two methods. We find that a straightforward implementation of parallel tempering can outperform simulated annealing in several crucial respects. When parameters are chosen appropriately, both methods yield close approximation to the actual minimum distance for an instance with 200 nodes. However, parallel tempering yields more consistently accurate results when a series of independent simulations are performed. Our results suggest that parallel tempering might offer a simple but powerful alternative to simulated annealing for combinatorial optimization problems.

  8. Influence of environmental factors on phage-bacteria interaction and on the efficacy and infectivity of phage P100

    Directory of Open Access Journals (Sweden)

    Susanne Fister

    2016-07-01

    Full Text Available When using bacteriophages to control food-borne bacteria in food production plants and processed food, it is crucial to consider that environmental conditions influence their stability. These conditions can also affect the physiological state of bacteria and consequently host-virus interaction and the effectiveness of the phage ability to reduce bacteria numbers. In this study we investigated the stability, binding and replication capability of phage P100 and its efficacy to control L. monocytogenes under conditions typically encountered in dairy plants. The influences of SDS, Lutensol AO 7, salt, smear water and different temperatures were investigated. Results indicate that phage P100 is stable and able to bind to the host under most conditions tested. Replication was dependent upon the growth of L. monocytogenes and efficacy was higher when bacterial growth was reduced by certain environmental conditions. In long-term experiments at different temperatures phages were initially able to reduce bacteria up to seven log10 units after two weeks at 4 °C. However, thereafter re-growth and development of phage-resistant L. monocytogenes isolates were encountered.

  9. Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase.

    Science.gov (United States)

    Mandali, Sridhar; Gupta, Kushol; Dawson, Anthony R; Van Duyne, Gregory D; Johnson, Reid C

    2017-06-01

    The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes , but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine. IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that

  10. PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data.

    Science.gov (United States)

    Garneau, Julian R; Depardieu, Florence; Fortier, Louis-Charles; Bikard, David; Monot, Marc

    2017-08-15

    The worrying rise of antibiotic resistance in pathogenic bacteria is leading to a renewed interest in bacteriophages as a treatment option. Novel sequencing technologies enable description of an increasing number of phage genomes, a critical piece of information to understand their life cycle, phage-host interactions, and evolution. In this work, we demonstrate how it is possible to recover more information from sequencing data than just the phage genome. We developed a theoretical and statistical framework to determine DNA termini and phage packaging mechanisms using NGS data. Our method relies on the detection of biases in the number of reads, which are observable at natural DNA termini compared with the rest of the phage genome. We implemented our method with the creation of the software PhageTerm and validated it using a set of phages with well-established packaging mechanisms representative of the termini diversity, i.e. 5'cos (Lambda), 3'cos (HK97), pac (P1), headful without a pac site (T4), DTR (T7) and host fragment (Mu). In addition, we determined the termini of nine Clostridium difficile phages and six phages whose sequences were retrieved from the Sequence Read Archive. PhageTerm is freely available (https://sourceforge.net/projects/phageterm), as a Galaxy ToolShed and on a Galaxy-based server (https://galaxy.pasteur.fr).

  11. A fast method for large-scale isolation of phages from hospital ...

    African Journals Online (AJOL)

    Yomi

    2012-03-15

    Mar 15, 2012 ... Adsorption of phage to its host bacteria was performed with 0.1. MOI, then the bacteria was cultured at 37°C, at 200 rpm. An aliquot of 1 μl of culture medium was collected at the indicated time and the phage titer was measured by serial dilution. Identification of the phage genome and morphology.

  12. Multidimensional metrics for estimating phage abundance, distribution, gene density, and sequence coverage in metagenomes

    Directory of Open Access Journals (Sweden)

    Ramy Karam Aziz

    2015-05-01

    Full Text Available Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set of publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. We propose adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution.

  13. Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

    DEFF Research Database (Denmark)

    Sørensen, Martine C. Holst; Gencay, Yilmaz Emre; Birk, Tina

    2015-01-01

    In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated...

  14. Selective posttranslational modification of phage-displayed polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-11-19

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2] cycloaddition reactions and Staudinger modifications.

  15. Selective posttranslational modification of phage-displayed polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-02-05

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2]cycloaddition reactions and Staudinger modifications.

  16. Quantifying the significance of phage attack on starter cultures: a mechanistic model for population dynamics of phage and their hosts isolated from fermenting sauerkraut.

    Science.gov (United States)

    Mudgal, P; Breidt, F; Lubkin, S R; Sandeep, K P

    2006-06-01

    We investigated the possibility of using starter cultures in sauerkraut fermentation and thereby reducing the quantity of salt used in the process. This, in turn, would reduce the amount of waste salt that would enter in our water resources. Phage, naturally present in sauerkraut fermentation, could potentially affect the starter cultures introduced. Thus, a mechanistic mathematical model was developed to quantify the growth kinetics of the phage and starter cultures. The model was validated by independent experiments with two Leuconostoc mesenteroides strains isolated from sauerkraut and their corresponding phage. Model simulations and experimental evidence showed the presence of phage-resistant cell populations in starter cultures which replaced phage-sensitive cells, even when the initial phage density (P(0)) and multiplicity of infection (MOI) were low (P(0) temperature and pH, should be considered to make the model applicable to commercial fermentations.

  17. Lactococcal Abortive Infection Protein AbiV Interacts Directly with the Phage Protein SaV and Prevents Translation of Phage Proteins

    DEFF Research Database (Denmark)

    Haaber, Jakob Brandt Borup; Samson, J.E.; Labrie, S.J.

    2010-01-01

    RNAs and proteins suggested that AbiV blocks the activation of late gene transcription, probably by a general inhibition of translation. Using size exclusion chromatography coupled with on-line static light scattering and refractometry, as well as fluorescence quenching experiments, we also demonstrated that both......AbiV is an abortive infection protein that inhibits the lytic cycle of several virulent phages infecting Lactococcus lactis, while a mutation in the phage gene sav confers insensitivity to AbiV. In this study, we have further characterized the effects of the bacterial AbiV and its interaction...... with the phage p2 protein SaV. First, we showed that during phage infection of lactococcal AbiV(+) cells, AbiV rapidly inhibited protein synthesis. Among early phage transcripts, sav gene transcription was slightly inhibited while the SaV protein could not be detected. Analyses of other phage p2 m...

  18. Phage-host interaction in Lactococcus and Leuconostoc

    DEFF Research Database (Denmark)

    Kot, Witold Piotr

    , bacteriophages infecting Leuconostoc species were characterized. A large group of Leuconostoc phages was characterized regarding the morphology, host range variation and genetic relatedness. A classification system for dairy Leuconostoc phages was proposed. dditionally, a PCR test for the dairy Leuconostoc...... phages was developed and tested. The complete genomic DNA sequences of nine Leuconostoc phages were determined. The genomic sequences were analyzed with a focus on the gene content, gene organization and comparative genomics. Sequenced phages of Leuconostoc could be classified into two groups, which...

  19. Probing Tumor Microenvironment with In Vivo Phage Display

    Science.gov (United States)

    2014-09-01

    isolated from Stage II invasive lobular breast cancer using human telomerase reverse transcriptase (hTERT). The resulting phage pool bound to i6011...presence of an excess of soluble RGD as an inhibitor to reduce the frequency of peptides containing the integrin-binding RGD motif in the output

  20. Inhibition of phage infection in capsule-producing Streptococcus ...

    African Journals Online (AJOL)

    SERVER

    2007-10-04

    Oct 4, 2007 ... Prazak A, Murano E, Mercado I, Acuff G (2002). Prevalence of Listeria monocytogenes during production and post harvest processing on cabbage. J. Food Prot. 65: 1728-1734. Quiberoni A, Stiefel J, Reinheimer J (2000). Characterization of phage receptors in Streptococcus thermophilus using purified cell ...

  1. What Can Phages Tell Us about Host-Pathogen Coevolution?

    Directory of Open Access Journals (Sweden)

    John J. Dennehy

    2012-01-01

    Full Text Available The outcomes of host-parasite interactions depend on the coevolutionary forces acting upon them, but because every host-parasite relation is enmeshed in a web of biotic and abiotic interactions across a heterogeneous landscape, host-parasite coevolution has proven difficult to study. Simple laboratory phage-bacteria microcosms can ameliorate this difficulty by allowing controlled, well-replicated experiments with a limited number of interactors. Genetic, population, and life history data obtained from these studies permit a closer examination of the fundamental correlates of host-parasite coevolution. In this paper, I describe the results of phage-bacteria coevolutionary studies and their implications for the study of host-parasite coevolution. Recent experimental studies have confirmed phage-host coevolutionary dynamics in the laboratory and have shown that coevolution can increase parasite virulence, specialization, adaptation, and diversity. Genetically, coevolution frequently proceeds in a manner best described by the Gene for Gene model, typified by arms race dynamics, but certain contexts can result in Red Queen dynamics according to the Matching Alleles model. Although some features appear to apply only to phage-bacteria systems, other results are broadly generalizable and apply to all instances of antagonistic coevolution. With laboratory host-parasite coevolutionary studies, we can better understand the perplexing array of interactions that characterize organismal diversity in the wild.

  2. Strain diversity and phage resistance in complex dairy starter cultures

    NARCIS (Netherlands)

    Spus, M.; Alexeeva, S.V.; Wolkers-Rooijackers, J.C.M.; Zwietering, M.H.; Abee, T.; Smid, E.J.

    2015-01-01

    The compositional stability of the complex Gouda cheese starter culture Ur is thought to be influenced by diversity in phage resistance of highly related strains that co-exist together with bacteriophages. To analyze the role of bacteriophages in maintaining culture diversity at the level of genetic

  3. Kullback Leibler divergence in complete bacterial and phage genomes.

    Science.gov (United States)

    Akhter, Sajia; Aziz, Ramy K; Kashef, Mona T; Ibrahim, Eslam S; Bailey, Barbara; Edwards, Robert A

    2017-01-01

    The amino acid content of the proteins encoded by a genome may predict the coding potential of that genome and may reflect lifestyle restrictions of the organism. Here, we calculated the Kullback-Leibler divergence from the mean amino acid content as a metric to compare the amino acid composition for a large set of bacterial and phage genome sequences. Using these data, we demonstrate that (i) there is a significant difference between amino acid utilization in different phylogenetic groups of bacteria and phages; (ii) many of the bacteria with the most skewed amino acid utilization profiles, or the bacteria that host phages with the most skewed profiles, are endosymbionts or parasites; (iii) the skews in the distribution are not restricted to certain metabolic processes but are common across all bacterial genomic subsystems; (iv) amino acid utilization profiles strongly correlate with GC content in bacterial genomes but very weakly correlate with the G+C percent in phage genomes. These findings might be exploited to distinguish coding from non-coding sequences in large data sets, such as metagenomic sequence libraries, to help in prioritizing subsequent analyses.

  4. Dispersal and Survival of Flavobacterium psychrophilum Phages In Vivo in Rainbow Trout and In Vitro under Laboratory Conditions: Implications for Their Use in Phage Therapy

    DEFF Research Database (Denmark)

    Madsen, Lone; Bertelsen, Sif K.; Dalsgaard, Inger

    2013-01-01

    and bacterium was most prevalent in the kidney and spleen, with only minor occurrence in the brain. The experiment showed that injected phages were rapidly spread in the internal organs of the fish, also in the absence of bacteria. Parallel examination of the regulation of bacteriophage infectivity...... in controlled laboratory experiments at various environmental conditions showed that pH had only minor effects on long-term (3 months) phage infectivity within a pH range of 4.5 to 7.5, whereas phage infectivity was immediately lost at pH 3. In the absence of host cells, phage infectivity decreased by a factor...

  5. The Effect of Tempering on Strength Properties and Seed Coat ...

    African Journals Online (AJOL)

    ... tempering on seed coat adhesion strength and mechanical strength of sorghum and millet grain kernels was investigated at different tempering durations. Tempering reduced the kernel breaking strength and had significant effect on seed coat adhesion strength. Tempering the grain for 60 minutes at ambient temperature ...

  6. A highly specific phage defense system is a conserved feature of the Vibrio cholerae mobilome.

    Directory of Open Access Journals (Sweden)

    Brendan J O'Hara

    2017-06-01

    Full Text Available Vibrio cholerae-specific bacteriophages are common features of the microbial community during cholera infection in humans. Phages impose strong selective pressure that favors the expansion of phage-resistant strains over their vulnerable counterparts. The mechanisms allowing virulent V. cholerae strains to defend against the ubiquitous threat of predatory phages have not been established. Here, we show that V. cholerae PLEs (phage-inducible chromosomal island-like elements are widespread genomic islands dedicated to phage defense. Analysis of V. cholerae isolates spanning a 60-year collection period identified five unique PLEs. Remarkably, we found that all PLEs (regardless of geographic or temporal origin respond to infection by a myovirus called ICP1, the most prominent V. cholerae phage found in cholera patient stool samples from Bangladesh. We found that PLE activity reduces phage genome replication and accelerates cell lysis following ICP1 infection, killing infected host cells and preventing the production of progeny phage. PLEs are mobilized by ICP1 infection and can spread to neighboring cells such that protection from phage predation can be horizontally acquired. Our results reveal that PLEs are a persistent feature of the V. cholerae mobilome that are adapted to providing protection from a single predatory phage and advance our understanding of how phages influence pathogen evolution.

  7. A highly specific phage defense system is a conserved feature of the Vibrio cholerae mobilome

    Science.gov (United States)

    O’Hara, Brendan J.

    2017-01-01

    Vibrio cholerae-specific bacteriophages are common features of the microbial community during cholera infection in humans. Phages impose strong selective pressure that favors the expansion of phage-resistant strains over their vulnerable counterparts. The mechanisms allowing virulent V. cholerae strains to defend against the ubiquitous threat of predatory phages have not been established. Here, we show that V. cholerae PLEs (phage-inducible chromosomal island-like elements) are widespread genomic islands dedicated to phage defense. Analysis of V. cholerae isolates spanning a 60-year collection period identified five unique PLEs. Remarkably, we found that all PLEs (regardless of geographic or temporal origin) respond to infection by a myovirus called ICP1, the most prominent V. cholerae phage found in cholera patient stool samples from Bangladesh. We found that PLE activity reduces phage genome replication and accelerates cell lysis following ICP1 infection, killing infected host cells and preventing the production of progeny phage. PLEs are mobilized by ICP1 infection and can spread to neighboring cells such that protection from phage predation can be horizontally acquired. Our results reveal that PLEs are a persistent feature of the V. cholerae mobilome that are adapted to providing protection from a single predatory phage and advance our understanding of how phages influence pathogen evolution. PMID:28594826

  8. Significance of phage-host interactions for biocontrol of Campylobacter jejuni in food

    DEFF Research Database (Denmark)

    Athina, Zampara; Sørensen, Martine Camilla Holst; Elsser-Gravesen, Anne

    2017-01-01

    Poultry meat is the main source of Campylobacter jejuni foodborne disease. Currently, no effective control measures prevent C. jejuni from contaminating poultry meat. However, post-harvest phage treatment is a promising biocontrol strategy that has not yet been explored. Here we identified phages....... A thorough understanding of phage-host interactions is prerequisite to further advance phage application as a post-harvest biocontrol strategy against C. jejuni....... most effective phages (F356 showing 0.49 and F357 showing 0.55 log reductions, respectively) led to a 0.73 log reduction of C. jejuni on artificially contaminated chicken skin. Our study shows that poly-phage treatment at 5 °C can be more effective against C. jejuni compared to single phage application...

  9. Comparisons of invasive plants in southern Africa originating from southern temperate, northern temperate and tropical regions

    Directory of Open Access Journals (Sweden)

    L. Henderson

    2006-08-01

    Full Text Available A subset of invasive alien plant species in southern Africa was analysed in terms of their history of introduction, rate of spread, countries/region of origin, taxonomy, growth forms, cultivated uses, weed status and current distribution in southern Africa, and comparisons made of those originating from south of the tropic of Capricorn, north of the tropic of Cancer and from the tropics. The subset of 233 species, belonging to 58 families, includes all important declared species and some potentially important species. Almost as many species originate from temperate regions (112 as from the tropics (121. Most southern temperate species came from Australia (28/36, most tropical species from tropical America (92/121 and most northern temperate species from Europe (including the Mediterranean and Asia (58/76. Transformers account for 33% of  all species. More transformers are of tropical origin (36 than of northern temperate (24 and southern temperate origin (18. However. 50% of southern temperate species are transformers, compared to 32% of northern temperate and 29% of tropical species. Southern temperate transformer species are mainly woody trees and shrubs that were established on a grand scale as silvicultural crops, barriers (hedges, windbreaks and screens and cover/binders. Most aquatics, herbs, climbers and succulent shrubs an. trom the tropics. Ornamentals are the single largest category of plants from all three regions, the tropics having contributed twice as many species as temperate regions.

  10. Comparisons of invasive plants in southern Africa originating from southern temperate, northern temperate and tropical regions

    Directory of Open Access Journals (Sweden)

    L. Henderson

    2006-12-01

    Full Text Available A subset of invasive alien plant species in southern Africa was analysed in terms of their history of introduction, rate of spread, countries/region of origin, taxonomy, growth forms, cultivated uses, weed status and current distribution in southern Africa, and comparisons made of those originating from south of the tropic of Capricorn, north of the tropic of Cancer and from the tropics. The subset of 233 species, belonging to 58 families, includes all important declared species and some potentially important species. Almost as many species originate from temperate regions (112 as from the tropics (121. Most southern temperate species came from Australia (28/36, most tropical species from tropical America (92/121 and most northern temperate species from Europe (including the Mediterranean and Asia (58/76. Transformers account for 33% of  all species. More transformers are of tropical origin (36 than of northern temperate (24 and southern temperate origin (18. However. 50% of southern temperate species are transformers, compared to 32% of northern temperate and 29% of tropical species. Southern temperate transformer species are mainly woody trees and shrubs that were established on a grand scale as silvicultural crops, barriers (hedges, windbreaks and screens and cover/binders. Most aquatics, herbs, climbers and succulent shrubs an. trom the tropics. Ornamentals are the single largest category of plants from all three regions, the tropics having contributed twice as many species as temperate regions.

  11. Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species

    DEFF Research Database (Denmark)

    Szymczak, Paula; Janzen, Thomas; Neves, Ana Rute

    2017-01-01

    Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S....... thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had...... shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus...

  12. Effect of Exopolysaccharides on Phage-Host Interactions in Lactococcus lactis

    Science.gov (United States)

    Deveau, Hélène; Van Calsteren, Marie-Rose; Moineau, Sylvain

    2002-01-01

    In this study, we report that Lactococcus lactis strains producing exopolysaccharides (EPS) are sensitive to virulent phages. Eight distinct lytic phages (Q61 to Q68) specifically infecting Eps+ strains were isolated in 47 buttermilk samples obtained from 13 North American factories. The eight phages were classified within the 936 species by the multiplex PCR method, indicating that these phages are not fundamentally distinct from those infecting Eps− L. lactis strains. The host range of these phages was determined with 19 Lactococcus strains, including 7 Eps+ and 12 Eps− cultures. Three phages (Q62, Q63, and Q64) attacked only the Eps+ strain SMQ-419, whereas the five other phages (Q61, Q65, Q66, Q67, and Q68) infected only the Eps+ strain SMQ-420. The five other Eps+ strains (H414, MLT2, MLT3, SMQ-461, and SMQ-575) as well as the 12 Eps− strains were insensitive to these phages. The monosaccharide composition of the polymer produced by the seven Eps+ strains was determined. The EPS produced by strains MLT3, SMQ-419, and SMQ-575 contained glucose, galactose, and rhamnose. The EPS fabricated by H414 contained only galactose. The EPS made by MLT2, SMQ-420, and SMQ-461 contained glucose and galactose. These findings indicate that the sugar composition of the EPS has no effect on phage sensitivity. The plasmid encoding the eps operon was cured from the two phage-sensitive strains. The cured derivatives were still phage sensitive, which indicates that EPS are not necessary for phage infection. Phage adsorption assays showed that the production of EPS does not confer a significant phage resistance phenotype. PMID:12200288

  13. Comparison of sampling efficiency between simulated tempering and replica exchange

    OpenAIRE

    Zhang, Cheng; Ma, Jianpeng

    2008-01-01

    We compared the sampling efficiency of simulated tempering and replica exchange. Our results indicate that simulated tempering is superior to replica exchange if the parameters for temperature transition in simulated tempering are adjusted to be proportional to the partition function. It is shown that, in simulated tempering, the rate of traversing energy space of different temperatures is much higher than that in replica exchange, especially in the case of low tempering frequency and∕or larg...

  14. Comparison of sampling efficiency between simulated tempering and replica exchange.

    Science.gov (United States)

    Zhang, Cheng; Ma, Jianpeng

    2008-10-07

    We compared the sampling efficiency of simulated tempering and replica exchange. Our results indicate that simulated tempering is superior to replica exchange if the parameters for temperature transition in simulated tempering are adjusted to be proportional to the partition function. It is shown that, in simulated tempering, the rate of traversing energy space of different temperatures is much higher than that in replica exchange, especially in the case of low tempering frequency and/or larger temperature separations.

  15. PhiSiGns: an online tool to identify signature genes in phages and design PCR primers for examining phage diversity

    Directory of Open Access Journals (Sweden)

    Dwivedi Bhakti

    2012-03-01

    Full Text Available Abstract Background Phages (viruses that infect bacteria have gained significant attention because of their abundance, diversity and important ecological roles. However, the lack of a universal gene shared by all phages presents a challenge for phage identification and characterization, especially in environmental samples where it is difficult to culture phage-host systems. Homologous conserved genes (or "signature genes" present in groups of closely-related phages can be used to explore phage diversity and define evolutionary relationships amongst these phages. Bioinformatic approaches are needed to identify candidate signature genes and design PCR primers to amplify those genes from environmental samples; however, there is currently no existing computational tool that biologists can use for this purpose. Results Here we present PhiSiGns, a web-based and standalone application that performs a pairwise comparison of each gene present in user-selected phage genomes, identifies signature genes, generates alignments of these genes, and designs potential PCR primer pairs. PhiSiGns is available at (http://www.phantome.org/phisigns/; http://phisigns.sourceforge.net/ with a link to the source code. Here we describe the specifications of PhiSiGns and demonstrate its application with a case study. Conclusions PhiSiGns provides phage biologists with a user-friendly tool to identify signature genes and design PCR primers to amplify related genes from uncultured phages in environmental samples. This bioinformatics tool will facilitate the development of novel signature genes for use as molecular markers in studies of phage diversity, phylogeny, and evolution.

  16. Characterization of JG024, a pseudomonas aeruginosa PB1-like broad host range phage under simulated infection conditions

    Directory of Open Access Journals (Sweden)

    Rohde Manfred

    2010-11-01

    Full Text Available Abstract Background Pseudomonas aeruginosa causes lung infections in patients suffering from the genetic disorder Cystic Fibrosis (CF. Once a chronic lung infection is established, P. aeruginosa cannot be eradicated by antibiotic treatment. Phage therapy is an alternative to treat these chronic P. aeruginosa infections. However, little is known about the factors which influence phage infection of P. aeruginosa under infection conditions and suitable broad host range phages. Results We isolated and characterized a phage, named JG024, which infects a broad range of clinical and environmental P. aeruginosa strains. Sequencing of the phage genome revealed that the phage JG024 is highly related to the ubiquitous and conserved PB1-like phages. The receptor of phage JG024 was determined as lipopolysaccharide. We used an artificial sputum medium to study phage infection under conditions similar to a chronic lung infection. Alginate production was identified as a factor reducing phage infectivity. Conclusions Phage JG024 is a suitable broad host range phage which could be used in phage therapy. Phage infection experiments under simulated chronic lung infection conditions showed that alginate production reduces phage infection efficiency.

  17. Synergistic Interaction Between Phage Therapy and Antibiotics Clears Pseudomonas Aeruginosa Infection in Endocarditis and Reduces Virulence.

    Science.gov (United States)

    Oechslin, Frank; Piccardi, Philippe; Mancini, Stefano; Gabard, Jérôme; Moreillon, Philippe; Entenza, José M; Resch, Gregory; Que, Yok-Ai

    2017-03-01

    Increasing antibiotic resistance warrants therapeutic alternatives. Here we investigated the efficacy of bacteriophage-therapy (phage) alone or combined with antibiotics against experimental endocarditis (EE) due to Pseudomonas aeruginosa, an archetype of difficult-to-treat infection. In vitro fibrin clots and rats with aortic EE were treated with an antipseudomonas phage cocktail alone or combined with ciprofloxacin. Phage pharmacology, therapeutic efficacy, and resistance were determined. In vitro, single-dose phage therapy killed 7 log colony-forming units (CFUs)/g of fibrin clots in 6 hours. Phage-resistant mutants regrew after 24 hours but were prevented by combination with ciprofloxacin (2.5 × minimum inhibitory concentration). In vivo, single-dose phage therapy killed 2.5 log CFUs/g of vegetations in 6 hours (P 6 log CFUs/g of vegetations in 6 hours and successfully treating 64% (n = 7/11) of rats. Phage-resistant mutants emerged in vitro but not in vivo, most likely because resistant mutations affected bacterial surface determinants important for infectivity (eg, the pilT and galU genes involved in pilus motility and LPS formation). Single-dose phage therapy was active against P. aeruginosa EE and highly synergistic with ciprofloxacin. Phage-resistant mutants had impaired infectivity. Phage-therapy alone or combined with antibiotics merits further clinical consideration.

  18. Comparative genomic and morphological analyses of Listeria phages isolated from farm environments.

    Science.gov (United States)

    Denes, Thomas; Vongkamjan, Kitiya; Ackermann, Hans-Wolfgang; Moreno Switt, Andrea I; Wiedmann, Martin; den Bakker, Henk C

    2014-08-01

    The genus Listeria is ubiquitous in the environment and includes the globally important food-borne pathogen Listeria monocytogenes. While the genomic diversity of Listeria has been well studied, considerably less is known about the genomic and morphological diversity of Listeria bacteriophages. In this study, we sequenced and analyzed the genomes of 14 Listeria phages isolated mostly from New York dairy farm environments as well as one related Enterococcus faecalis phage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. These Listeria phages, based on gene orthology and morphology, together with previously sequenced Listeria phages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large genome (~135-kb) myoviruses belonging to the genus “Twort-like viruses,” three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (~66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) of E. faecalis phages, with medium-sized genomes (~56 kb), was identified, which grouped together and shares morphological features with the novel Listeria phage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes.

  19. Phage Encoded H-NS: A Potential Achilles Heel in the Bacterial Defence System

    Science.gov (United States)

    Skennerton, Connor T.; Angly, Florent E.; Breitbart, Mya; Bragg, Lauren; He, Shaomei; McMahon, Katherine D.; Hugenholtz, Philip; Tyson, Gene W.

    2011-01-01

    The relationship between phage and their microbial hosts is difficult to elucidate in complex natural ecosystems. Engineered systems performing enhanced biological phosphorus removal (EBPR), offer stable, lower complexity communities for studying phage-host interactions. Here, metagenomic data from an EBPR reactor dominated by Candidatus Accumulibacter phosphatis (CAP), led to the recovery of three complete and six partial phage genomes. Heat-stable nucleoid structuring (H-NS) protein, a global transcriptional repressor in bacteria, was identified in one of the complete phage genomes (EPV1), and was most similar to a homolog in CAP. We infer that EPV1 is a CAP-specific phage and has the potential to repress up to 6% of host genes based on the presence of putative H-NS binding sites in the CAP genome. These genes include CRISPR associated proteins and a Type III restriction-modification system, which are key host defense mechanisms against phage infection. Further, EPV1 was the only member of the phage community found in an EBPR microbial metagenome collected seven months prior. We propose that EPV1 laterally acquired H-NS from CAP providing it with a means to reduce bacterial defenses, a selective advantage over other phage in the EBPR system. Phage encoded H-NS could constitute a previously unrecognized weapon in the phage-host arms race. PMID:21625595

  20. Phage encoded H-NS: a potential achilles heel in the bacterial defence system.

    Directory of Open Access Journals (Sweden)

    Connor T Skennerton

    Full Text Available The relationship between phage and their microbial hosts is difficult to elucidate in complex natural ecosystems. Engineered systems performing enhanced biological phosphorus removal (EBPR, offer stable, lower complexity communities for studying phage-host interactions. Here, metagenomic data from an EBPR reactor dominated by Candidatus Accumulibacter phosphatis (CAP, led to the recovery of three complete and six partial phage genomes. Heat-stable nucleoid structuring (H-NS protein, a global transcriptional repressor in bacteria, was identified in one of the complete phage genomes (EPV1, and was most similar to a homolog in CAP. We infer that EPV1 is a CAP-specific phage and has the potential to repress up to 6% of host genes based on the presence of putative H-NS binding sites in the CAP genome. These genes include CRISPR associated proteins and a Type III restriction-modification system, which are key host defense mechanisms against phage infection. Further, EPV1 was the only member of the phage community found in an EBPR microbial metagenome collected seven months prior. We propose that EPV1 laterally acquired H-NS from CAP providing it with a means to reduce bacterial defenses, a selective advantage over other phage in the EBPR system. Phage encoded H-NS could constitute a previously unrecognized weapon in the phage-host arms race.

  1. Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries

    Directory of Open Access Journals (Sweden)

    Pottel Hans

    2009-03-01

    Full Text Available Abstract Background Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library. Results Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. E. coli cells were applied on the column for infection with the specifically bound phages. Conclusion Chromato-panning allows combining several steps of the panning procedure resulting in 4–8 fold decrease of total time needed for phage selection.

  2. Englacial Hydrology of Temperate Glaciers

    Science.gov (United States)

    Fountain, A. G.; Creyts, T. T.

    2015-12-01

    The englacial region of temperate glaciers is generally treated as a passive conveyor of water from the surface to the bed. Consequently, few studies have examined this region and relatively little is known. This is an important issue because englacial processes probably exert a first order control on the distribution of water to the subglacial hydraulic system. Controlling the water distribution probably controls the type of subglacial hydraulic features present and therefore sliding behavior. Certainly, englacial conduits play a major, if not primary, role in conveying water in the ablation zone. In regions of over-deepenings, areas highly crevassed, or in the accumulation zone, the importance of englacial conduits is less clear. Field studies have shown that intersecting englacial passageways in these regions are relatively common, implying that large water fluxes can drain efficiently through a network of fractures. Hypothetically, efficient drainage systems composed of englacial conduits develop in response to point input of large surface water fluxes. Where input is small and distributed, common to highly crevassed areas or the accumulation zone, water is probably routed through a network of englacial fractures. Glacier geometry may also play a role. Conduits may not develop in the over-deepened (closed basins) regions of a glacier requiring another flow pathway. That englacial fractures exist and can convey water presents a promising alternative. Measured rates of flow in fractures strongly suggest laminar conditions and a sufficient fracture density exists to accommodate the estimated water flux generated upstream by surface melt. The slow flow rates do not generate sufficient viscous heat to compensate expected rates of closure by freezing, however field observations and seismic evidence point to spontaneous fracture formation at depth that must regenerate the fracture network. It is unfortunate that englacial investigations are ignored in favor of

  3. Characterization and genome sequencing of two Propionibacterium acnes phages displaying pseudolysogeny

    Directory of Open Access Journals (Sweden)

    Collin Mattias

    2011-04-01

    Full Text Available Abstract Background Propionibacterium acnes is a Gram positive rod inhabiting the human skin that also infects orthopaedic implants and is associated with acne vulgaris. Previously, one lytic bacteriophage, PA6, from P. acnes has been sequenced and partially characterized. We recently isolated several inducible phages from P. acnes classified as Siphoviruses based on morphology and partial genome sequencing. Results In this study we sequenced the inducible P. acnes phages PAD20 and PAS50, isolated from deep infection and from skin, respectively. The genomes of PAD20 and PAS50 are 29,074 and 29,017 bp, respectively, compared with the 29,739 bp of PA6. The phage genomes have 87.3-88.7% nucleotide sequence identity. The genes are divided into clusters with different levels of similarity between the phages. PAD20 and PAS50 share four genes encoding identical amino acid sequences. Some deletions and insertions in the genomes have occurred, resulting in lack of genes, frame shifts, and possible regulatory differences. No obvious virulence factor gene candidates were found. The phages are inducible, but bacteria can be cured of phages by serial colony isolations and lose their phages during stationary phase, but are still sensitive to new phage infections. Construction of a phylogenetic tree based on more than 459 phage genomes, suggested that P. acnes phages represent a new lineage of Siphoviruses. Conclusions The investigated P. acnes Siphovirus genomes share a high degree of homology to other P. acnes phages sequenced, but not to genomes of other phages isolated from Propionibacteria. The phage genomes are not integrated in the bacterial genome, but instead, most likely have a pseudolysogenic life cycle.

  4. Dynamics of adaptive immunity against phage in bacterial populations

    CERN Document Server

    Bradde, Serena; Tesileanu, Tiberiu; Balasubramanian, Vijay

    2015-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats) mechanism allows bacteria to adaptively defend against phages by acquiring short genomic sequences (spacers) that target specific sequences in the viral genome. We propose a population dynamical model where immunity can be both acquired and lost. The model predicts regimes where bacterial and phage populations can co-exist, others where the populations oscillate, and still others where one population is driven to extinction. Our model considers two key parameters: (1) ease of acquisition and (2) spacer effectiveness in conferring immunity. Analytical calculations and numerical simulations show that if spacers differ mainly in ease of acquisition, or if the probability of acquiring them is sufficiently high, bacteria develop a diverse population of spacers. On the other hand, if spacers differ mainly in their effectiveness, their final distribution will be highly peaked, akin to a "winner-take-all" scenario, leading to a specialized spacer ...

  5. Zinc binding to lambda phage DNA studied by voltammetric techniques

    Directory of Open Access Journals (Sweden)

    Souza Jurandir R. de

    2000-01-01

    Full Text Available Binding of zinc to lambda phage DNA was investigated by differential pulse voltammetry and cyclic voltammetry. These methods rely on the direct monitoring of reduction and oxidation current of zinc in the absence and presence of this virion DNA. Titration graphs of Zn2+ with DNA were obtained in the concentration ranges from 3.57 x 10-12 to 3.92 x 10-11 mol L-1 and 6.97 x 10-12 to 5.56 x 10-11 mol L-1. These data were used to calculate the dissociation constant of the complex and the stoichiometry. The mechanism of this reaction was studied by cyclic voltammetry through curves I (oxidation current of Zn2+ versus v½ (square root of scan rate. These curves showed that the oxidation-reduction process of Zn2+ was not controlled by diffusion in the presence of lambda phage DNA.

  6. Autodeformation of Carburized Steel during Tempering

    Directory of Open Access Journals (Sweden)

    Regita BENDIKIENĖ

    2013-03-01

    Full Text Available The article analyses the results of autodeformation registered during tempering of carburized steel. Test pieces for the tests were carburized till the different depth in order to examine influence of depth of carburization on the deformation of steel during heat treatment operation. Carburization was performed on the one surface of test pieces seeking to analyze extent of acted normal stresses to autodeformation of steel. Different bending loads were applied for analyzed steel from 5 MPa to 100 MPa. Deflection of test pieces was analyzed. The obtained results proved that size and direction of deflection were affected by depth of carburization. Particular results of stretched and compressed surface examination showed different behavior of test pieces during tempering process. Test pieces, which undergo deformation at the beginning of martensitic transformation, after unloading bend further. When tempered test pieces with assimetrically carburized layer bend during hardening, its direction and extent of autodeformation depend on depth of carburization and tempering temperature. Kinetics of autodeformation (during tempering is affected by difference of volume changes in the carburized part and in the unaffected low carbon part of specimen, and similarly by decomposition of retained austenite in the carburized part.DOI: http://dx.doi.org/10.5755/j01.ms.19.1.3820

  7. First Temperate Exoplanet Sized Up

    Science.gov (United States)

    2010-03-01

    Combining observations from the CoRoT satellite and the ESO HARPS instrument, astronomers have discovered the first "normal" exoplanet that can be studied in great detail. Designated Corot-9b, the planet regularly passes in front of a star similar to the Sun located 1500 light-years away from Earth towards the constellation of Serpens (the Snake). "This is a normal, temperate exoplanet just like dozens we already know, but this is the first whose properties we can study in depth," says Claire Moutou, who is part of the international team of 60 astronomers that made the discovery. "It is bound to become a Rosetta stone in exoplanet research." "Corot-9b is the first exoplanet that really does resemble planets in our solar system," adds lead author Hans Deeg. "It has the size of Jupiter and an orbit similar to that of Mercury." "Like our own giant planets, Jupiter and Saturn, the planet is mostly made of hydrogen and helium," says team member Tristan Guillot, "and it may contain up to 20 Earth masses of other elements, including water and rock at high temperatures and pressures." Corot-9b passes in front of its host star every 95 days, as seen from Earth [1]. This "transit" lasts for about 8 hours, and provides astronomers with much additional information on the planet. This is fortunate as the gas giant shares many features with the majority of exoplanets discovered so far [2]. "Our analysis has provided more information on Corot-9b than for other exoplanets of the same type," says co-author Didier Queloz. "It may open up a new field of research to understand the atmospheres of moderate- and low-temperature planets, and in particular a completely new window in our understanding of low-temperature chemistry." More than 400 exoplanets have been discovered so far, 70 of them through the transit method. Corot-9b is special in that its distance from its host star is about ten times larger than that of any planet previously discovered by this method. And unlike all such

  8. Borrelia burgdorferi adhesins identified using in vivo phage display.

    Science.gov (United States)

    Antonara, Styliani; Chafel, Rebecca M; LaFrance, Michelle; Coburn, Jenifer

    2007-10-01

    Borrelia burgdorferi, the agent of Lyme disease, disseminates from the site of deposition by Ixodes ticks to cause systemic infection. Dissemination occurs through the circulation and through tissue matrices, but the B. burgdorferi molecules that mediate interactions with the endothelium in vivo have not yet been identified. In vivo selection of filamentous phage expressing B. burgdorferi protein fragments on the phage surface identified several new candidate adhesins, and verified the activity of one adhesin that had been previously characterized in vitro. P66, a B. burgdorferi ligand for beta(3)-chain integrins, OspC, a protein that is essential for the establishment of infection in mammals, and Vls, a protein that undergoes antigenic variation in the mammal, were all selected for binding to the murine endothelium in vivo. Additional B. burgdorferi proteins for which no functions have been identified, including all four members of the OspF family and BmpD, were identified as candidate adhesins. The use of in vivo phage display is one approach to the identification of adhesins in pathogenic bacteria that are not easily grown in the laboratory, or for which genetic manipulations are not straightforward.

  9. Advances in phage display technology for drug discovery.

    Science.gov (United States)

    Omidfar, Kobra; Daneshpour, Maryam

    2015-06-01

    Over the past decade, several library-based methods have been developed to discover ligands with strong binding affinities for their targets. These methods mimic the natural evolution for screening and identifying ligand-target interactions with specific functional properties. Phage display technology is a well-established method that has been applied to many technological challenges including novel drug discovery. This review describes the recent advances in the use of phage display technology for discovering novel bioactive compounds. Furthermore, it discusses the application of this technology to produce proteins and peptides as well as minimize the use of antibodies, such as antigen-binding fragment, single-chain fragment variable or single-domain antibody fragments like VHHs. Advances in screening, manufacturing and humanization technologies demonstrate that phage display derived products can play a significant role in the diagnosis and treatment of disease. The effects of this technology are inevitable in the development pipeline for bringing therapeutics into the market, and this number is expected to rise significantly in the future as new advances continue to take place in display methods. Furthermore, a widespread application of this methodology is predicted in different medical technological areas, including biosensing, monitoring, molecular imaging, gene therapy, vaccine development and nanotechnology.

  10. Intragenus generalized transduction in Staphylococcus spp. by a novel giant phage.

    Science.gov (United States)

    Uchiyama, Jumpei; Takemura-Uchiyama, Iyo; Sakaguchi, Yoshihiko; Gamoh, Keiji; Kato, Shin-ichiro; Daibata, Masanori; Ujihara, Takako; Misawa, Naoaki; Matsuzaki, Shigenobu

    2014-09-01

    Bacteriophage (phage)-mediated generalized transduction is expected to contribute to the emergence of drug-resistant staphylococcal clones in various environments. In this study, novel phage S6 was isolated from sewage and used to test generalized transduction in human- and animal-derived staphylococci. Phage S6 was a novel type of giant myophage, which possessed a DNA genome that contained uracil instead of thymine, and it could infect all of the tested staphylococcal species. The phage S6 appeared to be similar to the transducing phage PBS1, which infects Bacillus spp. Moreover, phage S6 facilitated the transduction of a plasmid in Staphylococcus aureus and from S. aureus to non-aureus staphylococcal species, as well as vice versa. Transduction of methicillin resistance also occurred in S. aureus. This is the first report of successful intragenus generalized transduction among staphylococci.

  11. Advancement and applications of peptide phage display technology in biomedical science.

    Science.gov (United States)

    Wu, Chien-Hsun; Liu, I-Ju; Lu, Ruei-Min; Wu, Han-Chung

    2016-01-19

    Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

  12. The phage-host arms race: Shaping the evolution of microbes

    Energy Technology Data Exchange (ETDEWEB)

    Stern, Adi [Weizmann Inst. of Science, Rehovot (Israel). Dept. of Molecular Genetics; Sorek, Rotem [Weizmann Inst. of Science, Rehovot (Israel). Dept. of Molecular Genetics

    2010-10-26

    Bacteria, the most abundant organisms on the planet, are outnumbered by a factor of 10 to 1 by phages that infect them. Faced with the rapid evolution and turnover of phage particles, bacteria have evolved various mechanisms to evade phage infection and killing, leading to an evolutionary arms race. The extensive co-evolution of both phage and host has resulted in considerable diversity on the part of both bacterial and phage defensive and offensive strategies. In this paper, we discuss the unique and common features of phage resistance mechanisms and their role in global biodiversity. Finally, the commonalities between defense mechanisms suggest avenues for the discovery of novel forms of these mechanisms based on their evolutionary traits.

  13. Enrichment of an in vivo phage display repertoire by subtraction for easy identification of pathology biomarkers

    Directory of Open Access Journals (Sweden)

    karina Vargas Sanchez

    2015-03-01

    Conclusion. This physical subtraction discarded from a complex repertoire the non-specific selected ligands. STRATEGY 1 Three rounds of in vivo phage peptide selection in EAE female Lewis rats ("EAE repertoire" vs controls ("HEALTHY repertoire". 2 DNA subtraction of the most common sequences between «HEALTHY» and «EAE» phage repertoires to obtain a third EAE specific «SUBTRACTION » phage repertoire. 3 Massive sequencing of the three repertoires and bioinformatic analysis to identify the peptides sequences with high EAE specificity. 4 Biological tests of potential EAE specific phage clones with CNS tissues from EAE and Healthy control rats. 5 Biological tests of the EAE specific peptide and phage clones on the BBB in vitro model (hCMEC/D3 cells under inflammatory conditions (IL-1β stimulation. 6 Target separation and identification by cross-link between the selected phage clones and hMEC/D3 endothelial cells targets under IL-1β stimulation vs controls.

  14. Tempering of Low-Temperature Bainite

    Science.gov (United States)

    Peet, Mathew J.; Babu, Sudarsanam Suresh; Miller, Mike K.; Bhadeshia, H. K. D. H.

    2017-07-01

    Electron microscopy, X-ray diffraction, and atom probe tomography have been used to identify the changes which occur during the tempering of a carbide-free bainitic steel transformed at 473 K (200 °C). Partitioning of solute between ferrite and thin-films of retained austenite was observed on tempering at 673 K (400 °C) for 30 minutes. After tempering at 673 K (400 °C) and 773 K (500 °C) for 30 minutes, cementite was observed in the form of nanometre scale precipitates. Proximity histograms showed that the partitioning of solutes other than silicon from the cementite was slight at 673 K (400 °C) and more obvious at 773 K (500 °C). In both cases, the nanometre scale carbides are greatly depleted in silicon.

  15. The T7-Related Pseudomonas putida Phage ϕ15 Displays Virion-Associated Biofilm Degradation Properties

    OpenAIRE

    Cornelissen, Anneleen; Ceyssens, Pieter-Jan; T'Syen, Jeroen; Van Praet, Helena; Noben, Jean-Paul; Shaburova, Olga V; Krylov, Victor N; Volckaert, Guido; Lavigne, Rob

    2011-01-01

    Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage phi 15, a 'T7-like virus' with a virion-associated exopolysaccharide (EPS) depolymerase. Phage phi 15 forms plaques surrounded by grow...

  16. An efficient method for isolating antibody fragments against small peptides by antibody phage display

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

  17. Isolation of Dermatophilus congolensis phage from the 'lumpy wool' of sheep in Western Australia.

    Science.gov (United States)

    Patten, K M; Kurtböke, D I; Lindsay, D R

    1995-04-01

    A lytic phage with species-specific activity was isolated from wool samples infected with the actinomycete Dermatophilus congolensis, the agent of 'lumpy wool', collected from properties in Western Australia. The physiochemical properties, plaque morphology, host range and particle morphology of the phage isolated were characterized. The isolated phage reduced the cell numbers of Dermatophilus congolensis on infected wool samples in vitro. It may therefore have potential as a biocontrol agent of dermatophilosis.

  18. In vitro management of hospital Pseudomonas aeruginosa biofilm using indigenous T7-like lytic phage.

    Science.gov (United States)

    Ahiwale, Sangeeta; Tamboli, Nilofer; Thorat, Kiran; Kulkarni, Rajendra; Ackermann, Hans; Kapadnis, Balasaheb

    2011-02-01

    Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24 h. It could also disperse pre-formed biofilms of all hospital isolates (HS1-HS6) on the pegs within 24 h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.

  19. Phage-display libraries of murine and human antibody Fab fragments

    DEFF Research Database (Denmark)

    Engberg, J; Andersen, P S; Nielsen, L K

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...... phages from libraries, and for selecting antigen binders by panning are presented. The latter protocol includes a procedure for trypsin elution of bound phage....

  20. Development of expanded host range phage active on biofilms of multi-drug resistant Pseudomonas aeruginosa

    Science.gov (United States)

    Mapes, Abigail C.; Trautner, Barbara W.; Liao, Kershena S.; Ramig, Robert F.

    2016-01-01

    ABSTRACT Phage therapy is a promising treatment of multi-drug resistant (MDR) bacterial infections but is limited by the narrow host range of phage. To overcome this limitation, we developed a host range expansion (HRE) protocol that expands the host range of Pseudomonas aeruginosa-specific phage by cycles of co-incubation of phage with multiple P. aeruginosa strains. Application of the HRE protocol to a mixture of 4 phages, using 16 P. aeruginosa strains for development, resulted in undefined phage mixtures with greatly expanded host range. Individual phage clones derived from the undefined mixture had expanded host ranges but no individual clone could lyse all of the strains covered by the undefined mixture from which it was isolated. Reconstituting host range-characterized clones into cocktails produced defined cocktails with predictable and broad host ranges. The undefined mixture from the 30th cycle of the mixed-phage HRE (4ϕC30) showed a dose-dependent ability to prevent biofilm formation by, and to reduce a pre-existing biofilm of, 3 P. aeruginosa clinical isolates that produced high amounts of biofilm. A defined cocktail reconstituted from 3 host range-characterized clones had activity on high biofilm-formers susceptible to the phage. Phage therapy was superior to antibiotic therapy (levofloxacin) in a strain of P. aeruginosa that was resistant to levofloxacin. The HRE protocol establishes a rapid approach to create libraries of phage clones and phage cocktails with broad host range, defined composition and anti-biofilm activity. PMID:27144083

  1. NEW Fe-C-Mn-Si-Cr BEARING ALLOY: TEMPERING CURVES AND TEMPERED MARTENSITE EMBRITTLEMENT

    Directory of Open Access Journals (Sweden)

    José Benedito Marcomini

    2012-12-01

    Full Text Available SAE 52100 steel is not only used as a rolled raw material for bearing manufacturing but for building some rolling devices as well, such as guide rollers and straightener rollers. The purpose of this work is the characterization of a Fe-C-Mn-Si-Cr bearing alloy (SAE 52100 steel, modified with 1.74% Si by plotting the variation of quenched and tempered hardness curve (tempering curve and tempered martensite embrittlement susceptibility. The present application is based on the same idea as 300M steel regarding SAE 4340 steel. The effect of silicon on the kinetics of cementite precipitation leads to a rise in temperature of tempered martensite embrittlement. Quench and temper treatments were carried out and impact tests were performed with modified and commercial steels and the results were compared. Microstructure aspects are studied by scanning electron microscopy and x-ray diffraction analysis. The silicon alloyed steel presents great resistance to softening after tempering and no tempered martensite embrittlement.

  2. Identification of gliadin-binding peptides by phage display

    Directory of Open Access Journals (Sweden)

    Östman Sofia

    2011-02-01

    Full Text Available Abstract Background Coeliac disease (CD is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products. Results Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 1011 to 1012 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptide-expressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro. By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin. Conclusions We believe that several of

  3. A Transnational Temperance Discourse? William Wells Brown, Creole Civilization, and Temperate Manners

    Directory of Open Access Journals (Sweden)

    Carole Lynn Stewart

    2011-03-01

    Full Text Available

    In the nineteenth century, temperance movements provided the occasion for a transnational discourse. These conversations possessed an intensity throughout Britain and the United States. In America temperance often became associated with strongly nationalistic Euro-American forms of identity and internal purity. Nonetheless, African American reformers and abolitionists bound themselves to temperance ideals in forming civil societies that would heal persons and provide communal modes of democratic freedom in the aftermath and recovery from chattel slavery. This paper explores the possibilities of temperance as a transnational discourse by considering its meaning in the life and work of the African American author and activist, William Wells Brown. Brown expressed a “creole civilization” that employed the stylistics of the trickster as a unique mode of restraint that revealed a peculiar power of passivity that was able to claim efficacy over one’s life and community. This meaning of temperance diverges from and dovetails with certain European meanings of civilization that were being forged in the nineteenth century. Brown was in conversation with temperance reformers in America, Britain, and Europe. He imagined the possible meaning of temperance in African, Egyptian, Christian, and Islamic civilizations. He speculated upon the possibility of temperance as a defining characteristic of a transnational civilization and culture that would provide spaces for the expression of democratic freedom. Brown reimagined temperance as a form of corporeal restraint that offered a direct and sacred relation to the land, space, people that appeared in between an ethnic nationalist ethos and the European imperialistic civilization.

  4. Activation and Transfer of the Chromosomal Phage Resistance Mechanism AbiV in Lactococcus lactis

    DEFF Research Database (Denmark)

    Haaber, J.; Moineau, S.; Hammer, Karin

    2009-01-01

    AbiV is a chromosomally encoded phage resistance mechanism that is silent in the wild-type phage-sensitive strain Lactococcus lactis subsp. cremoris MG1363. Spontaneous phage-resistant mutants of L. lactis MG1363 were analyzed by reverse transcriptase PCR and shown to express AbiV. This expression...... was related to a reorganization in the upstream region of abiV. Transfer of abiV between two lactococcal strains, most likely by conjugation, was also demonstrated. To our knowledge, this is the first report of natural transfer of a chromosomally encoded phage resistance mechanism....

  5. Directed synthesis of bio-inorganic vanadium oxide composites using genetically modified filamentous phage

    Science.gov (United States)

    Mueller, Michael; Baik, Seungyun; Jeon, Hojeong; Kim, Yuchan; Kim, Jungtae; Kim, Young Jun

    2015-05-01

    The growth of crystalline vanadium oxide using a filamentous bacteriophage template was investigated using sequential incubation in a V2O5 precursor. Using the genetic modification of the bacteriophage, we displayed two cysteines that constrained the RSTB-1 peptide on the major coat protein P8, resulting in vanadium oxide crystallization. The phage-driven vanadium oxide crystals with different topologies, microstructures, photodegradation and vanadium oxide composites were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), quartz microbalance and dissipation (QCM-D) and X-ray photoelectron spectroscopy (XPS). Non-specific electrostatic attraction between a wild-type phage (wt-phage) and vanadium cations in the V2O5 precursor caused phage agglomeration and fiber formation along the length of the viral scaffold. As a result, the addition of recombinant phage (re-phage) in V2O5 precursors formed heterogeneous structures, which led to efficient condensation of vanadium oxide crystal formation in lines, shown by QCM-D analysis. Furthermore, re-phage/VxOx composites showed significantly enhanced photodegradation activities compared with the synthesized wt-phage-V2O5 composite under illumination. This study demonstrates that peptide-mediated vanadium oxide mineralization is governed by a complicated interplay of peptide sequence, local structure, kinetics and the presence of a mineralizing aid, such as the two cysteine-constrained peptides on the phage surface, and has potential for use in nanotechnology applications.

  6. Surface topological differences of phage infected uropathogenic Escherichia coli (UPEC) strains, revealed by atomic force microscopy.

    Science.gov (United States)

    Hanif, Bassamah; Jamil, Nusrat; Shah, Muhammad Raza

    2016-01-01

    Atomic force microscopy (AFM) is an advance microscopic technique that provides three dimensional structures of cell surfaces with high resolution. In the present study AFM was used for comparative analysis of surface topology of phage infected and uninfected Uropathogenic Escherichia coli (UPEC) cells. Two UPEC strains NE and HN were isolated from urine samples of Urinary tract infection patients and their specific narrow host range lytic phages 3S and HNΦ were isolated from the sewage of different areas. On the basis of one step growth curve both phages characterized as short latent period phages with latency period of about 30 min. On AFM analysis significant difference in topology of healthy and infected cells were observed. It was hypothesized that progeny of both lytic phages released out from their respective host cells in different manner. The image of 3S infected UPEC host cells (NE) revealed multiple internal projections which showed progeny phages released out from host cells through these multiple sites. Whereas images of HNΦ infected HN host cells showed central depression which illustrated that new phages released out through single exit point from the middle of cell. These results are significant to extend future studies on isolated phages as an effective tool for phage therapy.

  7. Phage Therapy Is Effective in a Mouse Model of Bacterial Equine Keratitis.

    Science.gov (United States)

    Furusawa, Takaaki; Iwano, Hidetomo; Hiyashimizu, Yutaro; Matsubara, Kazuki; Higuchi, Hidetoshi; Nagahata, Hajime; Niwa, Hidekazu; Katayama, Yoshinari; Kinoshita, Yuta; Hagiwara, Katsuro; Iwasaki, Tomohito; Tanji, Yasunori; Yokota, Hiroshi; Tamura, Yutaka

    2016-09-01

    Bacterial keratitis of the horse is mainly caused by staphylococci, streptococci, and pseudomonads. Of these bacteria, Pseudomonas aeruginosa sometimes causes rapid corneal corruption and, in some cases, blindness. Antimicrobial resistance can make treatment very difficult. Therefore, new strategies to control bacterial infection are required. A bacteriophage (phage) is a virus that specifically infects and kills bacteria. Since phage often can lyse antibiotic-resistant bacteria because the killing mechanism is different, we examined the use of phage to treat horse bacterial keratitis. We isolated Myoviridae or Podoviridae phages, which together have a broad host range. They adsorb efficiently to host bacteria; more than 80% of the ΦR18 phage were adsorbed to host cells after 30 s. In our keratitis mouse model, the administration of phage within 3 h also could kill bacteria and suppress keratitis. A phage multiplicity of infection of 100 times the host bacterial number could kill host bacteria effectively. A cocktail of two phages suppressed bacteria in the keratitis model mouse. These data demonstrated that the phages in this study could completely prevent the keratitis caused by P. aeruginosa in a keratitis mouse model. Furthermore, these results suggest that phage may be a more effective prophylaxis for horse keratitis than the current preventive use of antibiotics. Such treatment may reduce the use of antibiotics and therefore antibiotic resistance. Further studies are required to assess phage therapy as a candidate for treatment of horse keratitis. Antibiotic-resistant bacteria are emerging all over the world. Bacteriophages have great potential for resolution of this problem. A bacteriophage, or phage, is a virus that infects bacteria specifically. As a novel therapeutic strategy against racehorse keratitis caused by Pseudomonas aeruginosa, we propose the application of phages for treatment. Phages isolated in this work had in vitro effectiveness for a broad

  8. Novel phage-based bio-processing of pathogenic Escherichia coli and its biofilms.

    Science.gov (United States)

    Jassim, S A A; Abdulamir, A S; Abu Bakar, F

    2012-01-01

    To explore new approaches of phage-based bio-process of specifically pathogenic Escherichia coli bacteria in food products within a short period. One hundred and forty highly lytic designed coliphages were used. Escherichia coli naturally contaminated and Enterohemorrhagic Escherichia coli experimentally inoculated samples of lettuce, cabbage, meat, and egg were used. In addition, experimentally produced biofilms of E. coli were tested. A phage concentration of 10(3) PFU/ml was used for food products immersion, and for spraying of food products, 10(5) PFU/ml of a phage cocktail was used by applying a 20-s optimal dipping time in a phage cocktail. Food samples were cut into pieces and were either sprayed with or held in a bag immersed in lambda buffer containing a cocktail of 140 phages. Phage bio-processing was successful in eliminating completely E. coli in all processed samples after 48 h storage at 4°C. Partial elimination of E. coli was observed in earlier storage periods (7 and 18 h) at 24° and 37°C. Moreover, E. coli biofilms were reduced >3 log cycles upon using the current phage bio-processing. The use of a phage cocktail of 140 highly lytic designed phages proved highly effective in suppressing E. coli contaminating food products. Proper decontamination/prevention methods of pathogenic E. coli achieved in this study can replace the current chemically less effective decontamination methods.

  9. Cost-effective HRMA pre-sequence typing of clone libraries; application to phage display selection

    National Research Council Canada - National Science Library

    Pepers, Barry A; Schut, Menno H; Vossen, Rolf Ham; van Ommen, Gert-Jan B; den Dunnen, Johan T; van Roon-Mom, Willeke Mc

    2009-01-01

    Methodologies like phage display selection, in vitro mutagenesis and the determination of allelic expression differences include steps where large numbers of clones need to be compared and characterised...

  10. On-demand isolation of bacteriophages against drug-resistant bacteria for personalized phage therapy

    Directory of Open Access Journals (Sweden)

    Sari eMattila

    2015-11-01

    Full Text Available Bacteriophages are bacterial viruses, capable of killing even multi-drug resistant bacterial cells. For this reason, therapeutic use of phages is considered as a possible alternative to conventional antibiotics. However, phages are very host specific in comparison to wide-spectrum antibiotics and thus preparation of phage-cocktails beforehand against pathogens can be difficult. In this study, we evaluate whether it may be possible to isolate phages on-demand from environmental reservoir. We attempted to enrich infectious bacteriophages from sewage against nosocomial drug-resistant bacterial strains of different medically important species in order to evaluate the probability of discovering novel therapeutic phages. Stability and host-range were determined for the acquired phages. Our results suggest that on-demand isolation of phages is possible against Pseudomonas aeruginosa, Salmonella and extended spectrum beta-lactamase (ESBL Escherichia coli and Klebsiella pneumoniae. The probability of finding suitable phages was less than 40% against vancomycin resistant Enterococcus (VRE and Acinetobacter baumannii strains. Furthermore, isolation of new phages against methicillin resistant Staphylococcus aureus (MRSA strains was found to be very difficult.

  11. On-Demand Isolation of Bacteriophages Against Drug-Resistant Bacteria for Personalized Phage Therapy.

    Science.gov (United States)

    Mattila, Sari; Ruotsalainen, Pilvi; Jalasvuori, Matti

    2015-01-01

    Bacteriophages are bacterial viruses, capable of killing even multi-drug resistant bacterial cells. For this reason, therapeutic use of phages is considered as a possible alternative to conventional antibiotics. However, phages are very host specific in comparison to wide-spectrum antibiotics and thus preparation of phage-cocktails beforehand against pathogens can be difficult. In this study, we evaluate whether it may be possible to isolate phages on-demand from environmental reservoir. We attempted to enrich infectious bacteriophages from sewage against nosocomial drug-resistant bacterial strains of different medically important species in order to evaluate the probability of discovering novel therapeutic phages. Stability and host-range were determined for the acquired phages. Our results suggest that on-demand isolation of phages is possible against Pseudomonas aeruginosa, Salmonella and extended spectrum beta-lactamase Escherichia coli and Klebsiella pneumoniae. The probability of finding suitable phages was less than 40% against vancomycin resistant Enterococcus and Acinetobacter baumannii strains. Furthermore, isolation of new phages against methicillin resistant Staphylococcus aureus strains was found to be very difficult.

  12. Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

    Science.gov (United States)

    Regeimbal, James M; Jacobs, Anna C; Corey, Brendan W; Henry, Matthew S; Thompson, Mitchell G; Pavlicek, Rebecca L; Quinones, Javier; Hannah, Ryan M; Ghebremedhin, Meron; Crane, Nicole J; Zurawski, Daniel V; Teneza-Mora, Nimfa C; Biswas, Biswajit; Hall, Eric R

    2016-10-01

    Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulated A. baumannii bacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor for A. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in a Galleria mellonella model. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe.

  13. Phage morphology recapitulates phylogeny: the comparative genomics of a new group of myoviruses.

    Directory of Open Access Journals (Sweden)

    André M Comeau

    Full Text Available Among dsDNA tailed bacteriophages (Caudovirales, members of the Myoviridae family have the most sophisticated virion design that includes a complex contractile tail structure. The Myoviridae generally have larger genomes than the other phage families. Relatively few "dwarf" myoviruses, those with a genome size of less than 50 kb such as those of the Mu group, have been analyzed in extenso. Here we report on the genome sequencing and morphological characterization of a new group of such phages that infect a diverse range of Proteobacteria, namely Aeromonas salmonicida phage 56, Vibrio cholerae phages 138 and CP-T1, Bdellovibrio phage φ1422, and Pectobacterium carotovorum phage ZF40. This group of dwarf myoviruses shares an identical virion morphology, characterized by usually short contractile tails, and have genome sizes of approximately 45 kb. Although their genome sequences are variable in their lysogeny, replication, and host adaption modules, presumably reflecting differing lifestyles and hosts, their structural and morphogenesis modules have been evolutionarily constrained by their virion morphology. Comparative genomic analysis reveals that these phages, along with related prophage genomes, form a new coherent group within the Myoviridae. The results presented in this communication support the hypothesis that the diversity of phages may be more structured than generally believed and that the innumerable phages in the biosphere all belong to discrete lineages or families.

  14. Multisystem altruistic metadynamics—Well-tempered variant

    Science.gov (United States)

    Hošek, Petr; Kříž, Pavel; Toulcová, Daniela; Spiwok, Vojtěch

    2017-03-01

    Metadynamics method has been widely used to enhance sampling in molecular simulations. Its original form suffers two major drawbacks, poor convergence in complex (especially biomolecular) systems and its serial nature. The first drawback has been addressed by introduction of a convergent variant known as well-tempered metadynamics. The second was addressed by introduction of a parallel multisystem metadynamics referred to as altruistic metadynamics. Here, we combine both approaches into well-tempered altruistic metadynamics. We provide mathematical arguments and trial simulations to show that it accurately predicts free energy surfaces.

  15. Multisystem altruistic metadynamics-Well-tempered variant.

    Science.gov (United States)

    Hošek, Petr; Kříž, Pavel; Toulcová, Daniela; Spiwok, Vojtěch

    2017-03-28

    Metadynamics method has been widely used to enhance sampling in molecular simulations. Its original form suffers two major drawbacks, poor convergence in complex (especially biomolecular) systems and its serial nature. The first drawback has been addressed by introduction of a convergent variant known as well-tempered metadynamics. The second was addressed by introduction of a parallel multisystem metadynamics referred to as altruistic metadynamics. Here, we combine both approaches into well-tempered altruistic metadynamics. We provide mathematical arguments and trial simulations to show that it accurately predicts free energy surfaces.

  16. Warm-temperate deciduous forests around the Northern Hemisphere

    National Research Council Canada - National Science Library

    Box E.O; Fujiwara K

    2015-01-01

    Warm-temperate deciduous forests are "southern", mainly oak-dominated deciduous forests, as found over the warmer southern parts of the temperate deciduous forest regions of East Asia, Europe and eastern North America...

  17. The Temperance Movement and Social Work

    Science.gov (United States)

    Murdach, Allison D.

    2009-01-01

    This article examines a forgotten episode in social work history: the involvement of the profession in the temperance movement in the late 19th and early 20th centuries. Though some notable social workers such as Jane Addams, Robert A. Woods, and Representative Jeannette Rankin (the first woman elected to the U.S. Congress), championed the…

  18. Simulation of quenching and tempering of steels

    Science.gov (United States)

    Jin, Long

    An efficient simulation method, which includes microstructure, temperature and stress analysis applicable to both quenching and tempering processes, is developed and implemented using the commercial FEM package ABAQUS. This simulation encompasses phase transformations and their effects on the temperature distribution and stress/strain evolution, including the dependency of material properties on temperature and microstructure, transformation strains, latent heats and transformation plasticity. Three different multi-phase constitutive models, namely the average property model, the Voigt model and the Reuss model, have been implemented. The average property model is based on the linear mixture of material properties of different phase, while the Voigt model assumes the same strain field in all phases and the Reuss model assumes the iso-stress field. The simulation model has been applied to quenching and tempering of modified 4320 steel. Experiments of tempering and quenching on carburized circular plates of the same steel have been performed. The calculated distortion and residual stress profiles are in good agreement with corresponding measurements made in experiments and thus verifies the correctness of the model. The simulation model developed in this study is a useful design tool for quenching and tempering as well as machining of steels.

  19. Morphological characterization and biocontrol effects of Vibrio vulnificus phages against Vibriosis in the shrimp aquaculture environment.

    Science.gov (United States)

    Srinivasan, Pappu; Ramasamy, Palaniappan

    2017-10-01

    The re-emerging field of phage therapy is the potential biocontrol agents for the transfer of virulence factor and to kill their bacterial hosts. In this study, the lytic Vibrio vulnificus phages were studied to provide a better understanding of phage-host interactions and development of phage therapy. Four new V. vulnificus phages were detected from shrimp aquaculture system, named VV1, VV2, VV3 and VV4. All lytic V. vulnificus phages are the Tectiviruses of the family Tectiviridae with typical double layered elongated icosahedral head and tailless morphology. Lytic V. vulnificus phages which infect other Vibrio isolates were further characterized by long term storage, enzymes treatment, organic solvents treatment, detergents treatment, pH stability, temperature stability, agar bioassay method and one-step growth experiment. The effects of chloroform, acetone, ethyl alcohol, methyl alcohol, ribonuclease (RNase), trypsin, protease, Triton-X100 treatments were not affected the growth of VV1, VV2, VV3 and VV4 phages. These phages (VV1-VV4) were inactivated completely with temperature (over 60 °C), pH (12), lysozyme and sodium dodecyl sulfate (SDS) treatment. One-step growth experiments indicated that the latent period was at 3 h and burst size was at 37 °C. Agar bioassay method indicated that the percentage of inhibition was 75% (VV1) and 70% (VV2, VV3 & VV4), respectively. SDS-PAGE analysis of all V. vulnificus phages had similar protein patterns with molecular weight masses of 260, 249, 204, 148, 63, 59, 22 and 15 kDa. Hence, it could be concluded that V. vulnificus phage had a broad lytic spectrum and potential biocontrol of luminous Vibriosis in the shrimp aquaculture system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean.

    Science.gov (United States)

    Tucker, Kimberly P; Parsons, Rachel; Symonds, Erin M; Breitbart, Mya

    2011-05-01

    Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem.

  1. Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice.

    Directory of Open Access Journals (Sweden)

    Andrey A Filippov

    Full Text Available BACKGROUND: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. CONCLUSIONS/SIGNIFICANCE: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.

  2. Analysis of high-throughput sequencing and annotation strategies for phage genomes.

    Directory of Open Access Journals (Sweden)

    Matthew R Henn

    Full Text Available BACKGROUND: Bacterial viruses (phages play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. METHODOLOGY/PRINCIPAL FINDINGS: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles, and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL or of a whole genome shotgun library (WGSL, or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. CONCLUSIONS/SIGNIFICANCE: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.

  3. Phages of dairy Leuconostoc mesenteroides: genomics and factors influencing their adsorption.

    Science.gov (United States)

    Pujato, Silvina A; Mercanti, Diego J; Guglielmotti, Daniela M; Rousseau, Geneviève M; Moineau, Sylvain; Reinheimer, Jorge A; Quiberoni, Andrea del L

    2015-05-18

    Phages infecting Leuconostoc mesenteroides strains can be overlooked during milk fermentation because they do not slowdown the acidification process. However, they can negatively impact the flavor profile of the final product. Yet, the information about these phages is still scarce. In this work, we investigated diverse factors influencing the adsorption of seven virulent Ln. mesenteroides phages, isolated from blue cheese manufacture in Argentina, to their host cells. The addition of calcium ions was generally necessary to observe complete cell lysis and plaque formation for four of the seven phages, but adsorption was very high even in the absence of this cation for all phages. The temperature barely influenced the adsorption process as it was high within the temperature range tested (0 to 50 °C). Moreover, the kinetics of adsorption were similar on viable and non-viable cells, revealing that phage adsorption does not depend on physiological state of the bacterial cells. The adsorption rates were also high at pH values from 4 to 9 for all Ln. mesenteroides phages. We also analyzed the complete genome sequences of two of these phages. Complete nucleotide analysis of phages Ln-8 and Ln-9 showed dsDNA genomes with sizes of 28.5 and 28.9 kb, and the presence of 45 and 48 open reading frames (ORFs), respectively. These genomes were highly similar to those of previously characterized Φ1-A4 (USA, sauerkraut, fermentation) and ΦLN25 (England, whey), both virulent Ln. mesenteroides phages. A detailed understanding of these phages will lead to better control strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Aggregation/dispersion transitions of T4 phage triggered by environmental ion availability.

    Science.gov (United States)

    Szermer-Olearnik, Bożena; Drab, Marek; Mąkosa, Mateusz; Zembala, Maria; Barbasz, Jakub; Dąbrowska, Krystyna; Boratyński, Janusz

    2017-04-24

    Bacteriophage survives in at least two extremes of ionic environments: bacterial host (high ionic-cytosol) and that of soil (low ionic-environmental water). The impact of ionic composition in the micro- and macro-environments has not so far been addressed in phage biology. Here, we discovered a novel mechanism of aggregation/disaggregation transitions by phage virions. When normal sodium levels in phage media (150 mM) were lowered to 10 mM, advanced imaging by scanning electron microscopy, atomic force microscopy and dynamic light scattering all revealed formation of viral packages, each containing 20-100 virions. When ionic strength was returned from low to high, the aggregated state of phage reversed to a dispersed state, and the change in ionic strength did not substantially affect infectivity of the phage. By providing the direct evidence, that lowering of the sodium ion below the threshold of 20 mM causes rapid aggregation of phage while returning Na+ concentration to the values above this threshold causes dispersion of phage, we identified a biophysical mechanism of phage aggregation. Our results implicate operation of group behavior in phage and suggest a new kind of quorum sensing among its virions that is mediated by ions. Loss of ionic strength may act as a trigger in an evolutionary mechanism to improve the survival of bacteriophage by stimulating aggregation of phage when outside a bacterial host. Reversal of phage aggregation is also a promising breakthrough in biotechnological applications, since we demonstrated here the ability to retain viable virion aggregates on standard micro-filters.

  5. Longitudinal monitoring of Listeria monocytogenes and Listeria phages in seafood processing environments in Thailand.

    Science.gov (United States)

    Vongkamjan, Kitiya; Benjakul, Soottawat; Kim Vu, Hue Thi; Vuddhakul, Varaporn

    2017-09-01

    Listeria monocytogenes is a foodborne pathogen commonly found in environments of seafood processing, thus presenting a challenge for eradication from seafood processing facilities. Monitoring the prevalence and subtype diversity of L. monocytogenes together with phages that are specific to Listeria spp. ("Listeria phages") will provide knowledge on the bacteria-phage ecology in food processing plants. In this work, a total of 595 samples were collected from raw material, finished seafood products and environmental samples from different sites of a seafood processing plant during 17 sampling visits in 1.5 years of study. L. monocytogenes and Listeria spp. (non-monocytogenes) were found in 22 (3.7%) and 43 (7.2%) samples, respectively, whereas 29 Listeria phages were isolated from 9 (1.5%) phage-positive samples. DNA fingerprint analysis of L. monocytogenes isolates revealed 11 Random Amplified Polymorphic DNA (RAPD) profiles, with two subtypes were frequently observed over time. Our data reveal a presence of Listeria phages within the same seafood processing environments where a diverse set of L. monocytogenes subtypes was also found. Although serotype 4b was observed at lower frequency, data indicate that isolates from this seafood processing plant belonged to both epidemiologically important serotypes 1/2a and 4b, which may suggest a potential public health risk. Phages (all showed a unique genome size of 65 ± 2 kb) were classified into 9 host range groups, representing both broad- and narrow-host range. While most L. monocytogenes isolates from this facility were susceptible to phages, five isolates showed resistance to 12-20 phages. Variations in phage host range among Listeria phages isolated from food processing plant may affect a presence of a diverse set of L. monocytogenes isolates derived from the same processing environment in Thailand. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Characterization of Pseudomonas aeruginosa phage KPP21 belonging to family Podoviridae genus N4-like viruses isolated in Japan.

    Science.gov (United States)

    Shigehisa, Ryu; Uchiyama, Jumpei; Kato, Shin-Ichiro; Takemura-Uchiyama, Iyo; Yamaguchi, Kotoe; Miyata, Reina; Ujihara, Takako; Sakaguchi, Yoshihiko; Okamoto, Noriaki; Shimakura, Hidekatsu; Daibata, Masanori; Sakaguchi, Masahiro; Matsuzaki, Shigenobu

    2016-01-01

    Bacteriophages (phages) belonging to the family Podoviridae genus N4-like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4-like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4-like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7. © 2015 The Societies and John Wiley & Sons Australia, Ltd.

  7. Novel chitosan film embedded with liposome-encapsulated phage for biocontrol of Escherichia coli O157:H7 in beef.

    Science.gov (United States)

    Cui, Haiying; Yuan, Lu; Lin, Lin

    2017-12-01

    In recent years, phages used for the reduction of pathogenic bacteria have fostered many attentions, but they are liable to lost bioactivity in food due to the presence of acidic compounds, enzymes and evaporite materials. To improve the stability of phages, a chitosan edible film containing liposome-encapsulated phage was engineered in the present study. The characteristics of liposome-encapsulated phage and the chitosan film containing liposome-encapsulated phage were investigated. The encapsulation efficiency of phages in liposome reached 57.66±0.12%. Besides, the desirable physical properties of chitosan film were obtained. The chitosan film embedded with liposome-encapsulated phage exhibited high antibacterial activity against Escherichia coli O157:H7, without the impact on the sensory properties of beef. Hence, chitosan film containing liposome-encapsulated phage could be a promising antibacterial packaging for beef preservation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. MIMOX: a web tool for phage display based epitope mapping

    Directory of Open Access Journals (Sweden)

    Honda Wataru

    2006-10-01

    Full Text Available Abstract Background Phage display is widely used in basic research such as the exploration of protein-protein interaction sites and networks, and applied research such as the development of new drugs, vaccines, and diagnostics. It has also become a promising method for epitope mapping. Research on new algorithms that assist and automate phage display based epitope mapping has attracted many groups. Most of the existing tools have not been implemented as an online service until now however, making it less convenient for the community to access, utilize, and evaluate them. Results We present MIMOX, a free web tool that helps to map the native epitope of an antibody based on one or more user supplied mimotopes and the antigen structure. MIMOX was coded in Perl using modules from the Bioperl project. It has two sections. In the first section, MIMOX provides a simple interface for ClustalW to align a set of mimotopes. It also provides a simple statistical method to derive the consensus sequence and embeds JalView as a Java applet to view and manage the alignment. In the second section, MIMOX can map a single mimotope or a consensus sequence of a set of mimotopes, on to the corresponding antigen structure and search for all of the clusters of residues that could represent the native epitope. NACCESS is used to evaluate the surface accessibility of the candidate clusters; and Jmol is embedded to view them interactively in their 3D context. Initial case studies show that MIMOX can reproduce mappings from existing tools such as FINDMAP and 3DEX, as well as providing novel, rational results. Conclusion A web-based tool called MIMOX has been developed for phage display based epitope mapping. As a publicly available online service in this area, it is convenient for the community to access, utilize, and evaluate, complementing other existing programs. MIMOX is freely available at http://web.kuicr.kyoto-u.ac.jp/~hjian/mimox.

  9. SNP-PHAGE – High throughput SNP discovery pipeline

    Directory of Open Access Journals (Sweden)

    Cregan Perry B

    2006-10-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs as defined here are single base sequence changes or short insertion/deletions between or within individuals of a given species. As a result of their abundance and the availability of high throughput analysis technologies SNP markers have begun to replace other traditional markers such as restriction fragment length polymorphisms (RFLPs, amplified fragment length polymorphisms (AFLPs and simple sequence repeats (SSRs or microsatellite markers for fine mapping and association studies in several species. For SNP discovery from chromatogram data, several bioinformatics programs have to be combined to generate an analysis pipeline. Results have to be stored in a relational database to facilitate interrogation through queries or to generate data for further analyses such as determination of linkage disequilibrium and identification of common haplotypes. Although these tasks are routinely performed by several groups, an integrated open source SNP discovery pipeline that can be easily adapted by new groups interested in SNP marker development is currently unavailable. Results We developed SNP-PHAGE (SNP discovery Pipeline with additional features for identification of common haplotypes within a sequence tagged site (Haplotype Analysis and GenBank (-dbSNP submissions. This tool was applied for analyzing sequence traces from diverse soybean genotypes to discover over 10,000 SNPs. This package was developed on UNIX/Linux platform, written in Perl and uses a MySQL database. Scripts to generate a user-friendly web interface are also provided with common queries for preliminary data analysis. A machine learning tool developed by this group for increasing the efficiency of SNP discovery is integrated as a part of this package as an optional feature. The SNP-PHAGE package is being made available open source at http://bfgl.anri.barc.usda.gov/ML/snp-phage/. Conclusion SNP-PHAGE provides a bioinformatics

  10. Development of a Phage Cocktail to Control Proteus mirabilis Catheter-associated Urinary Tract Infections.

    Science.gov (United States)

    Melo, Luís D R; Veiga, Patrícia; Cerca, Nuno; Kropinski, Andrew M; Almeida, Carina; Azeredo, Joana; Sillankorva, Sanna

    2016-01-01

    Proteus mirabilis is an enterobacterium that causes catheter-associated urinary tract infections (CAUTIs) due to its ability to colonize and form crystalline biofilms on the catheters surface. CAUTIs are very difficult to treat, since biofilm structures are highly tolerant to antibiotics. Phages have been used widely to control a diversity of bacterial species, however, a limited number of phages for P. mirabilis have been isolated and studied. Here we report the isolation of two novel virulent phages, the podovirus vB_PmiP_5460 and the myovirus vB_PmiM_5461, which are able to target, respectively, 16 of the 26 and all the Proteus strains tested in this study. Both phages have been characterized thoroughly and sequencing data revealed no traces of genes associated with lysogeny. To further evaluate the phages' ability to prevent catheter's colonization by Proteus, the phages adherence to silicone surfaces was assessed. Further tests in phage-coated catheters using a dynamic biofilm model simulating CAUTIs, have shown a significant reduction of P. mirabilis biofilm formation up to 168 h of catheterization. These results highlight the potential usefulness of the two isolated phages for the prevention of surface colonization by this bacterium.

  11. Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library

    Directory of Open Access Journals (Sweden)

    McMahon James B

    2007-10-01

    Full Text Available Abstract Background Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis. Methods In this study, we evaluated and compared the diversity of a 12-mer T7 phage-displayed peptide library randomized using codon-corrected trinucleotide cassettes with a T7 and an M13 12-mer phage-displayed peptide library constructed using the degenerate codon randomization method. Results We herein demonstrate that the combination of trinucleotide cassette amino acid codon randomization and T7 phage display construction methods resulted in a significant enhancement to the functional diversity of a 12-mer peptide library. This novel library exhibited superior amino acid uniformity and order-of-magnitude increases in amino acid sequence diversity as compared to degenerate codon randomized peptide libraries. Comparative analyses of the biophysical characteristics of the 12-mer peptide libraries revealed the trinucleotide cassette-randomized library to be a unique resource. Conclusion The combination of T7 phage display and trinucleotide cassette randomization resulted in a novel resource for the potential isolation of binding peptides for new and previously studied molecular targets.

  12. Treatment of in vitro enterohemorrhagic Escherichia coli infection using phage and probiotics.

    Science.gov (United States)

    Dini, C; Bolla, P A; de Urraza, P J

    2016-07-01

    To assay the combination of phage and probiotics against EHEC in vitro on infected Hep-2 cells. Phage and probiotics treatments on EHEC O157:H7-infected Hep-2 cells were assayed individually or combined. The effect of freeze-drying on phage and probiotic antimicrobial activity was also studied. While treatment with phage alone increased cell detachment caused by EHEC infection, the treatments with MM alone or in combination with phage proved to effectively diminish cell damage caused by EHEC infection. Combined treatment showed a decrease in apoptotic cell count of 57·3% and a reduction in EHEC adhesion to cell monolayer of 1·2 log CFU. The simultaneous use of phage and probiotics showed no antagonistic effect, and freeze-drying did not affect their antipathogenic activity. The combination of phage and probiotics has great potential for reducing the number of pathogens adhered to epithelial cells during EHEC O157:H7 infection and attenuating the cytotoxic effect derived from it. Further in vivo assays are needed for assessing the actual effectiveness of the treatment. This study presents a freeze-dried formulation of phage and probiotics capable of controlling EHEC infections and reducing epithelial cell damage in vitro. © 2016 The Society for Applied Microbiology.

  13. Phage inactivation of foodborne Shigella on ready-to-eat spiced chicken.

    Science.gov (United States)

    Zhang, Hui; Wang, Ran; Bao, Hongduo

    2013-01-01

    Shigellosis, also called bacillary dysentery, is an infectious disease caused by Shigella species, including Shigella flexneri, Shigella dysenteriae, Shigella sonnei, and Shigella boydii. Infection with S. flexneri can result in epidemics, and Shigella-contaminated food is often the source of infection, such as ready-to-eat spiced chicken and duck. Therefore, we investigated the ability of Shigella phages to inhibit pathogenic Shigella spp. in ready-to-eat spiced chicken. Food samples were inoculated with individual species (1 × 10(4) cfu/g) or a mixture (S. flexneri 2a, S. dysenteriae, and S. sonnei) to a total concentration of 3 × 10(4) cfu/g. Single phages or a phage cocktail were added thereafter (1 × 10(8) pfu/g or 3 × 10(8) pfu/g), respectively, and samples were incubated at 4°C for 72 h. In general, the application of more phages (3 × 10(8) pfu/g) was the most effective treatment. Phages could reduce bacterial counts by up to 2 log(10)/g after 48 h incubation when treated with the cocktail, and after 72 h the host could not be detected. Similarly, the host in spiced chicken treated with single phage was also sharply reduced after 72 h incubation. The results suggest that an obligately virulent phage cocktail, such as S. flexneri, S. dysenteriae, and S. sonnei phages, can effectively reduce potential contamination of Shigella spp. in ready-to-eat chicken products.

  14. Characterization of Pseudomonas aeruginosa phage K5 genome and identification of its receptor related genes.

    Science.gov (United States)

    Li, Lingyan; Pan, Xuewei; Cui, Xiaoli; Sun, Qinghui; Yang, Xiaojing; Yang, Hongjiang

    2016-12-01

    Phage genomic information and the nature of host-phage interactions are important for phage applications. In this study, Pseudomonas aeruginosa phage K5 is characterized as a linear double-stranded genomic DNA molecule of 93,754 bp with identical 1182-bp direct terminal repeats. Comparative genomic analysis reveals that phage K5 is highly homologous to the "PaP1-like" phages. Thirteen mutants resistant to phage K5 are screened in a transposon mutant library. The disrupted genetic loci are identified as gene Y880_RS05480 encoding a putative O-antigen polymerase Wzy and gene wapH encoding a glycosyltransferase. The mutants are confirmed by the complementation experiment. The production of biofilm and the profile of lipopolysaccharide (LPS) are further analyzed in the Y880_RS05480 mutant. Our data indicate that LPS is the receptor of phage K5. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Salmonella-Typhimurium phage types from human salmonellosis in denmark 1988 to 1993

    DEFF Research Database (Denmark)

    Wegener, Henrik Caspar; Baggesen, Dorte Lau; Gaarslev, K.

    1994-01-01

    A total of 989 isolates of Salmonella enterica ssp. enterica serovar Typhimurium from cases of human salmonellosis were investigated by phage typing, The isolates comprised all isolates recovered during the month of August in each of the years from 1988 to 1993. Phage typing assigned 82.......6% of the strains to 36 different definitive types, 11.9% of the strains belonged to types of unknown lysis pattern (RDNC), and 5.5% could not be typed by the phages used (NT). Three phage types (12, 66 and 110) made up approximately 50% of the isolates in each of the years investigated. During the period...... in question these types showed major changes in prevalence: phage type 12 increased from 4.0% in 1988 to a maximum of 55.2% in 1992, and phage type 66 and phage type 110 were reduced from 40.1% and 27.8% to a minimum of 3.9% and 4.8% in 1993 and 1992, respectively. The increasing prevalence of phage type 12...

  16. Bioinformatic analysis of phage AB3, a phiKMV-like virus infecting Acinetobacter baumannii.

    Science.gov (United States)

    Zhang, J; Liu, X; Li, X-J

    2015-01-16

    The phages of Acinetobacter baumannii has drawn increasing attention because of the multi-drug resistance of A. baumanni. The aim of this study was to sequence Acinetobacter baumannii phage AB3 and conduct bioinformatic analysis to lay a foundation for genome remodeling and phage therapy. We isolated and sequenced A. baumannii phage AB3 and attempted to annotate and analyze its genome. The results showed that the genome is a double-stranded DNA with a total length of 31,185 base pairs (bp) and 97 open reading frames greater than 100 bp. The genome includes 28 predicted genes, of which 24 are homologous to phage AB1. The entire coding sequence is located on the negative strand, representing 90.8% of the total length. The G+C mol% was 39.18%, without areas of high G+C content over 200 bp in length. No GC island, tRNA gene, or repeated sequence was identified. Gene lengths were 120-3099 bp, with an average of 1011 bp. Six genes were found to be greater than 2000 bp in length. Genomic alignment and phylogenetic analysis of the RNA polymerase gene showed that similar to phage AB1, phage AB3 is a phiKMV-like virus in the T7 phage family.

  17. New perspective for phage display as an efficient and versatile technology of functional proteomics.

    Science.gov (United States)

    Li, Wei; Caberoy, Nora B

    2010-01-01

    Phage display with antibody libraries has been widely used with versatile applications. However, phage display with cDNA libraries is rare and inefficient. Because of uncontrollable reading frames and stop codons in cDNA repertoires, high percentage of phage clones identified from conventional cDNA libraries are non-open reading frames (non-ORFs) encoding unnatural short peptides with minimal implications in protein networks. Consequently, phage display has not been used as a technology of functional proteomics to elucidate protein-protein interactions like yeast two-hybrid system and mass spectrometry-based technologies. Several strategies, including C-terminal display and ORF cDNA libraries, have been explored to circumvent the technical problem. The accumulative endeavors eventually led to the efficient elucidation of a large number of tubby- and phosphatidylserine-binding proteins in recent studies by ORF phage display with minimal reading frame issue. ORF phage display inherits all the versatile applications of antibody phage display, but enables efficient identification of real endogenous proteins with efficiency, sensitivity, and accuracy comparable to other technologies of functional proteomics. Its ELISA-like procedure can be conveniently adapted by individual laboratories or fully automated for high-throughput screening. Thus, ORF phage display is an efficient, sensitive, versatile, and convenient technology of functional proteomics for elucidation of global and pathway-specific protein-protein interactions, disease mechanisms, or therapeutic targets.

  18. Bacteriophage phi11 lysin: physicochemical characterization and comparison with phage phi80a lysin

    Science.gov (United States)

    Phage lytic enzymes are promising antimicrobial agents. Lysins of phage phi11 (LysPhi11) and phi80a (LysPhi80a) can lyse (destroy) biofilms and cells of antibiotic-resistant strains of Staphylococcus aureus. Stability of enzymes is one of the parameters making their practical use possible. The obj...

  19. Phages of non-dairy lactococci: isolation and characterisation of ФL47, a phage infecting the grass isolate Lactococcus lactis ssp. cremoris DPC6860

    Directory of Open Access Journals (Sweden)

    Daniel eCavanagh

    2014-01-01

    Full Text Available Lactococci isolated from non-dairy sources have been found to possess enhanced metabolic activity when compared to dairy strains. These capabilities may be harnessed through the use of these strains as starter or adjunct cultures to produce more diverse flavour profiles in cheese and other dairy products. To understand the interactions between these organisms and the phages that infect them, a number of phages were isolated against lactococcal strains of non-dairy origin. One such phage, ФL47, was isolated from a sewage sample using the grass isolate L. lactis ssp. cremoris DPC6860 as a host. Visualisation of phage virions by transmission electron microscopy established that this phage belongs to the family Siphoviridae and possesses a long tail fiber, previously unseen in dairy lactococcal phages. Determination of the lytic spectrum revealed a broader than expected host range, with ФL47 capable of infecting 4 industrial dairy strains, including ML8, HP and 310, and 3 additional non-dairy isolates. Whole genome sequencing of ФL47 revealed a dsDNA genome of 128, 546 bp, making it the largest sequenced lactococcal phage to date. In total, 190 open reading frames (ORFs were identified, and comparative analysis revealed that the predicted products of 117 of these ORFs shared greater than 50% amino acid identity with those of L. lactis phage Ф949, a phage isolated from cheese whey. Despite their different ecological niches, the genomic content and organisation of ФL47 and Ф949 are quite similar, with both containing 4 gene clusters oriented in different transcriptional directions. Other features that distinguish ФL47 from Ф949 and other lactococcal phages, in addition to the presence of the tail fiber and the genome length, include a low GC content (32.5% and a high number of predicted tRNA genes (8. Comparative genome analysis supports the conclusion that ФL47 is a new member of the 949 lactococcal phage group which currently includes the

  20. Assessment of the Effects of Various UV Sources on Inactivation and Photoproduct Induction in Phage T7 Dosimeter

    NARCIS (Netherlands)

    Fekete, A.; Vink, A.A.; Gaspar, S.; Berces, A.; Modos, K.; Ronto, Gy.; Roza, L.

    1998-01-01

    The correlation between the biologically effective dose (BED) of a phage T7 biological dosimeter and the induction of cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts ((6-4)PD) in the phage DNA was determined using seven various UV sources. The BED is the inactivation rate of phage T7

  1. Phage Therapy an Effective Remedy Against Drug-Resistant Bugs and Hard to Treat Bacterial Infections-A Review

    Directory of Open Access Journals (Sweden)

    Mudasir Ali Rather

    2012-04-01

    Full Text Available The injudicious use of antibiotics not only in medicine but also to promote the growth of farm animals has led to the development of antibiotic resistance against many bacterial diseases. One of the remedy against such drug resistant bacterial infections is the application of phage (Bacteriophage therapy. Phage therapy involves using phages or their products as bioagents for the treatment or prophylaxis of bacterial infections. There are two types of phages based on their type of life cycle: the lytic and the lysogenic phages. Only the lytic phages are used in phage therapy, because of the disadvantages of lysogenic pahges (Superinfection immunity, lysogenic conversion, specialized transduction. Apart from live phages the phage byproducts like phage lysins can also be used specifically against certain bacterial infections. The reports indicate that appropriate administration of living phages can be used to treat lethal infectious diseases caused by bacteria, like Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella spp. and Staphylococcus aureus etc. In the coming time the phage therapy will compensate for unavoidable complications of antimicrobial therapy, particularly the appearance of multidrug resistance bacteria (super bugs.

  2. Cultivated single stranded DNA phages that infect marine Bacteroidetes prove difficult to detect with DNA binding stains

    DEFF Research Database (Denmark)

    Holmfeldt, Karin; Odic, Dusko; Sullivan, Matthew B.

    2012-01-01

    This is the first description of cultivated icosahedral single stranded DNA (ssDNA) phages isolated on heterotrophic marine bacterioplankton and with Bacteroidetes hosts. None of the 8 phages stained well with DNA binding stains, suggesting that in situ abundances of ssDNA phages are drastically...

  3. Phage based green chemistry for gold ion reduction and gold retrieval.

    Science.gov (United States)

    Setyawati, Magdiel I; Xie, Jianping; Leong, David T

    2014-01-22

    The gold mining industry has taken its toll on the environment, triggering the development of more environmentally benign processes to alleviate the waste load release. Here, we demonstrate the use of bacteriophages (phages) for biosorption and bioreduction of gold ions from aqueous solution, which potentially can be applied to remediate gold ions from gold mining waste effluent. Phage has shown a remarkably efficient sorption of gold ions with a maximum gold adsorption capacity of 571 mg gold/g dry weight phage. The product of this phage mediated process is gold nanocrystals with the size of 30-630 nm. Biosorption and bioreduction processes are mediated by the ionic and covalent interaction between gold ions and the reducing groups on the phage protein coat. The strategy offers a simple, ecofriendly and feasible option to recover of gold ions to form readily recoverable products of gold nanoparticles within 24 h.

  4. SIMULTANEOUS EFFECTS OF SHAKING AND TEMPERATURE ON VEROTOXIN1 PHAGE INDUCTION FROM VEROTOXIGENIC ESCHERICHIA COLI STRAINS

    Directory of Open Access Journals (Sweden)

    H. Hosain Zadegan, M. Sattari, M. H. Zahir, A. A. Allame

    2006-01-01

    Full Text Available Induction of lambda phage carring verotoxin1 gene from a verotoxigenic strains of Escherichia coli and released verotoxin1 were studied under environmental factors of shaking and termperature. Verotoxin1 phage in Escherichia coli PA 101 and transductants was confirmed by bacteriophage detection assay. Shaking of culture media and increasing temperature until 42 ºC increased phage particles in supernatants of Escherichia coli PA 101. Our results indicate that environmental factors such as shaking movements in natural inhabitates of bacteria such as river or sewage streams and temperature rise in summer season could be factors in induce and release free verotoxin1 – producing phage particles in nature that in turn could be the source of phage spreading to other related bacteria , and responsible for increased outbreaks of food borne diseases with verotoxigenic Escherichia coli in warm monthes of year in tropical areas.

  5. Resistance gene transfer: induction of transducing phage by sub-inhibitory concentrations of antimicrobials is not correlated to induction of lytic phage.

    Science.gov (United States)

    Stanczak-Mrozek, Kinga I; Laing, Ken G; Lindsay, Jodi A

    2017-06-01

    Horizontal gene transfer of antimicrobial resistance (AMR) genes between clinical isolates via transduction is poorly understood. MRSA are opportunistic pathogens resistant to all classes of antimicrobial agents but currently no strains are fully drug resistant. AMR gene transfer between Staphylococcus aureus isolates is predominantly due to generalized transduction via endogenous bacteriophage, and recent studies have suggested transfer is elevated during host colonization. The aim was to investigate whether exposure to sub-MIC concentrations of antimicrobials triggers bacteriophage induction and/or increased efficiency of AMR gene transfer. Isolates from MRSA carriers were exposed to nine antimicrobials and supernatants were compared for lytic phage particles and ability to transfer an AMR gene. A new technology, droplet digital PCR, was used to measure the concentration of genes in phage particles. All antibiotics tested induced lytic phage and AMR gene transduction, although the ratio of transducing particles to lytic particles differed substantially for each antibiotic. Mupirocin induced the highest ratio of transducing versus lytic particles. Gentamicin and novobiocin reduced UV-induced AMR transduction. The genes carried in phage particles correlated with AMR transfer or lytic particle activity, suggesting antimicrobials influence which DNA sequences are packaged into phage particles. Sub-inhibitory antibiotics induce AMR gene transfer between clinical MRSA, while combination therapy with an inhibiting antibiotic could potentially alter AMR gene packaging into phage particles, reducing AMR transfer. In a continually evolving environment, pathogens have an advantage if they can transfer DNA while lowering the risk of lytic death.

  6. Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas.

    Science.gov (United States)

    Ahern, Stephen J; Das, Mayukh; Bhowmick, Tushar Suvra; Young, Ry; Gonzalez, Carlos F

    2014-01-01

    The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ~4 × 10(-12) ml cell(-1) min(-1) for X. fastidiosa strain Temecula 1 and ~5 × 10(-10) to 7 × 10(-10) ml cell(-1) min(-1) for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa.

  7. Phage-driven loss of virulence in a fish pathogenic bacterium.

    Directory of Open Access Journals (Sweden)

    Elina Laanto

    2012-12-01

    Full Text Available Parasites provide a selective pressure during the evolution of their hosts, and mediate a range of effects on ecological communities. Due to their short generation time, host-parasite interactions may also drive the virulence of opportunistic bacteria. This is especially relevant in systems where high densities of hosts and parasites on different trophic levels (e.g. vertebrate hosts, their bacterial pathogens, and virus parasitizing bacteria co-exist. In farmed salmonid fingerlings, Flavobacterium columnare is an emerging pathogen, and phage that infect F. columnare have been isolated. However, the impact of these phage on their host bacterium is not well understood. To study this, four strains of F. columnare were exposed to three isolates of lytic phage and the development of phage resistance and changes in colony morphology were monitored. Using zebrafish (Danio rerio as a model system, the ancestral rhizoid morphotypes were associated with a 25-100% mortality rate, whereas phage-resistant rough morphotypes that lost their virulence and gliding motility (which are key characteristics of the ancestral types, did not affect zebrafish survival. Both morphotypes maintained their colony morphologies over ten serial passages in liquid culture, except for the low-virulence strain, Os06, which changed morphology with each passage. To our knowledge, this is the first report of the effects of phage-host interactions in a commercially important fish pathogen where phage resistance directly correlates with a decline in bacterial virulence. These results suggest that phage can cause phenotypic changes in F. columnare outside the fish host, and antagonistic interactions between bacterial pathogens and their parasitic phage can favor low bacterial virulence under natural conditions. Furthermore, these results suggest that phage-based therapies can provide a disease management strategy for columnaris disease in aquaculture.

  8. Isolation and characterization of a T7-like lytic phage for Pseudomonas fluorescens

    Directory of Open Access Journals (Sweden)

    Neubauer Peter

    2008-10-01

    Full Text Available Abstract Background Despite the proven relevance of Pseudomonas fluorescens as a spoilage microorganism in milk, fresh meats and refrigerated food products and the recognized potential of bacteriophages as sanitation agents, so far no phages specific for P. fluorescens isolates from dairy industry have been closely characterized in view of their lytic efficiency. Here we describe the isolation and characterization of a lytic phage capable to infect a variety of P. fluorescens strains isolated from Portuguese and United States dairy industries. Results Several phages were isolated which showed a different host spectrum and efficiency of lysis. One of the phages, phage ϕIBB-PF7A, was studied in detail due to its efficient lysis of a wide spectrum of P. fluorescens strains and ribotypes. Phage ϕIBB-PF7A with a head diameter of about 63 nm and a tail size of about 13 × 8 nm belongs morphologically to the Podoviridae family and resembles a typical T7-like phage, as analyzed by transmission electron microscopy (TEM. The phage growth cycle with a detected latent period of 15 min, an eclipse period of 10 min, a burst size of 153 plaque forming units per infected cell, its genome size of approximately 42 kbp, and the size and N-terminal sequence of one of the protein bands, which gave similarity to the major capsid protein 10A, are consistent with this classification. Conclusion The isolated T7-like phage, phage ϕIBB-PF7A, is fast and efficient in lysing different P. fluorescens strains and may be a good candidate to be used as a sanitation agent to control the prevalence of spoilage causing P. fluorescens strains in dairy and food related environments.

  9. Manipulating or superseding host recombination functions: a dilemma that shapes phage evolvability.

    Directory of Open Access Journals (Sweden)

    Louis-Marie Bobay

    Full Text Available Phages, like many parasites, tend to have small genomes and may encode autonomous functions or manipulate those of their hosts'. Recombination functions are essential for phage replication and diversification. They are also nearly ubiquitous in bacteria. The E. coli genome encodes many copies of an octamer (Chi motif that upon recognition by RecBCD favors repair of double strand breaks by homologous recombination. This might allow self from non-self discrimination because RecBCD degrades DNA lacking Chi. Bacteriophage Lambda, an E. coli parasite, lacks Chi motifs, but escapes degradation by inhibiting RecBCD and encoding its own autonomous recombination machinery. We found that only half of 275 lambdoid genomes encode recombinases, the remaining relying on the host's machinery. Unexpectedly, we found that some lambdoid phages contain extremely high numbers of Chi motifs concentrated between the phage origin of replication and the packaging site. This suggests a tight association between replication, packaging and RecBCD-mediated recombination in these phages. Indeed, phages lacking recombinases strongly over-represent Chi motifs. Conversely, phages encoding recombinases and inhibiting host recombination machinery select for the absence of Chi motifs. Host and phage recombinases use different mechanisms and the latter are more tolerant to sequence divergence. Accordingly, we show that phages encoding their own recombination machinery have more mosaic genomes resulting from recent recombination events and have more diverse gene repertoires, i.e. larger pan genomes. We discuss the costs and benefits of superseding or manipulating host recombination functions and how this decision shapes phage genome structure and evolvability.

  10. Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas

    Science.gov (United States)

    Ahern, Stephen J.; Das, Mayukh; Bhowmick, Tushar Suvra; Young, Ry

    2014-01-01

    The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ∼4 × 10−12 ml cell−1 min−1 for X. fastidiosa strain Temecula 1 and ∼5 × 10−10 to 7 × 10−10 ml cell−1 min−1 for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa. PMID:24214944

  11. The filamentous phages fd and IF1 use different mechanisms to infect Escherichia coli.

    Science.gov (United States)

    Lorenz, Stefan H; Jakob, Roman P; Weininger, Ulrich; Balbach, Jochen; Dobbek, Holger; Schmid, Franz X

    2011-01-28

    The filamentous phage fd uses its gene 3 protein (G3P) to target Escherichia coli cells in a two-step process. First, the N2 domain of G3P attaches to an F pilus, and then the N1 domain binds to TolA-C. N1 and N2 are tightly associated, rendering the phage robust but noninfectious because the binding site for TolA-C is buried at the domain interface. Binding of N2 to the F pilus initiates partial unfolding, domain disassembly, and prolyl cis-to-trans isomerization in the hinge between N1 and N2. This activates the phage, and trans-Pro213 maintains this state long enough for N1 to reach TolA-C. Phage IF1 targets I pili, and its G3P contains also an N1 domain and an N2 domain. The pilus-binding N2 domains of the phages IF1 and fd are unrelated, and the N1 domains share a 31% sequence identity. We show that N2 of phage IF1 mediates binding to the I pilus, and that N1 targets TolA. Crystallographic and NMR analyses of the complex between N1 and TolA-C indicate that phage IF1 interacts with the same site on TolA-C as phage fd. In IF1-G3P, N1 and N2 are independently folding units, however, and the TolA binding site on N1 is permanently accessible. Activation by unfolding and prolyl isomerization, as in the case of phage fd, is not observed. In IF1-G3P, the absence of stabilizing domain interactions is compensated for by a strong increase in the stabilities of the individual domains. Apparently, these closely related filamentous phages evolved different mechanisms to reconcile robustness with high infectivity. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Dynamics of adaptive immunity against phage in bacterial populations

    Science.gov (United States)

    Bradde, Serena; Vucelja, Marija; Tesileanu, Tiberiu; Balasubramanian, Vijay

    The CRISPR (clustered regularly interspaced short palindromic repeats) mechanism allows bacteria to adaptively defend against phages by acquiring short genomic sequences (spacers) that target specific sequences in the viral genome. We propose a population dynamical model where immunity can be both acquired and lost. The model predicts regimes where bacterial and phage populations can co-exist, others where the populations oscillate, and still others where one population is driven to extinction. Our model considers two key parameters: (1) ease of acquisition and (2) spacer effectiveness in conferring immunity. Analytical calculations and numerical simulations show that if spacers differ mainly in ease of acquisition, or if the probability of acquiring them is sufficiently high, bacteria develop a diverse population of spacers. On the other hand, if spacers differ mainly in their effectiveness, their final distribution will be highly peaked, akin to a ``winner-take-all'' scenario, leading to a specialized spacer distribution. Bacteria can interpolate between these limiting behaviors by actively tuning their overall acquisition rate.

  13. Structural Conservation of the Myoviridae Phage Tail Sheath Protein Fold

    Energy Technology Data Exchange (ETDEWEB)

    Aksyuk, Anastasia A.; Kurochkina, Lidia P.; Fokine, Andrei; Forouhar, Farhad; Mesyanzhinov, Vadim V.; Tong, Liang; Rossmann, Michael G. (SOIBC); (Purdue); (Columbia)

    2012-02-21

    Bacteriophage phiKZ is a giant phage that infects Pseudomonas aeruginosa, a human pathogen. The phiKZ virion consists of a 1450 {angstrom} diameter icosahedral head and a 2000 {angstrom}-long contractile tail. The structure of the whole virus was previously reported, showing that its tail organization in the extended state is similar to the well-studied Myovirus bacteriophage T4 tail. The crystal structure of a tail sheath protein fragment of phiKZ was determined to 2.4 {angstrom} resolution. Furthermore, crystal structures of two prophage tail sheath proteins were determined to 1.9 and 3.3 {angstrom} resolution. Despite low sequence identity between these proteins, all of these structures have a similar fold. The crystal structure of the phiKZ tail sheath protein has been fitted into cryo-electron-microscopy reconstructions of the extended tail sheath and of a polysheath. The structural rearrangement of the phiKZ tail sheath contraction was found to be similar to that of phage T4.

  14. Dynamics of adaptive immunity against phage in bacterial populations.

    Directory of Open Access Journals (Sweden)

    Serena Bradde

    2017-04-01

    Full Text Available The CRISPR (clustered regularly interspaced short palindromic repeats mechanism allows bacteria to adaptively defend against phages by acquiring short genomic sequences (spacers that target specific sequences in the viral genome. We propose a population dynamical model where immunity can be both acquired and lost. The model predicts regimes where bacterial and phage populations can co-exist, others where the populations exhibit damped oscillations, and still others where one population is driven to extinction. Our model considers two key parameters: (1 ease of acquisition and (2 spacer effectiveness in conferring immunity. Analytical calculations and numerical simulations show that if spacers differ mainly in ease of acquisition, or if the probability of acquiring them is sufficiently high, bacteria develop a diverse population of spacers. On the other hand, if spacers differ mainly in their effectiveness, their final distribution will be highly peaked, akin to a "winner-take-all" scenario, leading to a specialized spacer distribution. Bacteria can interpolate between these limiting behaviors by actively tuning their overall acquisition probability.

  15. Probabilistic and Flux Landscapes of the Phage $\\lambda$ Genetic Switch

    CERN Document Server

    Borggren, Nathan

    2012-01-01

    The phage $\\lambda$ infection of an \\textit{E. coli} cell has become a paradigm for understanding the molecular processes involved in gene expression and cell signaling. This system provides an example of a genetic switch, as cells with identical DNA choose either of two cell cycles: a lysogenic cycle, in which the phage genome is incorporated into the host and copied by the host; or a lytic cycle, resulting in the death of the cell and a burst of viruses. The robustness of this switch is remarkable; although the first stages of the lysogenic and lytic cycles are identical, a lysogen rarely spontaneously flips, and external stressors or instantaneous cell conditions are required to induce flipping. In particular, the cell fate decision can depend on the populations of two proteins, cI and Cro, as well as their oligomerization and subsequent binding affinities to three DNA sites. These processes in turn govern the rates at which RNAp transcribes the cI and Cro genes to produce more of their respective proteins...

  16. Regulation of a phage endolysin by disulfide caging.

    Science.gov (United States)

    Kuty, Gabriel F; Xu, Min; Struck, Douglas K; Summer, Elizabeth J; Young, Ry

    2010-11-01

    In contrast to canonical phage endolysins, which require holin-mediated disruption of the membrane to gain access to attack the cell wall, signal anchor release (SAR) endolysins are secreted by the host sec system, where they accumulate in an inactive form tethered to the membrane by their N-terminal SAR domains. SAR endolysins become activated by various mechanisms upon release from the membrane. In its inactive form, the prototype SAR endolysin, Lyz(P1), of coliphage P1, has an active-site Cys covalently blocked by a disulfide bond; activation involves a disulfide bond isomerization driven by a thiol in the newly released SAR domain, unblocking the active-site Cys. Here, we report that Lyz(103), the endolysin of Erwinia phage ERA103, is also a SAR endolysin. Although Lyz(103) does not have a catalytic Cys, genetic evidence suggests that it also is activated by a thiol-disulfide isomerization triggered by a thiol in the SAR domain. In this case, the inhibitory disulfide in nascent Lyz(103) is formed between cysteine residues flanking a catalytic glutamate, caging the active site. Thus, Lyz(P1) and Lyz(103) define subclasses of SAR endolysins that differ in the nature of their inhibitory disulfide, and Lyz(103) is the first enzyme found to be regulated by disulfide bond caging of its active site.

  17. Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex

    Directory of Open Access Journals (Sweden)

    Dennis Jonathan J

    2010-10-01

    Full Text Available Abstract Background The Burkholderia cepacia complex (BCC is comprised of at least seventeen Gram-negative species that cause infections in cystic fibrosis patients. Because BCC bacteria are broadly antibiotic resistant, phage therapy is currently being investigated as a possible alternative treatment for these infections. The purpose of our study was to sequence and characterize three novel BCC-specific phages: KS5 (vB_BceM-KS5 or vB_BmuZ-ATCC 17616, KS14 (vB_BceM-KS14 and KL3 (vB_BamM-KL3 or vB_BceZ-CEP511. Results KS5, KS14 and KL3 are myoviruses with the A1 morphotype. The genomes of these phages are between 32317 and 40555 base pairs in length and are predicted to encode between 44 and 52 proteins. These phages have over 50% of their proteins in common with enterobacteria phage P2 and so can be classified as members of the Peduovirinae subfamily and the "P2-like viruses" genus. The BCC phage proteins similar to those encoded by P2 are predominantly structural components involved in virion morphogenesis. As prophages, KS5 and KL3 integrate into an AMP nucleosidase gene and a threonine tRNA gene, respectively. Unlike other P2-like viruses, the KS14 prophage is maintained as a plasmid. The P2 E+E' translational frameshift site is conserved among these three phages and so they are predicted to use frameshifting for expression of two of their tail proteins. The lysBC genes of KS14 and KL3 are similar to those of P2, but in KS5 the organization of these genes suggests that they may have been acquired via horizontal transfer from a phage similar to λ. KS5 contains two sequence elements that are unique among these three phages: an ISBmu2-like insertion sequence and a reverse transcriptase gene. KL3 encodes an EcoRII-C endonuclease/methylase pair and Vsr endonuclease that are predicted to function during the lytic cycle to cleave non-self DNA, protect the phage genome and repair methylation-induced mutations. Conclusions KS5, KS14 and KL3 are the

  18. Following cell-fate in E. coli after infection by phage lambda.

    Science.gov (United States)

    Zeng, Lanying; Golding, Ido

    2011-10-14

    The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant). We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells. Here, we describe the full procedure for performing the infection experiments described in our earlier work. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35°C to trigger viral DNA injection. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome

  19. CRISPR-Cas-Mediated Phage Resistance Enhances Horizontal Gene Transfer by Transduction.

    Science.gov (United States)

    Watson, Bridget N J; Staals, Raymond H J; Fineran, Peter C

    2018-02-13

    A powerful contributor to prokaryotic evolution is horizontal gene transfer (HGT) through transformation, conjugation, and transduction, which can be advantageous, neutral, or detrimental to fitness. Bacteria and archaea control HGT and phage infection through CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) adaptive immunity. Although the benefits of resisting phage infection are evident, this can come at a cost of inhibiting the acquisition of other beneficial genes through HGT. Despite the ability of CRISPR-Cas to limit HGT through conjugation and transformation, its role in transduction is largely overlooked. Transduction is the phage-mediated transfer of bacterial DNA between cells and arguably has the greatest impact on HGT. We demonstrate that in Pectobacterium atrosepticum , CRISPR-Cas can inhibit the transduction of plasmids and chromosomal loci. In addition, we detected phage-mediated transfer of a large plant pathogenicity genomic island and show that CRISPR-Cas can inhibit its transduction. Despite these inhibitory effects of CRISPR-Cas on transduction, its more common role in phage resistance promotes rather than diminishes HGT via transduction by protecting bacteria from phage infection. This protective effect can also increase transduction of phage-sensitive members of mixed populations. CRISPR-Cas systems themselves display evidence of HGT, but little is known about their lateral dissemination between bacteria and whether transduction can contribute. We show that, through transduction, bacteria can acquire an entire chromosomal CRISPR-Cas system, including cas genes and phage-targeting spacers. We propose that the positive effect of CRISPR-Cas phage immunity on enhancing transduction surpasses the rarer cases where gene flow by transduction is restricted. IMPORTANCE The generation of genetic diversity through acquisition of DNA is a powerful contributor to microbial evolution and occurs through

  20. World temperate fruit production: characteristics and challenges

    Directory of Open Access Journals (Sweden)

    Jorge B. Retamales

    2011-10-01

    Full Text Available In the last 30 years world population has increased 70% but per capita global fruit consumption is only 20% higher. Even though tropical and temperate fruit have similar contributions to the 50 kg/person/year of US consumption of fresh fruit, in the last 30 years this has been slightly greater for temperate fruit. Within fruit consumption, the largest expansion has been for organic fruit which increased more than 50% in the 2002-2006 period. The largest expansion of area planted in the 1996-2006 has been for kiwi (29% and blueberries (20%, while apples (-24% and sour cherries (-13% have had the largest reductions. Nearly 50% of the total global volume of fruit is produced by 5 countries: China, USA, Brazil, Italy and Spain. The main producer (China accounts for 23% of the total. While the main exporters are Spain, USA and Italy, the main importers are Germany, Russia and UK. Demands for the industry have evolved towards quality, food safety and traceability. The industry faces higher productions costs (labor, energy, agrichemicals. The retailers are moving towards consolidation while the customers are changing preferences (food for health. In this context there is greater pressure on growers, processors and retailers. Emerging issues are labor supply, climate change, water availability and sustainability. Recent developments in precision agriculture, molecular biology, phenomics, crop modelling and post harvest physiology should increase yields and quality, and reduce costs for temperate fruit production around the world.

  1. A tail of two phages: Genomic and functional analysis of Listeria monocytogenes phages vB_LmoS_188 and vB_LmoS_293 reveal the receptor-binding proteins involved in host specificity.

    Directory of Open Access Journals (Sweden)

    Aidan eCasey

    2015-10-01

    Full Text Available The physical characteristics of bacteriophages establish them as viable candidates for downstream development of pathogen detection assays and biocontrol measures. To utilize phages for such purposes, a detailed knowledge of their host interaction mechanisms is a prerequisite. There is currently a wealth of knowledge available concerning Gram-negative phage-host interaction, but little by comparison for Gram-positive phages and Listeria phages in particular. In this research, the lytic spectrum of two recently isolated Listeria monocytogenes phages (vB_LmoS_188 and vB_LmoS_293 was determined, and the genomic basis for their observed serotype 4b/4e host-specificity was investigated using comparative genomics. The late tail genes of these phages were identified to be highly conserved when compared to other serovar 4-specific Listeria phages. Spontaneous mutants of each of these phages with broadened host specificities were generated. Their late tail gene sequences were compared with their wild-type counterparts resulting in the putative identification of the products of ORF 19 of vB_LmoS_188 and ORF 20 of vB_LmoS_293 as the receptor binding proteins of these phages. The research findings also indicate that conserved baseplate architectures and host interaction mechanisms exist for Listeria siphoviruses with differing host-specificities, and further contribute to the current knowledge of phage-host interactions with regard to Listeria phages.

  2. Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species

    DEFF Research Database (Denmark)

    Szymczak, Paula; Janzen, Thomas; Neves, Ana Rute

    2017-01-01

    shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus...... the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires...

  3. Investigation of the Relationship between Lactococcal Host Cell Wall Polysaccharide Genotype and 936 Phage Receptor Binding Protein Phylogeny

    DEFF Research Database (Denmark)

    Mahony, Jennifer; Kot, Witold Piotr; Murphy, James

    2013-01-01

    Comparative genomics of 11 lactococcal 936-type phages combined with host range analysis allowed subgrouping of these phage genomes, particularly with respect to their encoded receptor binding proteins. The so-called pellicle or cell wall polysaccharide of Lactococcus lactis, which has been...... of tested 936-type phages. Such a correlation would allow prediction of the intrinsic 936-type phage sensitivity of a particular lactococcal strain and substantiates the notion that the lactococcal pellicle polysaccharide represents the receptor for (certain) 936-type phages while also partially explaining...

  4. Recent advances in bacteriophage therapy: how delivery routes, formulation, concentration and timing influence the success of phage therapy.

    Science.gov (United States)

    Ryan, Elizabeth M; Gorman, Sean P; Donnelly, Ryan F; Gilmore, Brendan F

    2011-10-01

    Bacteriophages are bacteria-specific viruses that infect and, in the case of obligately lytic phages, destroy their host bacteria. Phage therapy has been used therapeutically to combat bacterial infections since their discovery. This paper reviewed recent in-vivo phage therapy studies, with a distinct focus on the effect of delivery routes, phage concentration and timing of administration on the success of the therapy. It was found that the most successful route of administration for the treatment of systemic infections was via the parenteral route. Oral delivery is mainly used to treat gastrointestinal infections. However, in some cases phages can also reach the systemic circulation. Local delivery (skin, ears, teeth) has proved extremely successful in the treatment of topical infections, as has the inhalation of phages for the treatment of lung infections. The ability of phages to prevent biofilm formation on medical devices has received much attention, mainly in the area of catheter coatings. This review also highlights areas in which phage therapy needs substantial development. Many papers were lacking in formulation details, with crude phage stocks being used in most cases. No phage stability data were included in any of the papers. The review concluded that although phage therapy is an excellent alternative for the treatment of bacterial infections, optimisation of formulations and long-term stability data is required before it can be widely used within a clinical setting. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

  5. Viruses versus bacteria-novel approaches to phage therapy as a tool against multidrug-resistant pathogens.

    Science.gov (United States)

    Viertel, Tania Mareike; Ritter, Klaus; Horz, Hans-Peter

    2014-09-01

    Bacteriophage therapy (the application of phages to treat bacterial infections) has a tradition dating back almost a century, but interest in phage therapy slowed down in the West when antibiotics were discovered. With the emerging threat of infections caused by multidrug-resistant bacteria and scarce prospects of newly introduced antibiotics in the future, phages are currently being reconsidered as alternative therapeutics. Conventional phage therapy uses lytic bacteriophages for treatment and recent human clinical trials have revealed encouraging results. In addition, several other modern approaches to phages as therapeutics have been made in vitro and in animal models. Dual therapy with phages and antibiotics has resulted in significant reductions in the number of bacterial pathogens. Bioengineered phages have overcome many of the problems of conventional phage therapy, enabled targeted drug delivery or reversed the resistance of drug-resistant bacteria. The use of enzymes derived from phages, such as endolysin, as therapeutic agents has been efficient in the elimination of Gram-positive pathogens. This review presents novel strategies for phage-related therapies and describes our current knowledge of natural bacteriophages within the human microbiome. Our aim is to provide an overview of the high number of different methodological concepts, thereby encouraging further research on this topic, with the ultimate goal of using phages as therapeutic or preventative medicines in daily clinical practice. © 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.

  6. Safety analysis of a Russian phage cocktail: From MetaGenomic analysis to oral application in healthy human subjects

    Energy Technology Data Exchange (ETDEWEB)

    McCallin, Shawna, E-mail: semccallin@yahoo.com [Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Alam Sarker, Shafiqul, E-mail: sasarker@icddrb.org [International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212 (Bangladesh); Barretto, Caroline, E-mail: Caroline.Barretto@rdls.nestle.com [Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Sultana, Shamima, E-mail: shamima@icddrb.org [International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212 (Bangladesh); Berger, Bernard, E-mail: bernard.berger@rdls.nestle.com [Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Huq, Sayeda, E-mail: sayeeda@mail.icddrb.org [International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212 (Bangladesh); Krause, Lutz, E-mail: ltz.krause@gmail.com [Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Bibiloni, Rodrigo, E-mail: Rodrigo.Bibiloni@agresearch.co.nz [Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Schmitt, Bertrand, E-mail: bertrand.schmitt@rdls.nestle.com [Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Reuteler, Gloria, E-mail: gloria.reuteler@rdls.nestle.com [Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Brüssow, Harald, E-mail: harald.bruessow@rdls.nestle.com [Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)

    2013-09-01

    Phage therapy has a long tradition in Eastern Europe, where preparations are comprised of complex phage cocktails whose compositions have not been described. We investigated the composition of a phage cocktail from the Russian pharmaceutical company Microgen targeting Escherichia coli/Proteus infections. Electron microscopy identified six phage types, with numerically T7-like phages dominating over T4-like phages. A metagenomic approach using taxonomical classification, reference mapping and de novo assembly identified 18 distinct phage types, including 7 genera of Podoviridae, 2 established and 2 proposed genera of Myoviridae, and 2 genera of Siphoviridae. De novo assembly yielded 7 contigs greater than 30 kb, including a 147-kb Myovirus genome and a 42-kb genome of a potentially new phage. Bioinformatic analysis did not reveal undesired genes and a small human volunteer trial did not associate adverse effects with oral phage exposure. - Highlights: • We analyzed the composition of a commercial Russian phage cocktail. • The cocktail consists of at least 10 different phage genera. • No undesired genes were detected. • No adverse effects were seen upon oral application in a small human clinical trial.

  7. Oceanic temperate forest versus warm temperate rainforest: a reply to Grubb et al. (2017)

    DEFF Research Database (Denmark)

    McGlone, Matt S.; Buitenwerf, Robert; Richardson, Sarah J.

    2017-01-01

    that they ‘see no virtue in using climatic variables to define a [vegetation] formation type’, and then discuss problems with the climate- based global biome schemes of Holdridge (1947), Whittaker (1970), Box (1981) and Prentice et al. (1992). As our oceanic temperate forest (OTF) concept is underpinned......Grubb et al. (2017) point out that we (McGlone et al. 2016) erroneously stated that the definition of warm temperate rain forest (WTRF; Grubb et al. 2013) was based in part on climatic criteria. We apologise: their text made clear that this was not the case. However, they go on to say...... by climatic variables, and as they suggest that it largely falls within their WTRF and cool temperate rain forest (CTRF) concepts, we take this opportunity to further discuss the relative merits of these contrasting ways of classifying vegetation cover....

  8. Oral T4-like phage cocktail application to healthy adult volunteers from Bangladesh

    Energy Technology Data Exchange (ETDEWEB)

    Sarker, Shafiqul Alam, E-mail: sasarker@icddrb.org [International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212 (Bangladesh); McCallin, Shawna; Barretto, Caroline [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Berger, Bernard, E-mail: bernard.berger@rdls.nestle.com [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Pittet, Anne-Cecile [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Sultana, Shamima, E-mail: shamima@icddrb.org [International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212 (Bangladesh); Krause, Lutz, E-mail: ltz.krause@gmail.com [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Huq, Sayeda, E-mail: sayeeda@mail.icddrb.org [International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212 (Bangladesh); Bibiloni, Rodrigo, E-mail: Rodrigo.Bibiloni@agresearch.co.nz [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Bruttin, Anne, E-mail: anne.bruttin@rdls.nestle.com [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Reuteler, Gloria, E-mail: gloria.reuteler@rdls.nestle.com [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland); Bruessow, Harald, E-mail: harald.bruessow@rdls.nestle.com [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)

    2012-12-20

    The genomic diversity of 99 T4-like coliphages was investigated by sequencing an equimolar mixture with Illumina technology and screening them against different databases for horizontal gene transfer and undesired genes. A 9-phage cocktail was given to 15 healthy adults from Bangladesh at a dose of 3 Multiplication-Sign 10{sup 9} and 3 Multiplication-Sign 10{sup 7} plaque-forming units and placebo respectively. Phages were detected in 64% of the stool samples when subjects were treated with higher titer phage, compared to 30% and 28% with lower-titer phage and placebo, respectively. No Escherichia coli was present in initial stool samples, and no amplification of phage was observed. One percent of the administered oral phage was recovered from the feces. No adverse events were observed by self-report, clinical examination, or from laboratory tests for liver, kidney, and hematology function. No impact of oral phage was seen on the fecal microbiota composition with respect to bacterial 16S rRNA from stool.

  9. Selection of phage-displayed peptides for the detection of imidacloprid in water and soil.

    Science.gov (United States)

    Liu, Zhiping; Liu, Jianfeng; Wang, Kai; Li, Wenhui; Shelver, Weilin L; Li, Qing X; Li, Ji; Xu, Ting

    2015-09-15

    Imidacloprid is the most widely used neonicotinoid insecticide in the world and shows widespread environment and human exposures. A phage clone designated L7-1 that selectively binds to imidacloprid was selected from a commercial phage display library containing linear 7-mer randomized amino acid residues. Using the clone L7-1, a competitive enzyme-linked immunosorbent assay (ELISA) for imidacloprid was developed. The half-maximum signal inhibition concentration (IC50) and the limit of detection (LOD) of the phage ELISA for imidacloprid were 96 and 2.3 ng ml(-1), respectively. This phage ELISA showed relatively low cross-reactivity with all of the tested compounds structurally similar to imidacloprid, less than 2% with the exception of 6-chloronicotinic acid, a metabolite of imidacloprid that showed 11.5%. The average recoveries of the phage ELISA for imidacloprid in water and soil samples were in the ranges of 74.6 to 86.3% and 72.5 to 93.6%, respectively. The results of the competitive phage ELISA for imidacloprid in the fortified samples agreed well with those of a high-performance liquid chromatography (HPLC) method. The simple phage-displayed peptide technology has been proven to be a convenient and efficient method for the development of an alternative format of ELISA for small molecules. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Isolation and characterization of lytic phages TSE1-3 against Enterobacter cloacae

    Directory of Open Access Journals (Sweden)

    Khawaja Komal Ameer

    2016-01-01

    Full Text Available The emergence of antibiotic resistant bacterial pathogens is becoming a major challenge for patient care. The utilization of alternative therapies for infectious diseases other than antibiotics is an urgent need of today medical practice. The utilization of lytic bacteriophages and their gene products as therapeutic agents against antibiotic resistant bacteria is one of the convincing alternative approaches. Here we present the isolation and characterization of three lytic bacteriophages TSE1-3 against Enterobacter cloacae from sewage effluent. The isolates maintained antibacterial activity for 10 hours of incubation followed by the development of phage resistance. Their stability at different temperatures and pH, established their possible application in phage therapy. The highest activity of the phages was observed at 37°C and pH 7.0, while they gave lytic activity up to 60°C. The latent period of all the TSE phages was 20 minutes, while the burst size was 360 for TSE1, 270 for TSE2 and 311 for TSE3. The phages were harboring double-stranded DNA larger than 12kb in size. Further research into the phages genome and proteins, animal experiments, delivery parameters and clinical trials may lead to their utilization in phage therapy.

  11. Isolation and characterization of a rare waterborne lytic phage of Salmonella enterica serovar Paratyphi B.

    Science.gov (United States)

    Ahiwale, Sangeeta S; Bankar, Ashok V; Tagunde, Sujata N; Zinjarde, Smita; Ackermann, Hans W; Kapadnis, B P

    2013-05-01

    A lytic phage of Salmonella serovar Paratyphi B, named φSPB, was isolated from surface waters of the Pavana River in India. Phage φSPB is a member of the Podoviridae family and is morphologically similar to the 7-11 phages of the C3 morphotype of tailed phages, characterized by a very long, cigar-shaped head. The head measured approximately 153 × 57 nm, and the tail size was 12 × 7 nm. The phage was stable over a wide range of pH (4-9) and temperature (4-40 °C). The adsorption rate constant was 4.7 × 10(-10). Latent and eclipse periods were 10 and 15 min, respectively, and the burst size was 100 plaque-forming units/infected cell after 25 min at 37 °C. The phage DNA was 59 kb in size. Ten major proteins were observed on SDS-PAGE, although some of these proteins could be bacterial contaminants. This is the first report of Salmonella enterica subsp. enterica serovar Paratyphi B phage of C3 morphotype from India that has many unique features, such as high replication potential, short replication time, and stability over a wide range of pH and temperature, making it a promising biocontrol agent against the drug-resistant strains of Salmonella Paratyphi B.

  12. Screening and identification of a renal carcinoma specific peptide from a phage display peptide library

    Directory of Open Access Journals (Sweden)

    Tu Xiangan

    2011-11-01

    Full Text Available Abstract Background Specific peptide ligands to cell surface receptors have been extensively used in tumor research and clinical applications. Phage display technology is a powerful tool for the isolation of cell-specific peptide ligands. To screen and identify novel markers for renal cell carcinoma, we evaluated a peptide that had been identified by phage display technology. Methods A renal carcinoma cell line A498 and a normal renal cell line HK-2 were used to carry out subtractive screening in vitro with a phage display peptide library. After three rounds of panning, there was an obvious enrichment for the phages specifically binding to the A498 cells, and the output/input ratio of phages increased about 100 fold. A group of peptides capable of binding specifically to the renal carcinoma cells were obtained, and the affinity of these peptides to the targeting cells and tissues was studied. Results Through a cell-based ELISA, immunocytochemical staining, immunohistochemical staining, and immunofluorescence, the Phage ZT-2 and synthetic peptide ZT-2 were shown to specifically bind to the tumor cell surfaces of A498 and incision specimens, but not to normal renal tissue samples. Conclusion A peptide ZT-2, which binds specifically to the renal carcinoma cell line A498 was selected from phage display peptide libraries. Therefore, it provides a potential tool for early diagnosis of renal carcinoma or targeted drug delivery in chemotherapy.

  13. Phage applications for improving food safety and infection control in Egypt.

    Science.gov (United States)

    El-Shibiny, A; El-Sahhar, S; Adel, M

    2017-08-01

    The study investigated the use of bacteriophages to control bacterial contamination of chicken skin, eggs, tomatoes and meat. Experiments were performed to test the host ranges and killing potential of two isolated phages, ZCSE1 and ZCEC1, with hosts Salmonella and Escherichia coli respectively. The efficacy of both phages was determined by comparing the viable counts of recovered bacteria from treatment and phage-free control samples. In vitro experiments showed that phage ZCSE1 was able to reduce the numbers of Salmonella enterica ATCC 25566 to below 4·0 log 10 CFU per ml (3·4 log 10 CFU per ml reduction) in 240 min postinfection and phage ZCEC1 reduced the number of E. coli ATCC 8739 to undetectable levels (6·45 log 10 CFU per ml reduction) during the first hour of infection at 37°C. When applied to chicken skin and the surface of eggs, phage ZCSE1 treatment reduced the number of S. enterica ATCC 25566 by 2 log 10 and to undetectable levels (foods. The results suggest that phages could be effective treatments for pathogenic bacteria in food relevant contexts in Egypt. © 2017 The Society for Applied Microbiology.

  14. [Phage display peptide library for screening the peptides that specifically bind to osteoblasts cells].

    Science.gov (United States)

    Wu, Mingyue; Zhou, Yuqin; Du, Mingliang; Qian, Haiyan; Li, Quanli

    2015-07-01

    To explore the experimental methods that the phage peptide library technology screening human osteoblast specificity polypeptide, which will provide the basis of the experiment of the Ti surface biolization modification. Human calvarial osteoblasts were used as the target cells for whole-cell biopanning from a 12-mer peptide phage-display library. Cell eluent and cell lysis buffer were cultivate and count respectively after washing. Then the target cells were analyzed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence detection to authenticate the positive phage clones by human gingival fibroblast as the absorber cells. The positive phage clones were deduced by DNA sequencing. After four rounds of screening, twenty-two positive phage clones were found out from randomly selected phage monoclonals, whose single-strand DNA were extracted and sequenced. Amino acid sequence of the highest frequency peptide was MGWSWWPETWPM. The specific peptide against human osteoblasts can be obtained from a phage-display peptide library for use as a new research approach and experimental basis of the biolization modification of the titanium surface.

  15. New semi-pilot-scale reactor to study the photocatalytic inactivation of phages contained in aerosol.

    Science.gov (United States)

    Briggiler Marcó, Mariángeles; Negro, Antonio Carlos; Alfano, Orlando Mario; Quiberoni, Andrea Del Luján

    2017-04-12

    The aims of this work were to design and build a photocatalytic reactor (UV-A/TiO 2 ) to study the inactivation of phages contained in bioaerosols, which constitute the main dissemination via phages in industrial environments. The reactor is a close system with recirculation that consists of a stainless steel camera (cubic form, side of 60 cm) in which air containing the phage particles circulates and an acrylic compartment with six borosilicate plates covered with TiO 2 . The reactor is externally illuminated by 20 UV-A lamps. Both compartments are connected by a fan to facilitate the sample circulation. Samples are injected into the camera using two piston nebulizers working in series whereas several methodologies for sampling (impinger/syringe, sampling on photocatalytic plates, and impact of air on slide) were assayed. The reactor setup was carried out using phage B1 (Lactobacillus plantarum), and assays demonstrated a decrease of phage counts of 2.7 log orders after 1 h of photocatalytic treatment. Photonic efficiencies of inactivation were assessed by phage sampling on the photocatalytic plates or by impact of air on a glass slide at the photocatalytic reactor exit. Efficiencies of the same order of magnitude were observed using both sampling methods. This study demonstrated that the designed photocatalytic reactor is effective to inactivate phage B1 (Lb. plantarum) contained in bioaerosols.

  16. ORIGINAL ARTICLE: Multidrug Resistance and Phage Pattern of Staphylococcus aureus in Pyoderma Cases

    Directory of Open Access Journals (Sweden)

    Sanjay M. Wavare

    2012-01-01

    Full Text Available Background: Pyoderma is common in India and other tropical countries. Staphylococcus aureus is the commonest causative agent ofpyoderma. Aims and Objectives: To know the antibiotic susceptibility and bacteriophage pattern of Staphylococcus aureus isolated from pyoderma infection. Materials and Methods: One hundred clinically diagnosed pyoderma cases were investigated bacteriologically. A total of 59 isolates of S. aureus were subjected to antibioticsusceptibility testing by Kirby Bauer’s disk diffusion method and phage typing by routine test dilution X 100 bacteriophages. Results: Most of the strains were resistant to penicillin, ampicillin and were susceptible to gentamicin, streptomycin and erythromycin. Multidrug resistance was also high among these strains. Regarding the phage types, Phage type 52 (15 strains, 96 (8 strains and 71(16strains were predominant among the typed strains (55.95% of S. aureus. The most common group was mixed phage group (17% followed by phage group I (13.55%. Conclusion: Knowledge of antibioticsusceptibility pattern is essential to give proper antibiotic therapy and avoid unnecessary medication with non-effective drugs, which may increase resistance. Gentamicin, streptomycin and erythromycin are the drugs of choice in that order. Association of phage typing and antibiotic sensitivity of S. aureus showed the predominance of phage group III with greater frequency of penicillin resistance.

  17. Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display

    Directory of Open Access Journals (Sweden)

    Dragana eGagic

    2016-04-01

    Full Text Available Microbial surface and secreted proteins (the secretome contain a large number of proteins that interact with other microbes, host and/or environment. These proteins are exported by the coordinated activities of the protein secretion machinery present in the cell. A group of phage, called filamentous phage, have the ability to hijack the cellular protein secretion machinery in order to amplify and assemble via a secretion-like process. This ability has been harnessed in the use of filamentous phage of Escherichia coli in biotechnology applications, including screening large libraries of variants for binding to bait of interest, from tissues in vivo to pure proteins or even inorganic substrates. In this review we discuss the roles of secretome proteins in pathogenic and non-pathogenic bacteria and corresponding secretion pathways. We describe the basics of phage display technology and its variants applied to discovery of bacterial proteins that have functions of interest for bacterial colonization and pathogenesis, through filamentous phage display library screening. Published literature also shows that phage display is suitable for secretome protein display as a tool for identification immunogenic peptides and can be used for discovery of vaccine candidates. Secretome selection aided by next-generation sequence analysis can also be used for selective display of the secretome at a microbial community scale, the latter revealing the richness of secretome functions of interest and surprising versatility in filamentous phage display of secretome proteins from large number of Gram-negative as well as Gram-positive bacteria and archaea.

  18. Development of an engineered bioluminescent reporter phage for detection of bacterial blight of crucifers.

    Science.gov (United States)

    Schofield, David A; Bull, Carolee T; Rubio, Isael; Wechter, W Patrick; Westwater, Caroline; Molineux, Ian J

    2012-05-01

    Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant "light"-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis.

  19. Genomic and proteomic analyses of the terminally redundant genome of the Pseudomonas aeruginosa phage PaP1: establishment of genus PaP1-like phages.

    Directory of Open Access Journals (Sweden)

    Shuguang Lu

    Full Text Available We isolated and characterized a new Pseudomonas aeruginosa myovirus named PaP1. The morphology of this phage was visualized by electron microscopy and its genome sequence and ends were determined. Finally, genomic and proteomic analyses were performed. PaP1 has an icosahedral head with an apex diameter of 68-70 nm and a contractile tail with a length of 138-140 nm. The PaP1 genome is a linear dsDNA molecule containing 91,715 base pairs (bp with a G+C content of 49.36% and 12 tRNA genes. A strategy to identify the genome ends of PaP1 was designed. The genome has a 1190 bp terminal redundancy. PaP1 has 157 open reading frames (ORFs. Of these, 143 proteins are homologs of known proteins, but only 38 could be functionally identified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-mass spectrometry allowed identification of 12 ORFs as structural protein coding genes within the PaP1 genome. Comparative genomic analysis indicated that the Pseudomonas aeruginosa phage PaP1, JG004, PAK_P1 and vB_PaeM_C2-10_Ab1 share great similarity. Besides their similar biological characteristics, the phages contain 123 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore propose that these four phages be classified as PaP1-like phages, a new phage genus of Myoviridae that infects Pseudomonas aeruginosa.

  20. Intra-domain phage display (ID-PhD) of peptides and protein mini-domains censored from canonical pIII phage display.

    Science.gov (United States)

    Tjhung, Katrina F; Deiss, Frédérique; Tran, Jessica; Chou, Ying; Derda, Ratmir

    2015-01-01

    In this paper, we describe multivalent display of peptide and protein sequences typically censored from traditional N-terminal display on protein pIII of filamentous bacteriophage M13. Using site-directed mutagenesis of commercially available M13KE phage cloning vector, we introduced sites that permit efficient cloning using restriction enzymes between domains N1 and N2 of the pIII protein. As infectivity of phage is directly linked to the integrity of the connection between N1 and N2 domains, intra-domain phage display (ID-PhD) allows for simple quality control of the display and the natural variations in the displayed sequences. Additionally, direct linkage to phage propagation allows efficient monitoring of sequence cleavage, providing a convenient system for selection and evolution of protease-susceptible or protease-resistant sequences. As an example of the benefits of such an ID-PhD system, we displayed a negatively charged FLAG sequence, which is known to be post-translationally excised from pIII when displayed on the N-terminus, as well as positively charged sequences which suppress production of phage when displayed on the N-terminus. ID-PhD of FLAG exhibited sub-nanomolar apparent Kd suggesting multivalent nature of the display. A TEV-protease recognition sequence (TEVrs) co-expressed in tandem with FLAG, allowed us to demonstrate that 99.9997% of the phage displayed the FLAG-TEVrs tandem and can be recognized and cleaved by TEV-protease. The residual 0.0003% consisted of phage clones that have excised the insert from their genome. ID-PhD is also amenable to display of protein mini-domains, such as the 33-residue minimized Z-domain of protein A. We show that it is thus possible to use ID-PhD for multivalent display and selection of mini-domain proteins (Affibodies, scFv, etc.).

  1. The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties.

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    Anneleen Cornelissen

    Full Text Available Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a 'T7-like virus' with a virion-associated exopolysaccharide (EPS depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10(4 and 10(6 pfu and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.

  2. Specific and selective probes for Staphylococcus aureus from phage-displayed random peptide libraries.

    Science.gov (United States)

    De Plano, Laura M; Carnazza, Santina; Messina, Grazia M L; Rizzo, Maria Giovanna; Marletta, Giovanni; Guglielmino, Salvatore P P

    2017-09-01

    Staphylococcus aureus is a major human pathogen causing health care-associated and community-associated infections. Early diagnosis is essential to prevent disease progression and to reduce complications that can be serious. In this study, we selected, from a 9-mer phage peptide library, a phage clone displaying peptide capable of specific binding to S. aureus cell surface, namely St.au9IVS5 (sequence peptide RVRSAPSSS).The ability of the isolated phage clone to interact specifically with S. aureus and the efficacy of its bacteria-binding properties were established by using enzyme linked immune-sorbent assay (ELISA). We also demonstrated by Western blot analysis that the most reactive and selective phage peptide binds a 78KDa protein on the bacterial cell surface. Furthermore, we observed selectivity of phage-bacteria-binding allowing to identify clinical isolates of S. aureus in comparison with a panel of other bacterial species. In order to explore the possibility of realizing a selective bacteria biosensor device, based on immobilization of affinity-selected phage, we have studied the physisorbed phage deposition onto a mica surface. Atomic Force Microscopy (AFM) was used to determine the organization of phage on mica surface and then the binding performance of mica-physisorbed phage to bacterial target was evaluated during the time by fluorescent microscopy. The system is able to bind specifically about 50% of S. aureus cells after 15' and 90% after one hour. Due to specificity and rapidness, this biosensing strategy paves the way to the further development of new cheap biosensors to be used in developing countries, as lab-on-chip (LOC) to detect bacterial agents in clinical diagnostics applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Characterization of a ViI-like Phage Specific to Escherichia coli O157:H7

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    Kropinski Andrew M

    2011-09-01

    Full Text Available Abstract Phage vB_EcoM_CBA120 (CBA120, isolated against Escherichia coli O157:H7 from a cattle feedlot, is morphologically very similar to the classic phage ViI of Salmonella enterica serovar Typhi. Until recently, little was known genetically or physiologically about the ViI-like phages, and none targeting E. coli have been described in the literature. The genome of CBA120 has been fully sequenced and is highly similar to those of both ViI and the Shigella phage AG3. The core set of structural and replication-related proteins of CBA120 are homologous to those from T-even phages, but generally are more closely related to those from T4-like phages of Vibrio, Aeromonas and cyanobacteria than those of the Enterobacteriaceae. The baseplate and method of adhesion to the host are, however, very different from those of either T4 or the cyanophages. None of the outer baseplate proteins are conserved. Instead of T4's long and short tail fibers, CBA120, like ViI, encodes tail spikes related to those normally seen on podoviruses. The 158 kb genome, like that of T4, is circularly permuted and terminally redundant, but unlike T4 CBA120 does not substitute hmdCyt for cytosine in its DNA. However, in contrast to other coliphages, CBA120 and related coliphages we have isolated cannot incorporate 3H-thymidine (3H-dThd into their DNA. Protein sequence comparisons cluster the putative "thymidylate synthase" of CBA120, ViI and AG3 much more closely with those of Delftia phage φW-14, Bacillus subtilis phage SPO1, and Pseudomonas phage YuA, all known to produce and incorporate hydroxymethyluracil (hmdUra.

  4. PENGARUH VARIASI SUHU PADA PROSES SELF TEMPERING DAN VARIASI WAKTU TAHAN PADA PROSES TEMPERING TERHADAP SIFAT MEKANIS BAJA AISI 4140

    Directory of Open Access Journals (Sweden)

    Sunardi Sunardi

    2016-07-01

    Full Text Available Tujuan penelitian ini adalah untuk mengetahui sifat mekanis pada baja AISI 4140 dengan proses tempering dengan variasi waktu tahan dan variasi suhu pada proses self tempering. Material terlebih dahulu dipanaskan pada suhu 850°C, di tahan dengan waktu 14 dan 28 menit, media pendinginan adalah Oli SAE 20. Pada proses tempering baja dipanaskan kembali dengan suhu 200°C di tahan dengan waktu 30 dan 120 menit. Sedangkan untuk proses self tempering, baja di panaskan pada suhu 850°C di tahan dengan waktu 14 dan 28 menit kemudian didinginkan, suhu yang harus dicapai pada pendinginan adalah 200°C, 400°C dan 600°C. Proses tempering dengan variasi waktu tahan mempunyai nilai kekerasan terbesar 50,1 HRC dengan waktu tahan 120 menit, sedangkan nilai kekerasan terbesar pada proses self tempering dengan variasi suhu adalah 29,68 HRC pada suhu 200°C. Nilai ketangguhan terbesar pada saat proses tempering adalah 0,341 (J/mm2 dengan waktu tahan 120 menit, sedangkan pada saat proses self tempering ketangguhan terbesar pada suhu 600°C dengan nilai 0,375 (J/mm2. Laju korosi terbesar pada saat tempering adalah 0,055 (mpy dengan waktu tahan 30 menit, sedangkan pada saat proses self tempering laju korosi terbesar pada suhu 400°C dengan nilai 0,0388 (mpy. 

  5. Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with Pseudomonas aeruginosacystic fibrosis bacterial isolates

    DEFF Research Database (Denmark)

    Friman, Ville-Petri; Soanes-Brown, Daniel; Sierocinski, Pawel

    2016-01-01

    determined if in vitro experimental coevolution can increase the efficiency of phage therapy by limiting the resistance evolution of intermittent and chronic cystic fibrosis Pseudomonas aeruginosa lung isolates to four different phages. We first pre-adapted all phage strains against all bacterial strains...... and then compared the efficacy of pre-adapted and non-adapted phages against ancestral bacterial strains. We found that evolved phages were more efficient in reducing bacterial densities than ancestral phages. This was primarily because only 50% of bacterial strains were able to evolve resistance to evolved phages......Recent years have seen renewed interest in phage therapy - the use of viruses to specifically kill disease-causing bacteria – because of the alarming rise in antibiotic resistance. However, a major limitation of phage therapy is the ease at with bacteria can evolve resistance to phages. Here we...

  6. Bacteriophage T4 Infection of Stationary Phase E. coli: Life after Log from a Phage Perspective

    Directory of Open Access Journals (Sweden)

    Elizabeth Martin Kutter

    2016-09-01

    Full Text Available Virtually all studies of phage infections investigate bacteria growing exponentially in rich media. In nature, however, phages largely encounter non-growing cells. Bacteria entering stationary phase often activate well-studied stress defense mechanisms that drastically alter the cell, facilitating its long-term survival. An understanding of phage-host interactions in such conditions is of major importance from both an ecological and therapeutic standpoint. Here, we show that bacteriophage T4 can efficiently bind to, infect and kill E. coli in stationary phase, both in the presence and absence of a functional stationary-phase sigma factor, and explore the response of T4-infected stationary phase cells to the addition of fresh nutrients 5 or 24 hours after that infection. An unexpected new mode of response has been identified. Hibernation mode is a persistent but reversible dormant state in which the infected cells make at least some phage enzymes, but halt phage development until appropriate nutrients become available before producing phage particles. Our evidence indicates that the block in hibernation mode occurs after the middle-mode stage of phage development; host DNA breakdown and the incorporation of the released nucleotides into phage DNA indicate that the enzymes of the nucleotide synthesizing complex, under middle-mode control, have been made and assembled into a functional state. Once fresh glucose and amino acids become available, the standard lytic infection process rapidly resumes and concentrations of up to 1011 progeny phage (an average of about 40 phage per initially-present cell are produced. All evidence is consistent with the hibernation-mode control point lying between middle mode and late mode T4 gene expression. We have also observed a scavenger response, where the infecting phage takes advantage of whatever few nutrients are available to produce small quantities of progeny within 2 to 5 hours after infection. The scavenger

  7. Metagenomic Analysis of Therapeutic PYO Phage Cocktails from 1997 to 2014

    DEFF Research Database (Denmark)

    Villarroel, Julia; Larsen, Mette Voldby; Kilstrup, Mogens

    2017-01-01

    Phage therapy has regained interest in recent years due to the alarming spread of antibiotic resistance. Whilst phage cocktails are commonly sold in pharmacies in countries such as Georgia and Russia, this is not the case in western countries due to western regulatory agencies requiring a thorough...... production in the 1930s, the composition of the cocktail has been periodically modified to add phages effective against emerging pathogenic strains. In this paper, we compared the composition of three PYO cocktails from 1997 (PYO97), 2000 (PYO2000) and 2014 (PYO2014). Based on next generation sequencing, de...

  8. Emergence of a Competence-Reducing Filamentous Phage from the Genome of Acinetobacter baylyi ADP1.

    Science.gov (United States)

    Renda, Brian A; Chan, Cindy; Parent, Kristin N; Barrick, Jeffrey E

    2016-12-01

    Bacterial genomes commonly contain prophage sequences as a result of past infections with lysogenic phages. Many of these integrated viral sequences are believed to be cryptic, but prophage genes are sometimes coopted by the host, and some prophages may be reactivated to form infectious particles when cells are stressed or mutate. We found that a previously uncharacterized filamentous phage emerged from the genome of Acinetobacter baylyi ADP1 during a laboratory evolution experiment. This phage has a genetic organization similar to that of the Vibrio cholerae CTXϕ phage. The emergence of the ADP1 phage was associated with the evolution of reduced transformability in our experimental populations, so we named it the competence-reducing acinetobacter phage (CRAϕ). Knocking out ADP1 genes required for competence leads to resistance to CRAϕ infection. Although filamentous bacteriophages are known to target type IV pili, this is the first report of a phage that apparently uses a competence pilus as a receptor. A. baylyi may be especially susceptible to this route of infection because every cell is competent during normal growth, whereas competence is induced only under certain environmental conditions or in a subpopulation of cells in other bacterial species. It is possible that CRAϕ-like phages restrict horizontal gene transfer in nature by inhibiting the growth of naturally transformable strains. We also found that prophages with homology to CRAϕ exist in several strains of Acinetobacter baumannii These CRAϕ-like A. baumannii prophages encode toxins similar to CTXϕ that might contribute to the virulence of this opportunistic multidrug-resistant pathogen. We observed the emergence of a novel filamentous phage (CRAϕ) from the genome of Acinetobacter baylyi ADP1 during a long-term laboratory evolution experiment. CRAϕ is the first bacteriophage reported to require the molecular machinery involved in the uptake of environmental DNA for infection. Reactivation and

  9. Bxb1 phage recombinase assists genome engineering in Drosophila melanogaster.

    Science.gov (United States)

    Voutev, Roumen; Mann, Richard S

    2017-01-01

    Rapid and reliable genome modifications provide the basis for detailed in vivo functional analysis of any genomic entity (gene, regulatory DNA, non-coding RNA, etc). With the advent of CRISPR/Cas9 genome editing technology, manipulation of a particular genomic locus has become a routine undertaking in variety of model organisms, including the fruit fly Drosophila melanogaster. To further diversify the available tools for genome engineering, we successfully harnessed the phage recombinase Bxb1 to perform recombinase-mediated cassette exchange (RMCE) in D. melanogaster. We demonstrate that Bxb1 possesses highly efficient recombinase activity and could be used alone or in conjunction with other currently available recombinases for creating platforms for cassette exchange of targeted loci.

  10. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    Science.gov (United States)

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing.

  11. Construction of Synthetic Antibody Phage-Display Libraries.

    Science.gov (United States)

    Nilvebrant, Johan; Sidhu, Sachdev S

    2018-01-01

    Synthetic antibody libraries provide a vast resource of renewable antibody reagents that can rival or exceed those of natural antibodies and can be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of synthetic antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions. The resulting libraries can be used to generate recombinant domain antibodies for a wide range of protein antigens using phage display. Furthermore, analogous methods can be used to construct antibody libraries based on larger antibody fragments or second-generation libraries aimed to fine-tune antibody characteristics including affinity, specificity, and manufacturability. The procedures rely on standard reagents and equipment available in most molecular biology laboratories.

  12. Simulated Tempering and Swapping on Mean-Field Models

    OpenAIRE

    Bhatnagar, Nayantara; Randall, Dana

    2015-01-01

    Simulated and parallel tempering are families of Markov Chain Monte Carlo algorithms where a temperature parameter is varied during the simulation to overcome bottlenecks to convergence due to multimodality. In this work we introduce and analyze the convergence for a set of new tempering distributions which we call \\textit{entropy dampening}. For asymmetric exponential distributions and the mean field Ising model with and external field simulated tempering is known to converge slowly. We show...

  13. Phage lysis: three steps, three choices, one outcome

    Science.gov (United States)

    Young, Ry

    2014-01-01

    The lysis of bacterial hosts by double-strand DNA bacteriophages, once thought to reflect merely the accumulation of sufficient lysozyme activity during the infection cycle, has been revealed to recently been revealed to be a carefully regulated and temporally scheduled process. For phages of Gram-negative hosts, there are three steps, corresponding to subversion of each of the three layers of the cell envelope: inner membrane, peptidoglycan, and outer membrane. The pathway is controlled at the level of the cytoplasmic membrane. In canonical lysis, a phage encoded protein, the holin, accumulates harmlessly in the cytoplasmic membrane until triggering at an allele-specific time to form micron-scale holes. This allows the soluble endolysin to escape from the cytoplasm to degrade the peptidoglycan. Recently a parallel pathway has been elucidated in which a different type of holin, the pinholin, which, instead of triggering to form large holes, instead triggers to form small, heptameric channels that serve to depolarize the membrane. Pinholins are associated with SAR endolysins, which accumulate in the periplasm as inactive, membrane-tethered enzymes. Pinholin triggering collapses the proton motive force, allowing the SAR endolysins to refold to an active form and attack the peptidoglycan. Surprisingly, a third step, the disruption of the outer membrane is also required. This is usually achieved by a spanin complex, consisting of a small outer membrane lipoprotein and an integral cytoplasmic membrane protein, designated as o-spanins and i-spanins, respectively. Without spanin function, lysis is blocked and progeny virions are trapped in dead spherical cells, suggesting that the outer membrane has considerable tensile strength. In addition to two-component spanins, there are some single-component spanins, or u-spanins, that have an N-terminal outer-membrane lipoprotein signal and a C-terminal transmembrane domain. A possible mechanism for spanin function to disrupt the

  14. Trichinella in arctic, subarctic and temperate regions

    DEFF Research Database (Denmark)

    Kapel, C. M O

    1997-01-01

    and the human activity are all very important interacting factors affecting epidemiology. In Greenland, where only sylvatic trichinellosis is present, the high prevalence in wildlife appears closely connected with polar bear hunting. In the Scandinavian countries, the prevalence of both sylvatic and domestic......The transmission and occurrence of Trichinella spp according to the zoogeography of different climatic conditions, socioeconomy and human activity are discussed. Comparing arctic, subarctic and temperate regions, it appears that the species of Trichinella present, the composition of the fauna...... populations may have epidemiological importance in relation to the recent changes in production and infrastructure in these former Soviet states....

  15. Extended Hamiltonian approach to continuous tempering.

    Science.gov (United States)

    Gobbo, Gianpaolo; Leimkuhler, Benedict J

    2015-06-01

    We introduce an enhanced sampling simulation technique based on continuous tempering, i.e., on continuously varying the temperature of the system under investigation. Our approach is mathematically straightforward, being based on an extended Hamiltonian formulation in which an auxiliary degree of freedom, determining the effective temperature, is coupled to the physical system. The physical system and its temperature evolve continuously in time according to the equations of motion derived from the extended Hamiltonian. Due to the Hamiltonian structure, it is easy to show that a particular subset of the configurations of the extended system is distributed according to the canonical ensemble for the physical system at the correct physical temperature.

  16. A temperature predictor for parallel tempering simulations.

    Science.gov (United States)

    Patriksson, Alexandra; van der Spoel, David

    2008-04-21

    An algorithm is proposed that generates a set of temperatures for use in parallel tempering simulations (also known as temperature-replica exchange molecular dynamics simulations) of proteins to obtain a desired exchange probability Pdes. The input consists of the number of protein atoms and water molecules in the system, information about the use of constraints and virtual sites and the lower temperature limits. The temperatures generated yield probabilities which are very close to Pdes (correlation 97%), independent of force field and over a wide temperature range. To facilitate its use, the algorithm has been implemented as a web server at .

  17. Land Cover Analysis of Temperate Asia

    Science.gov (United States)

    Justice, Chris

    1998-01-01

    Satellite data from the advanced very high resolution radiometer (AVHRR) instrument were used to produce a general land cover distribution of temperate Asia (referred to hence as Central Asia) from 1982, starting with the NOAA-7 satellite, and continuing through 1991, ending with the NOAA-11 satellite. Emphasis was placed upon delineating the and and semi-arid zones of Central Asia (largely Mongolia and adjacent areas), mapping broad categories of aggregated land cover, and upon studying photosynthetic capacity increases in Central Asia from 1982 to 1991.

  18. Oceanic temperate forest versus warm temperate rainforest: a reply to Grubb et al. (2017)

    DEFF Research Database (Denmark)

    Buitenwerf, Robert

    2017-01-01

    Grubb et al. (2017) point out that we (McGlone et al. 2016) erroneously stated that the definition of warm temperate rain forest (WTRF; Grubb et al. 2013) was based in part on climatic criteria. We apologise: their text made clear that this was not the case. However, they go on to say that they ‘...

  19. Tetracycline consumption and occurrence of tetracycline resistance in Salmonella typhimurium phage types from Danish pigs

    DEFF Research Database (Denmark)

    Emborg, Hanne-Dorthe; Vigre, Håkan; Jensen, Vibeke Frøkjær

    2007-01-01

    The aims of the present study were to investigate at the farm-owner level the effect of prescribed tetracycline consumption in pigs and different Salmonella Typhimurium phage types on the probability that the S. Typhimurium was resistant to tetracycline. In this study, 1,307 isolates were included......, originating from 877 farm owners, and data were analyzed using logistic regression. The analysis showed that both the S. Typhimurium phage type (p resistance. In particular, the phage type...... was strongly associated with tetracycline resistance. A further analysis of data from the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) indicates that the tetracycline-susceptible phage types only slowly become tetracycline resistant, although tetracycline consumption...

  20. Binding mechanism and electrochemical properties of M13 phage-sulfur composite.

    Directory of Open Access Journals (Sweden)

    Dexian Dong

    Full Text Available Self-assembly of nanostructured materials has been proven a powerful technique in material design and synthesis. By phage display screening, M13 phage was found to strongly bind sulfur particles. Fourier transform infrared and X-ray photoelectron spectroscopy measurements indicated that the strong sulfur-binding ability of M13 phage derives from newly generated S-O and C-S bonds. Using this phage assembled sulfur composite in a lithium battery, the first discharge capacity reached 1117 mAh g(-1, which is more than twice that of the sulfur only cathode. Besides, the negative polysulfide shuttle effect in a lithium-sulfur battery was significantly suppressed.

  1. Theranostic applications of phage display to control leishmaniasis: selection of biomarkers for serodiagnostics, vaccination, and immunotherapy

    Directory of Open Access Journals (Sweden)

    Eduardo Antonio Ferraz Coelho

    2015-08-01

    Full Text Available AbstractPhage display is a high-throughput subtractive proteomic technology used for the generation and screening of large peptide and antibody libraries. It is based on the selection of phage-fused surface-exposed peptides that recognize specific ligands and demonstrate desired functionality for diagnostic and therapeutic purposes. Phage display has provided unmatched tools for controlling viral, bacterial, fungal, and parasitic infections, and allowed identification of new therapeutic targets to treat cancer, metabolic diseases, and other chronic conditions. This review presents recent advancements in serodiagnostics and prevention of leishmaniasis -an important tropical parasitic disease- achieved using phage display for the identification of novel antigens with improved sensitivity and specificity. Our focus is on theranostics of visceral leishmaniasis with the aim to develop biomarker candidates exhibiting both diagnostic and therapeutic potential to fight this important, yet neglected, tropical disease.

  2. Migration of Salmonella Enteritidis Phage Type 30 through Almond Hulls and Shells

    National Research Council Canada - National Science Library

    Danyluk, Michelle D; Brandl, Maria T; Harris, Linda J

    2008-01-01

    .... First, the outer surface of shell halves from five varieties of almonds that differed in shell hardness were placed in contact with a suspension of Salmonella enterica serovar Enteritidis phage type 30 for 24 h at 24°C...

  3. A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

    National Research Council Canada - National Science Library

    Kushwaha, Rekha; Schäfermeyer, Kim R; Downie, A. Bruce

    2014-01-01

    Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions...

  4. [Therapeutic effect of phages on extensively drug-resistant Acinetobacter baumannii-induced sepsis in mice].

    Science.gov (United States)

    Deng, L Y; Yang, Z C; Gong, Y L; Huang, G T; Yin, S P; Jiang, B; Peng, Y Z

    2016-09-20

    To study the therapeutic effect of phages on extensively drug-resistant Acinetobacter baumannii-induced sepsis in mice. (1) Sixty BALB/c mice were divided into blank control group, sepsis control group, antibiotics treatment group, phage treatment group, and phage control group according to the random number table, with 12 mice in each group. Mice in blank control group were intraperitoneally (the same injection position below) injected with 1 mL normal saline. Mice in sepsis control group, antibiotics treatment group, and phage treatment group were injected with 1 mL extensively drug-resistant Acinetobacter baumannii (the strain was isolated from the blood of a severely burned patient hospitalized in our unit) in the concentration of 5×10(7) colony-forming unit/mL to reproduce sepsis model. Two hours later, mice in sepsis control group, antibiotics treatment group, and phage treatment group were injected with 1 mL saline, 1 mg/mL imipenem/cilastatin, and 1×10(8) plaque-forming unit (PFU)/mL phages screened based on above-mentioned Acinetobacter baumannii (the same phages below) respectively. Mice in phage control group were injected with 1 mL phages in the titer of 1×10(8) PFU/mL. The injection was performed continuously for 7 days in each living mouse, and the survival situation of mice was observed each day to calculate the survival ratio in one week. (2) Another 60 BALB/c mice were grouped and treated as in experiment (1), and the injection was performed continuously for 5 days in each living mouse. On experiment day 2, 4, and 6, 3 mice from each group were selected (if the number of survived mouse in any group was less than 3 at sample collecting, all the survived mice were selected), and blood was drawn to determine white blood cell count (WBC, with 3 samples at each time point in each group). On experiment day 2, blood was drawn from the mice that had their blood taken earlier for bacterial culture, and lung, liver, kidney, and spleen tissue was collected

  5. Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections

    National Research Council Canada - National Science Library

    Regeimbal, James M; Jacobs, Anna C; Corey, Brendan W; Henry, Matthew S; Thompson, Mitchell G; Pavlicek, Rebecca L; Quinones, Javier; Hannah, Ryan M; Ghebremedhin, Meron; Crane, Nicole J; Zurawski, Daniel V; Teneza-Mora, Nimfa C; Biswas, Biswajit; Hall, Eric R

    2016-01-01

    .... In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model...

  6. Phage Display Technology in Biomaterials Engineering: Progress and Opportunities for Applications in Regenerative Medicine.

    Science.gov (United States)

    Martins, Ivone M; Reis, Rui L; Azevedo, Helena S

    2016-11-18

    The field of regenerative medicine has been gaining momentum steadily over the past few years. The emphasis in regenerative medicine is to use various in vitro and in vivo approaches that leverage the intrinsic healing mechanisms of the body to treat patients with disabling injuries and chronic diseases such as diabetes, osteoarthritis, and degenerative disorders of the cardiovascular and central nervous system. Phage display has been successfully employed to identify peptide ligands for a wide variety of targets, ranging from relatively small molecules (enzymes, cell receptors) to inorganic, organic, and biological (tissues) materials. Over the past two decades, phage display technology has advanced tremendously and has become a powerful tool in the most varied fields of research, including biotechnology, materials science, cell biology, pharmacology, and diagnostics. The growing interest in and success of phage display libraries is largely due to its incredible versatility and practical use. This review discusses the potential of phage display technology in biomaterials engineering for applications in regenerative medicine.

  7. Efficient production of antibodies against a mammalian integral membrane protein by phage display

    National Research Council Canada - National Science Library

    Hötzel, Isidro; Chiang, Vicki; Diao, Jingyu; Pantua, Homer; Maun, Henry R; Kapadia, Sharookh B

    2011-01-01

    The application of phage display technology to mammalian proteins with multiple transmembrane regions has had limited success due to the difficulty in generating these proteins in sufficient amounts and purity...

  8. Marine Phages As Tracers: Effects of Size, Morphology, and Physico-Chemical Surface Properties on Transport in a Porous Medium.

    Science.gov (United States)

    Ghanem, Nawras; Kiesel, Bärbel; Kallies, René; Harms, Hauke; Chatzinotas, Antonis; Wick, Lukas Y

    2016-12-06

    Although several studies examined the transport of viruses in terrestrial systems only few studies exist on the use of marine phages (i.e., nonterrestrial viruses infecting marine host bacteria) as sensitively detectable microbial tracers for subsurface colloid transport and water flow. Here, we systematically quantified and compared for the first time the effects of size, morphology and physicochemical surface properties of six marine phages and two coliphages (MS2, T4) on transport in sand-filled percolated columns. Phage-sand interactions were described by colloidal filtration theory and the extended Derjaguin-Landau-Verwey-Overbeek approach (XDLVO), respectively. The phages belonged to different families and comprised four phages never used in transport studies (i.e., PSA-HM1, PSA-HP1, PSA-HS2, and H3/49). Phage transport was influenced by size, morphology and hydrophobicity in an approximate order of size > hydrophobicity ≥ morphology. Two phages PSA-HP1, PSA-HS2 (Podoviridae and Siphoviridae) exhibited similar mass recovery as commonly used coliphage MS2 and were 7-fold better transported than known marine phage vB_PSPS-H40/1. Differing properties of the marine phages may be used to trace transport of indigenous viruses, natural colloids or anthropogenic nanomaterials and, hence, contribute to better risk analysis. Our results underpin the potential role of marine phages as microbial tracer for transport of colloidal particles and water flow.

  9. Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

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    Kuhn Andreas

    2011-09-01

    Full Text Available Abstract Background Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. Results The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Conclusions Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

  10. Effects of initial temperature and tempering medium on thermal tempering of dental porcelains.

    Science.gov (United States)

    Hojjatie, B; Anusavice, K J

    1993-03-01

    The objective of this study was to test the hypothesis that quenching of porcelain in silicone oil rather than in compressed air will significantly increase the flexure strength by reducing the potential for crack formation during transient cooling. A secondary hypothesis to be tested is that the initial tempering temperature can be reduced significantly below the porcelain maturing temperature of 982 degrees C but well above Tg without a decrease in strength. Opaque-body porcelain disks, 16 mm in diameter and 2 mm in thickness, with a thermal contraction mismatch (delta alpha) of -1.5, 0, and +3.2 ppm/degrees C were tempered from initial temperatures of 650, 750, 850, and 982 degrees C in silicone oil with kinematic viscosities of 50, 1000, and 5000 centistokes. Porcelain disks were also subjected to three cooling procedures in air: slow cooling in a furnace (SC), free convective cooling in a laboratory bench (FC), and tempering (T) by blasting the surface of body porcelain with air. The crack size induced by a Vickers microhardness indenter was measured within one minute after crack development. For determination of the influence of initial cooling temperature on biaxial flexure strength, six body porcelain disks (delta alpha = 0) were tempered in air from initial temperatures of 650, 750, 850, and 982 degrees C. The mean crack size of specimens tempered in oil was significantly smaller (p < or = 0.001) than that of specimens that were slowly-cooled or fast-cooled in air for all thermal contraction mismatch cases.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Isolation and characterization of a T7-like lytic phage for Pseudomonas fluorescens

    OpenAIRE

    Neubauer Peter; Sillankorva Sanna; Azeredo Joana

    2008-01-01

    Background: Despite the proven relevance of Pseudomonas fluorescens as a spoilage microorganism in milk, fresh meats and refrigerated food products and the recognized potential of bacteriophages as sanitation agents, so far no phages specific for P. fluorescens isolates from dairy industry have been closely characterized in view of their lytic efficiency. Here we describe the isolation and characterization of a lytic phage capable to infect a variety of P. fluorescens strains i...

  12. Complete genome sequence of a giant Vibrio phage ValKK3 infecting Vibrio alginolyticus

    Directory of Open Access Journals (Sweden)

    Tamrin M. Lal

    2016-06-01

    Full Text Available This paper describes the complete sequence of a giant lytic marine myophage, Vibrio phage ValKK3 that is specific to Vibrio alginolyticus ATCC® 17749™. Vibrio phage ValKK3 was subjected to whole genome sequencing on MiSeq sequencing platform and annotated using Blast2Go. The complete sequence of ValKK3 genome was deposited in DBBJ/EMBL/GenBank under accession number KP671755.

  13. Transport of Escherichia coli phage through saturated porous media considering managed aquifer recharge.

    Science.gov (United States)

    Zhang, Wenjing; Li, Shuo; Wang, Shuang; Lei, Liancheng; Yu, Xipeng; Ma, Tianyi

    2017-12-18

    Virus is one of the most potentially harmful microorganisms in groundwater. In this paper, the effects of hydrodynamic and hydrogeochemical conditions on the transportation of the colloidal virus considering managed aquifer recharge were systematically investigated. Escherichia coli phage, vB_EcoM-ep3, has a broad host range and was able to lyse pathogenic Escherichia coli. Bacteriophage with low risk to infect human has been found extensively in the groundwater environment, so it is considered as a representative model of groundwater viruses. Laboratory studies were carried out to analyze the transport of the Escherichia coli phage under varying conditions of pH, ionic strength, cation valence, flow rate, porous media, and phosphate buffer concentration. The results indicated that decreasing the pH will increase the adsorption of Escherichia coli phage. Increasing the ionic strength, either Na + or Ca 2+ , will form negative condition for the migration of Escherichia coli phage. A comparison of different cation valence tests indicated that changes in transport and deposition were more pronounced with divalent Ca 2+ than monovalent Na + . As the flow rate increases, the release of Escherichia coli phage increases and the retention of Escherichia coli phage in the aquifer medium reduces. Changes in porous media had a significant effect on Escherichia coli phage migration. With increase of phosphate buffer concentration, the suspension stability and migration ability of Escherichia coli phage are both increased. Based on laboratory-scale column experiments, a one-dimensional transport model was established to quantitatively describe the virus transport in saturated porous medium.

  14. The impact of orally administered phages on host immune response and surrounding microbial communities.

    Science.gov (United States)

    Hong, Yingying; Thimmapuram, Jyothi; Zhang, Jiayi; Collings, Clayton K; Bhide, Ketaki; Schmidt, Kyle; Ebner, Paul D

    2016-01-01

    Numerous studies have shown the efficacy of phage therapy in reducing foodborne pathogen carriage in food animals. Fewer studies have focused on host reactions, especially in terms of phage-mediated acute immune responses and effects on the gut microbiome. Here we administered E. coli O157:H7 phages in low (single dose of 105 PFU) or high (single dose of 107 PFU) quantities to mice. While there were time points at which cytokine levels in different treatment groups differed from one another, all cytokine levels remained within normal ranges for mice regardless of treatment. Similarly, the patterns of these differences were not dose related, indicating that phage treatment did not result in a strong acute immune response as measured here. In separate experiments, 3-week-old pigs received a diet containing an in-feed antibiotic or daily phage treatment. After two weeks, microbial DNA of ileal, cecal, and fecal contents was characterized using 16S rRNA sequencing. There were no statistical differences in performance among the different groups. Compared to control pigs (no antibiotic, no phage), antibiotic treatment significantly altered ileal microbiome composition (P < 0.05), with Bacilli being most affected (antibiotic treated: 22%; control: 76%; FDR = 0.0572). No significant differences were observed in cecal and fecal microbiome composition between antibiotic-treated and control pigs, and there were no differences in gut microbiome composition between phage treated and control pigs in any intestinal compartment. Significant abundance differences were observed at the OTU level, with OTUs belonging to genera such as Lactobacillus and Streptococcus being over- or under-represented in either antibiotic or phage treated groups compared to control pigs. Determining whether these changes are deleterious to host, however, requires further study.

  15. Complete genome sequence of a giant Vibrio phage ValKK3 infecting Vibrio alginolyticus.

    Science.gov (United States)

    Lal, Tamrin M; Sano, Motohiko; Hatai, Kishio; Ransangan, Julian

    2016-06-01

    This paper describes the complete sequence of a giant lytic marine myophage, Vibrio phage ValKK3 that is specific to Vibrio alginolyticus ATCC(®) 17749™. Vibrio phage ValKK3 was subjected to whole genome sequencing on MiSeq sequencing platform and annotated using Blast2Go. The complete sequence of ValKK3 genome was deposited in DBBJ/EMBL/GenBank under accession number KP671755.

  16. Diversity of Phage-Displayed Libraries of Peptides during Panning and Amplification

    Directory of Open Access Journals (Sweden)

    Mohammad R. Jafari

    2011-02-01

    Full Text Available The amplification of phage-displayed libraries is an essential step in the selection of ligands from these libraries. The amplification of libraries, however, decreases their diversity and limits the number of binding clones that a screen can identify. While this decrease might not be a problem for screens against targets with a single binding site (e.g., proteins, it can severely hinder the identification of useful ligands for targets with multiple binding sites (e.g., cells. This review aims to characterize the loss in the diversity of libraries during amplification. Analysis of the peptide sequences obtained in several hundred screens of peptide libraries shows explicitly that there is a significant decrease in library diversity that occurs during the amplification of phage in bacteria. This loss during amplification is not unique to specific libraries: it is observed in many of the phage display systems we have surveyed. The loss in library diversity originates from competition among phage clones in a common pool of bacteria. Based on growth data from the literature and models of phage growth, we show that this competition originates from growth rate differences of only a few percent for different phage clones. We summarize the findings using a simple two-dimensional “phage phase diagram”, which describes how the collapse of libraries, due to panning and amplification, leads to the identification of only a subset of the available ligands. This review also highlights techniques that allow elimination of amplification-induced losses of diversity, and how these techniques can be used to improve phage-display selection and enable the identification of novel ligands.

  17. Impedance Measurements Could Accelerate Phage-Based Identification of Bacillus anthracis and Other Bacteria

    Science.gov (United States)

    2016-09-01

    Methods Phage and spore preparations. Ɣ phage were propagated by infecting the lawns of B. anthracis CDC 684 vegetative cells on sheep’s blood agar...stress topics . Ann N Y Acad Sci, 2007. 1113: p. 95- 104. 5. Marles-Wright, J. and R.J. Lewis, Stress responses of bacteria. Curr Opin Struct Biol...impedance. Transfusion , 2011. 51(5): p. 1037-46. 7. Zavizion, B., et al., Rapid microbiological testing: monitoring the development of bacterial stress

  18. Whole genome sequencing provides possible explanations for the difference in phage susceptibility among two Salmonella Typhimurium phage types (DT8 and DT30) associated with a single foodborne outbreak.

    Science.gov (United States)

    Mohammed, Manal; Cormican, Martin

    2015-11-27

    Phage typing has been used for decades as a rapid, low cost approach for the epidemiological surveillance of Salmonella enterica subsp. enterica serovar Typhimurium. Although molecular methods are replacing phage typing the system is still in use and provides a valuable model for study of phage-host interaction. Phage typing depends on the pattern of bacterial resistance or sensitivity to a panel of specific bacteriophages. In the phage typing scheme, S. Typhimurium definitive phage types (DT) 8 and 30 differ greatly in their susceptibility to the 30 typing phages of S. Typhimurium; DT8 is susceptible to 11 phages whereas DT30 is resistant to all typing phages except one phage although both DT8 and DT30 were reported to be associated with a single foodborne salmonellosis outbreak in Ireland between 2009 and 2011. We wished to study the genomic correlates of the DT8 and DT30 difference in phage susceptibility using the whole genome sequence (WGS) of S. Typhimurium DT8 and DT30 representatives. Comparative genome analysis revealed that both S. Typhimurium DT8 and DT30 are lysogenic for three prophages including two S. Typhimurium associated prophages (Gifsy-2 and ST64B) and one S. Enteritidis associated prophage (Enteritidis lysogenic phage S) which has not been detected previously in S. Typhimurium. Furthermore, DT8 and DT30 contain identical clustered regularly interspaced short palindromic repeats (CRISPRs). Interestingly, S. Typhimurium DT8 harbours an accessory genome represented by a virulence plasmid that is highly related to the pSLT plasmid of S. Typhimurium strain LT2 (phage typed as DT4) and codes a unique methyltransferase (MTase); M.EcoGIX related MTase. This plasmid is not detected in DT30. On the other hand, DT30 carries a unique genomic island similar to the integrative and conjugative element (ICE) of Enterotoxigenic Escherichia coli (ETEC) and encodes type IV secretion pathway system (T4SS) and several hypothetical proteins. This genomic island is

  19. The bacteriophage WORiC is the active phage element in wRi of Drosophila simulans and represents a conserved class of WO phages

    Directory of Open Access Journals (Sweden)

    Biliske Jennifer A

    2011-11-01

    Full Text Available Abstract Background The alphaproteobacterium Wolbachia pipientis, the most common endosymbiont in eukaryotes, is found predominantly in insects including many Drosophila species. Although Wolbachia is primarily vertically transmitted, analysis of its genome provides evidence for frequent horizontal transfer, extensive recombination and numerous mobile genetic elements. The genome sequence of Wolbachia in Drosophila simulans Riverside (wRi is available along with the integrated bacteriophages, enabling a detailed examination of phage genes and the role of these genes in the biology of Wolbachia and its host organisms. Wolbachia is widely known for its ability to modify the reproductive patterns of insects. One particular modification, cytoplasmic incompatibility, has previously been shown to be dependent on Wolbachia density and inversely related to the titer of lytic phage. The wRi genome has four phage regions, two WORiBs, one WORiA and one WORiC. Results In this study specific primers were designed to distinguish between these four prophage types in wRi, and quantitative PCR was used to measure the titer of bacteriophages in testes, ovaries, embryos and adult flies. In all tissues tested, WORiA and WORiB were not found to be present in excess of their integrated prophages; WORiC, however, was found to be present extrachromosomally. WORiC is undergoing extrachromosomal replication in wRi. The density of phage particles was found to be consistent in individual larvae in a laboratory population. The WORiC genome is organized in conserved blocks of genes and aligns most closely with other known lytic WO phages, WOVitA and WOCauB. Conclusions The results presented here suggest that WORiC is the lytic form of WO in D. simulans, is undergoing extrachromosomal replication in wRi, and belongs to a conserved family of phages in Wolbachia.

  20. Not-so-well-tempered neutralino

    Science.gov (United States)

    Profumo, Stefano; Stefaniak, Tim; Stephenson-Haskins, Laurel

    2017-09-01

    Light electroweakinos, the neutral and charged fermionic supersymmetric partners of the standard model SU (2 )×U (1 ) gauge bosons and of the two SU(2) Higgs doublets, are an important target for searches for new physics with the Large Hadron Collider (LHC). However, if the lightest neutralino is the dark matter, constraints from direct dark matter detection experiments rule out large swaths of the parameter space accessible to the LHC, including in large part the so-called "well-tempered" neutralinos. We focus on the minimal supersymmetric standard model (MSSM) and explore in detail which regions of parameter space are not excluded by null results from direct dark matter detection, assuming exclusive thermal production of neutralinos in the early universe, and illustrate the complementarity with current and future LHC searches for electroweak gauginos. We consider both bino-Higgsino and bino-wino "not-so-well-tempered" neutralinos, i.e. we include models where the lightest neutralino constitutes only part of the cosmological dark matter, with the consequent suppression of the constraints from direct and indirect dark matter searches.

  1. A3R Phage and Staphylococcus aureus Lysate Do Not Induce Neutrophil Degranulation

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    Jan Borysowski

    2017-02-01

    Full Text Available The objective of this study was to evaluate the effects of A3R phage and Staphylococcus aureus lysate obtained after phage infection on neutrophil degranulation. The exocytosis of primary and secondary granules from neutrophils was investigated in vitro in whole blood specimens by flow cytometry based on the expression of specific markers of exocytosis (CD63 for primary granules and CD66b for secondary granules. We found that both A3R and S. aureus lysate had no significant effect on the exocytosis of primary and secondary granules. These data suggest that neither A3R virions nor any products of phage-induced lysis of S. aureus are likely to induce neutrophil degranulation in patients who are treated with phage preparations. Since neutrophil granules contain some potentially toxic proteins, our results provide an important argument for the safety of phage therapy. Moreover, these data indicate that the induction of neutrophil degranulation is not likely to contribute to antibacterial effects of phages.

  2. Isolation of a Novel Phage with Activity against Streptococcus mutans Biofilms.

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    Marion Dalmasso

    Full Text Available Streptococcus mutans is one of the principal agents of caries formation mainly, because of its ability to form biofilms at the tooth surface. Bacteriophages (phages are promising antimicrobial agents that could be used to prevent or treat caries formation by S. mutans. The aim of this study was to isolate new S. mutans phages and to characterize their antimicrobial properties. A new phage, ɸAPCM01, was isolated from a human saliva sample. Its genome was closely related to the only two other available S. mutans phage genomes, M102 and M102AD. ɸAPCM01 inhibited the growth of S. mutans strain DPC6143 within hours in broth and in artificial saliva at multiplicity of infections as low as 2.5x10-5. In the presence of phage ɸAPCM01 the metabolic activity of a S. mutans biofilm was reduced after 24 h of contact and did not increased again after 48 h, and the live cells in the biofilm decreased by at least 5 log cfu/ml. Despite its narrow host range, this newly isolated S. mutans phage exhibits promising antimicrobial properties.

  3. Formation of biofilms under phage predation: considerations concerning a biofilm increase.

    Science.gov (United States)

    Hosseinidoust, Zeinab; Tufenkji, Nathalie; van de Ven, Theo G M

    2013-01-01

    Bacteriophages are emerging as strong candidates for combating bacterial biofilms. However, reports indicating that host populations can, in some cases, respond to phage predation by an increase in biofilm formation are of concern. This study investigates whether phage predation can enhance the formation of biofilm and if so, if this phenomenon is governed by the emergence of phage-resistance or by non-evolutionary mechanisms (eg spatial refuge). Single-species biofilms of three bacterial pathogens (Pseudomonas aeruginosa, Salmonella enterica serotype Typhimurium, and Staphylococcus aureus) were pretreated and post-treated with species-specific phages. Some of the phage treatments resulted in an increase in the levels of biofilm of their host. It is proposed that the phenotypic change brought about by acquiring phage resistance is the main reason for the increase in the level of biofilm of P. aeruginosa. For biofilms of S. aureus and S. enterica Typhimurium, although resistance was detected, increased formation of biofilm appeared to be a result of non-evolutionary mechanisms.

  4. Emergence of new Salmonella Enteritidis phage types in Europe? Surveillance of infections in returning travellers

    Directory of Open Access Journals (Sweden)

    Andersson Yvonne

    2004-09-01

    Full Text Available Abstract Background Among human Salmonella Enteritidis infections, phage type 4 has been the dominant phage type in most countries in Western Europe during the last years. This is reflected in Salmonella infections among Swedish travellers returning from abroad. However, there are differences in phage type distribution between the countries, and this has also changed over time. Methods We used data from the Swedish infectious disease register and the national reference laboratory to describe phage type distribution of Salmonella Enteritidis infections in Swedish travellers from 1997 to 2002, and have compared this with national studies conducted in the countries visited. Results Infections among Swedish travellers correlate well with national studies conducted in the countries visited. In 2001 a change in phage type distribution in S. Enteritidis infections among Swedish travellers returning from some countries in southern Europe was observed, and a previously rare phage type (PT 14b became one of the most commonly diagnosed that year, continuing into 2002 and 2003. Conclusions Surveillance of infections among returning travellers can be helpful in detecting emerging infections and outbreaks in tourist destinations. The information needs to be communicated rapidly to all affected countries in order to expedite the implementation of appropriate investigations and preventive measures.

  5. Isolation of a Novel Phage with Activity against Streptococcus mutans Biofilms

    Science.gov (United States)

    Dalmasso, Marion; de Haas, Eric; Neve, Horst; Strain, Ronan; Cousin, Fabien J.; Stockdale, Stephen R.; Ross, R. Paul; Hill, Colin

    2015-01-01

    Streptococcus mutans is one of the principal agents of caries formation mainly, because of its ability to form biofilms at the tooth surface. Bacteriophages (phages) are promising antimicrobial agents that could be used to prevent or treat caries formation by S. mutans. The aim of this study was to isolate new S. mutans phages and to characterize their antimicrobial properties. A new phage, ɸAPCM01, was isolated from a human saliva sample. Its genome was closely related to the only two other available S. mutans phage genomes, M102 and M102AD. ɸAPCM01 inhibited the growth of S. mutans strain DPC6143 within hours in broth and in artificial saliva at multiplicity of infections as low as 2.5x10-5. In the presence of phage ɸAPCM01 the metabolic activity of a S. mutans biofilm was reduced after 24 h of contact and did not increased again after 48 h, and the live cells in the biofilm decreased by at least 5 log cfu/ml. Despite its narrow host range, this newly isolated S. mutans phage exhibits promising antimicrobial properties. PMID:26398909

  6. The Impact of Prophage on the Equilibria and Stability of Phage and Host

    Science.gov (United States)

    Yu, Pei; Nadeem, Alina; Wahl, Lindi M.

    2017-06-01

    In this paper, we present a bacteriophage model that includes prophage, that is, phage genomes that are incorporated into the host cell genome. The general model is described by an 18-dimensional system of ordinary differential equations. This study focuses on asymptotic behaviour of the model, and thus the system is reduced to a simple six-dimensional model, involving uninfected host cells, infected host cells and phage. We use dynamical system theory to explore the dynamic behaviour of the model, studying in particular the impact of prophage on the equilibria and stability of phage and host. We employ bifurcation and stability theory, centre manifold and normal form theory to show that the system has multiple equilibrium solutions which undergo a series of bifurcations, finally leading to oscillating motions. Numerical simulations are presented to illustrate and confirm the analytical predictions. The results of this study indicate that in some parameter regimes, the host cell population may drive the phage to extinction through diversification, that is, if multiple types of host emerge; this prediction holds even if the phage population is likewise diverse. This parameter regime is restricted, however, if infecting phage are able to recombine with prophage sequences in the host cell genome.

  7. Mimtags: the use of phage display technology to produce novel protein-specific probes.

    Science.gov (United States)

    Ahmed, Nayyar; Dhanapala, Pathum; Sadli, Nadia; Barrow, Colin J; Suphioglu, Cenk

    2014-03-01

    In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. DeltaPhage—a novel helper phage for high-valence pIX phagemid display

    Science.gov (United States)

    Nilssen, Nicolay R.; Frigstad, Terje; Pollmann, Sylvie; Roos, Norbert; Bogen, Bjarne; Sandlie, Inger; Løset, Geir Å.

    2012-01-01

    Phage display has been instrumental in discovery of novel binding peptides and folded domains for the past two decades. We recently reported a novel pIX phagemid display system that is characterized by a strong preference for phagemid packaging combined with low display levels, two key features that support highly efficient affinity selection. However, high diversity in selected repertoires are intimately coupled to high display levels during initial selection rounds. To incorporate this additional feature into the pIX display system, we have developed a novel helper phage termed DeltaPhage that allows for high-valence display on pIX. This was obtained by inserting two amber mutations close to the pIX start codon, but after the pVII translational stop, conditionally inactivating the helper phage encoded pIX. Until now, the general notion has been that display on pIX is dependent on wild-type complementation, making high-valence display unachievable. However, we found that DeltaPhage does facilitate high-valence pIX display when used with a non-suppressor host. Here, we report a side-by-side comparison with pIII display, and we find that this novel helper phage complements existing pIX phagemid display systems to allow both low and high-valence display, making pIX display a complete and efficient alternative to existing pIII phagemid display systems. PMID:22539265

  9. A Flagellar Glycan-Specific Protein Encoded by Campylobacter Phages Inhibits Host Cell Growth

    Directory of Open Access Journals (Sweden)

    Muhammad Afzal Javed

    2015-12-01

    Full Text Available We previously characterized a carbohydrate binding protein, Gp047, derived from lytic Campylobacter phage NCTC 12673, as a promising diagnostic tool for the identification of Campylobacter jejuni and Campylobacter coli. We also demonstrated that this protein binds specifically to acetamidino-modified pseudaminic acid residues on host flagella, but the role of this protein in the phage lifecycle remains unknown. Here, we report that Gp047 is capable of inhibiting C. jejuni growth both on solid and liquid media, an activity, which we found to be bacteriostatic. The Gp047 domain responsible for bacterial growth inhibition is localized to the C-terminal quarter of the protein, and this activity is both contact- and dose-dependent. Gp047 gene homologues are present in all Campylobacter phages sequenced to date, and the resulting protein is not part of the phage particle. Therefore, these results suggest that either phages of this pathogen have evolved an effector protein capable of host-specific growth inhibition, or that Campylobacter cells have developed a mechanism of regulating their growth upon sensing an impending phage threat.

  10. Comparative genomics of Bacillus thuringiensis phage 0305φ8-36: defining patterns of descent in a novel ancient phage lineage

    Directory of Open Access Journals (Sweden)

    Serwer Philip

    2007-10-01

    Full Text Available Abstract Background The recently sequenced 218 kb genome of morphologically atypical Bacillus thuringiensis phage 0305φ8-36 exhibited only limited detectable homology to known bacteriophages. The only known relative of this phage is a string of phage-like genes called BtI1 in the chromosome of B. thuringiensis israelensis. The high degree of divergence and novelty of phage genomes pose challenges in how to describe the phage from its genomic sequences. Results Phage 0305φ8-36 and BtI1 are estimated to have diverged 2.0 – 2.5 billion years ago. Positionally biased Blast searches aligned 30 homologous structure or morphogenesis genes between 0305φ8-36 and BtI1 that have maintained the same gene order. Functional clustering of the genes helped identify additional gene functions. A conserved long tape measure gene indicates that a long tail is an evolutionarily stable property of this phage lineage. An unusual form of the tail chaperonin system split to two genes was characterized, as was a hyperplastic homologue of the T4gp27 hub gene. Within this region some segments were best described as encoding a conservative array of structure domains fused with a variable component of exchangeable domains. Other segments were best described as multigene units engaged in modular horizontal exchange. The non-structure genes of 0305φ8-36 appear to include the remnants of two replicative systems leading to the hypothesis that the genome plan was created by fusion of two ancestral viruses. The case for a member of the RNAi RNA-directed RNA polymerase family residing in 0305φ8-36 was strengthened by extending the hidden Markov model of this family. Finally, it was noted that prospective transcriptional promoters were distributed in a gradient of small to large transcripts starting from a fixed end of the genome. Conclusion Genomic organization at a level higher than individual gene sequence comparison can be analyzed to aid in understanding large phage

  11. An Integrated Time-Temperature Approach for Predicting Mechanical Properties of Quenched and Tempered Steels

    OpenAIRE

    O'Connell, Corey James

    2014-01-01

    The purpose of this work was to develop a steel tempering model that is useful to the commercial heat treater. Most of the tempering models reported address isothermal conditions which are not typical of most heating methods used to perform the tempering heat treatment. In this work, a non-isothermal tempering model was developed based on the tempering response of four steel alloys. This tempering model employs the quantity resulting from the numerical integration of the time-temperature prof...

  12. The tempering quality evaluation of cocoa liquor during industrial ...

    African Journals Online (AJOL)

    The tempering quality evaluation of the crude cocoa liquor with average fat content 55.0 ± 0.3 % and an average acid value, 1.57 ± 0.34) has been established. The various parameters considered were recasting time (RT) and appearance (AP) of the tempered product on one hand and the flow (F) of the crude liquor on ...

  13. Spray mist cooling heat transfer in glass tempering process

    Science.gov (United States)

    Sozbir, Nedim; Yao, S. C.

    2017-05-01

    Energy saving is a very important issue in glass plants, especially in a glass tempering process, where very high velocity air jet impingement is applied during the cooling process of glass tempering. In fact, air compressor energy may be reduced by a spray cooling due to its high heat transfer capabilities. Presently, in this paper, both pure air and water mist spray cooling are investigated in the glass tempering process. The test results indicate that thin and low-cost tempered glass can be made by mist cooling without fracture. It is possible to find the optimal water flux and duration of mist application to achieve a desirable temperature distribution in the glass for deep penetration of the cooling front but without inducing cracking during the tempering. The use of mist cooling could give about 29 % air pressure reduction for 2-mm glass plate and 50 % reduction for both 3- and 4-mm glass plates.

  14. High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum

    DEFF Research Database (Denmark)

    Christiansen, Anders; Kringelum, Jens Vindahl; Hansen, Christian Skjødt

    2015-01-01

    Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high-throughput sequenc......Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high......-throughput sequencing. A bioinformatics approach was developed in order to identify peptide motifs of interest based on clustering and contrasting to control samples. Comparison of patient and control samples confirmed a major issue in phage display, namely the selection of unspecific peptides. The potential...... display by (i) enabling the analysis of complex biological samples, (ii) circumventing the traditional laborious picking and functional testing of individual phage clones and (iii) reducing the number of selection rounds....

  15. Phage adsorption to Lactobacillus plantarum: influence of physiological and environmental factors.

    Science.gov (United States)

    Marcó, M Briggiler; Reinheimer, J A; Quiberoni, A

    2010-04-15

    Bacteriophage infection of lactic acid bacteria (LAB) constitutes one of the major problems in the dairy industry, causing economic losses and a constant risk of low quality and/or unsafe foods. The first step in the phage biology is the adsorption on the host cell surface. In a previous study, a remarkable thermal, chemical and photocatalytic resistance was demonstrated by four phages of Lactobacillus plantarum (ATCC 8014-B1, ATCC 8014-B2, FAGK1 and FAGK2). In the present work, these phages were used to characterize the adsorption process on L. plantarum ATCC 8014. Clearly, the characterization of this process could increase the possibilities of design useful strategies in order to prevent phage infections. The influence of Ca(2+), temperature, pH and physiological cell state on phage adsorption was investigated. Burst sizes of phages ATCC 8014-B1 and ATCC 8014-B2 were 60 and 83 PFU/infective centre, respectively. The four phages exhibited a high infectivity even at pH 4 and pH 11. Calcium or magnesium ions were not indispensable for cell lysis and plaque formation, and more than 99% of phage particles were adsorbed either in the presence or absence of Ca(2+), after 15 min at 37 degrees C. Phage adsorption was only partially affected at 50 degrees C, while reached its maximum between 30 and 42 degrees C. The highest adsorption values (99.9%) were observed from pH 5 to 7, after 30 min at 37 degrees C. Adsorption rates decreased after the thermal inactivation of cells, though, when 20 microg/ml of chloramphenicol was used, adsorption values were similar on treated and untreated cells. All these results showed that the adsorption process was only partially affected by a few conditions: thermally killed host cells, an incubation temperature of 50 degrees C and pH values of 9 and 10. Nevertheless, and unfortunately, those conditions are not commonly applied during fermented food manufacturing, thus restricting highly the application of strategies currently available to

  16. The interactome of Streptococcus pneumoniae and its bacteriophages show highly specific patterns of interactions among bacteria and their phages

    OpenAIRE

    Rachelle Mariano; Stefan Wuchty; Vizoso-Pinto, Maria G.; Roman Häuser; Peter Uetz

    2016-01-01

    Although an abundance of bacteriophages exists, little is known about interactions between their proteins and those of their bacterial hosts. Here, we experimentally determined the phage-host interactomes of the phages Dp-1 and Cp-1 and their underlying protein interaction network in the host Streptococcus pneumoniae. We compared our results to the interaction patterns of E. coli phages lambda and T7. Dp-1 and Cp-1 target highly connected host proteins, occupy central network positions, and r...

  17. Genetic Evidence for O-Specific Antigen as Receptor of Pseudomonas aeruginosa Phage K8 and Its Genomic Analysis

    Directory of Open Access Journals (Sweden)

    Xuewei ePan

    2016-03-01

    Full Text Available Phage therapy requires the comprehensive understanding of the mechanisms underlying the host-phage interactions. In this work, to identify the genes related to Pseudomonas aeruginosa phage K8 receptor synthesis, 16 phage-resistant mutants were selected from a Tn5G transposon mutant library of strain PAK. The disrupted genetic loci were identified and they were related to O-specific antigen (OSA synthesis, including gene wbpR, ssg, wbpV, wbpO, and Y880_RS05480, which encoded a putative O-antigen polymerase Wzy. The LPS profile of the Y880_RS05480 mutant was analyzed and shown to lack the O-antigen. Therefore, the data from characterization of Y880_RS05480 by TMHMM and SDS-PAGE silver staining analysis suggest that this locus might encode Wzy. The complete phage K8 genome was characterized as 93879 bp in length and contained identical 1188-bp terminal direct repeats. Comparative genomic analysis showed that phage K8 was highly homologous to members of the genus PaP1-like phages. On the basis of our genetic findings, OSA of P. aeruginosa PAK is proven to be the receptor of phage K8. The highly conserved structural proteins among the genetic closely related phages suggest that they may recognize the same receptor.

  18. Recent Trends in Salmonella Outbreaks and Emerging Technology for Biocontrol of Salmonella Using Phages in Foods: A Review.

    Science.gov (United States)

    Oh, Jun-Hyun; Park, Mi-Kyung

    2017-12-28

    Salmonella is one of the principal causes of foodborne outbreaks. As traditional control methods have shown less efficacy against emerging Salmonella serotypes or antimicrobialresistant Salmonella , new approaches have been attempted. The use of lytic phages for the biocontrol of Salmonella in the food industry has become an attractive method owing to the many advantages offered by the use of phages as biocontrol agents. Phages are natural alternatives to traditional antimicrobial agents; they have proven effective in the control of bacterial pathogens in the food industry, which has led to the development of different phage products. The treatment with specific phages in the food industry can prevent the decay of products and the spread of bacterial diseases, and ultimately promotes safe environments for animal and plant food production, processing, and handling. After an extensive investigation of the current literature, this review focuses predominantly on the efficacy of phages for the successful control of Salmonella spp. in foods. This review also addresses the current knowledge on the pathogenic characteristics of Salmonella , the prevalence of emerging Salmonella outbreaks, the isolation and characterization of Salmonella -specific phages, the effectiveness of Salmonella -specific phages as biocontrol agents, and the prospective use of Salmonella -specific phages in the food industry.

  19. Two Novel Myoviruses from the North of Iraq Reveal Insights into Clostridium difficile Phage Diversity and Biology

    Directory of Open Access Journals (Sweden)

    Srwa J. Rashid

    2016-11-01

    Full Text Available Bacteriophages (phages are increasingly being explored as therapeutic agents to combat bacterial diseases, including Clostridium difficile infections. Therapeutic phages need to be able to efficiently target and kill a wide range of clinically relevant strains. While many phage groups have yet to be investigated in detail, those with new and useful properties can potentially be identified when phages from newly studied geographies are characterised. Here, we report the isolation of C. difficile phages from soil samples from the north of Iraq. Two myoviruses, CDKM15 and CDKM9, were selected for detailed sequence analysis on the basis of their broad and potentially useful host range. CDKM9 infects 25/80 strains from 12/20 C. difficile ribotypes, and CDKM15 infects 20/80 strains from 9/20 ribotypes. Both phages can infect the clinically relevant ribotypes R027 and R001. Phylogenetic analysis based on whole genome sequencing revealed that the phages are genetically distinct from each other but closely related to other long-tailed myoviruses. A comparative genomic analysis revealed key differences in the genes predicted to encode for proteins involved in bacterial infection. Notably, CDKM15 carries a clustered regularly interspaced short palindromic repeat (CRISPR array with spacers that are homologous to sequences in the CDKM9 genome and of phages from diverse localities. The findings presented suggest a possible shared evolutionary past for these phages and provides evidence of their widespread dispersal.

  20. Proposed Ancestors of Phage Nucleic Acid Packaging Motors (and Cells

    Directory of Open Access Journals (Sweden)

    Philip Serwer

    2011-07-01

    Full Text Available I present a hypothesis that begins with the proposal that abiotic ancestors of phage RNA and DNA packaging systems (and cells include mobile shells with an internal, molecule-transporting cavity. The foundations of this hypothesis include the conjecture that current nucleic acid packaging systems have imprints from abiotic ancestors. The abiotic shells (1 initially imbibe and later also bind and transport organic molecules, thereby providing a means for producing molecular interactions that are links in the chain of events that produces ancestors to the first molecules that are both information carrying and enzymatically active, and (2 are subsequently scaffolds on which proteins assemble to form ancestors common to both shells of viral capsids and cell membranes. Emergence of cells occurs via aggregation and merger of shells and internal contents. The hypothesis continues by using proposed imprints of abiotic and biotic ancestors to deduce an ancestral thermal ratchet-based DNA packaging motor that subsequently evolves to integrate a DNA packaging ATPase that provides a power stroke.

  1. Semi-Parametric Bayesian Inference for Phage Display Data

    Science.gov (United States)

    León-Novelo, Luis G.; Müller, Peter; Arap, Wadih; Kolonin, Mikhail; Sun, Jessica; Pasqualini, Renata; Do, Kim-Anh

    2012-01-01

    Summary We discuss inference for a human phage display experiment with three stages. The data are tripeptide counts by tissue and stage. The primary aim of the experiment is to identify ligands that bind with high affinity to a given tissue. We formalize the research question as inference about the monotonicity of mean counts over stages. The inference goal is then to identify a list of peptide-tissue pairs with significant increase over stages. We use a semi-parametric Dirichlet process mixture of Poisson model. The posterior distribution under this model allows the desired inference about the monotonicity of mean counts. However, the desired inference summary as a list of peptide-tissue pairs with significant increase involves a massive multiplicity problem. We consider two alternative approaches to address this multiplicity issue. First we propose an approach based on the control of the posterior expected false discovery rate. We notice that the implied solution ignores the relative size of the increase. This motivates a second approach based on a utility function that includes explicit weights for the size of the increase. PMID:23339534

  2. Temperate Ice Depth-Sounding Radar

    Science.gov (United States)

    Jara-Olivares, V. A.; Player, K.; Rodriguez-Morales, F.; Gogineni, P.

    2008-12-01

    Glaciers in several parts of the world are reported to be retreating and thinning rapidly over the last decade. Radar instruments can be used to provide a wealth of information regarding the internal and basal conditions of large and small ice masses. These instruments typically operate in the VHF and UHF regions of the electromagnetic spectrum. For temperate-ice sounding, however, the high water content produces scattering and attenuation in propagating radar waves at VHF and UHF frequencies, which significantly reduce the penetration depths. Radars operating in the HF band are better suited for systematic surveys of the thickness and sub-glacial topography of temperate-ice regions. We are developing a dual-frequency Temperate-Ice-Depth Sounding Radar (TIDSoR) that can penetrate through water pockets, thus providing more accurate measurements of temperate ice properties such as thickness and basal conditions. The radar is a light-weight, low power consumption portable system for surface-based observations in mountainous terrain or aerial surveys. TIDSoR operates at two different center frequencies: 7.7 MHz and 14 MHz, with a maximum output peak power of 20 W. The transmit waveform is a digitally generated linear frequency-modulated chirp with 1 MHz bandwidth. The radar can be installed on aircrafts such as the CReSIS UAV [1], DCH-6 (Twin Otter), or P-3 Orion for aerial surveys, where it could be supported by the airplane power system. For surface based experiments, TIDSoR can operate in a backpack configuration powered by a compact battery system. The system can also be installed on a sled towed by a motorized vehicle, in which case the power supply can be replaced by a diesel generator. The radar consists of three functional blocks: the digital section, the radio-frequency (RF) section, and the antenna, and is designed to weigh less than 2 kg, excluding the power supply. The digital section generates the transmit waveforms as well as timing and control signals

  3. Moist temperate forest butterflies of western Bhutan

    Directory of Open Access Journals (Sweden)

    Arun P. Singh

    2016-03-01

    Full Text Available Random surveys were carried out in moist temperate forests (1,860–3,116 m around Bunakha Village and Dochula Pass, near Thimphu in western Bhutan, recording 65 species of butterflies.  Of these, 11 species, viz., Straightwing Blue Orthomiella pontis pontis Elwes, Slate Royal Maneca bhotea bhotea Moore, Dull Green Hairstreak Esakiozephyrus icana Moore, Yellow Woodbrown Lethe nicetas Hewitson, Small Silverfork Zophoessa jalaurida elwesi Moore, Scarce Labyrinth, Neope pulahina (Evans, Chumbi Wall Chonala masoni Elwes, Pale Hockeystick Sailer Neptis manasa manasa Moore and White Commodore Parasarpa dudu dudu Westwood, are restricted to the eastern Himalaya, northeastern India and Myanmar.  Two other species, Tawny Mime Chiasa agestor agestor (Gray and Himalayan Spotted Flat Celaenorrhinus munda Moore have been only rarely recorded from Bhutan and a few individuals of the rare Bhutan Glory Bhutanitis lidderdalei Atkinson were also recorded near Bunakha.  

  4. Study on tempering behaviour of AISI 410 stainless steel

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Gopa, E-mail: gopa_mjs@igcar.gov.in [Metallurgy & Materials Group, Indira Gandhi Center for Atomic Research, Kalpakkam 603102 (India); Das, C.R.; Albert, S.K.; Bhaduri, A.K.; Thomas Paul, V. [Metallurgy & Materials Group, Indira Gandhi Center for Atomic Research, Kalpakkam 603102 (India); Panneerselvam, G. [Chemistry Group, Indira Gandhi Center for Atomic Research, Kalpakkam 603102 (India); Dasgupta, Arup [Metallurgy & Materials Group, Indira Gandhi Center for Atomic Research, Kalpakkam 603102 (India)

    2015-02-15

    Martensitic stainless steels find extensive applications due to their optimum combination of strength, hardness and wear-resistance in tempered condition. However, this class of steels is susceptible to embrittlement during tempering if it is carried out in a specific temperature range resulting in significant reduction in toughness. Embrittlement of as-normalised AISI 410 martensitic stainless steel, subjected to tempering treatment in the temperature range of 673–923 K was studied using Charpy impact tests followed by metallurgical investigations using field emission scanning electron and transmission electron microscopes. Carbides precipitated during tempering were extracted by electrochemical dissolution of the matrix and identified by X-ray diffraction. Studies indicated that temper embrittlement is highest when the steel is tempered at 823 K. Mostly iron rich carbides are present in the steel subjected to tempering at low temperatures of around 723 K, whereas chromium rich carbides (M{sub 23}C{sub 6}) dominate precipitation at high temperature tempering. The range 773–823 K is the transition temperature range for the precipitates, with both Fe{sub 2}C and M{sub 23}C{sub 6} types of carbides coexisting in the material. The nucleation of Fe{sub 2}C within the martensite lath, during low temperature tempering, has a definite role in the embrittlement of this steel. Embrittlement is not observed at high temperature tempering because of precipitation of M{sub 23}C{sub 6} carbides, instead of Fe{sub 2}C, preferentially along the lath and prior austenite boundaries. Segregation of S and P, which is widely reported as one of the causes for temper embrittlement, could not be detected in the material even through Auger electron spectroscopy studies. - Highlights: • Tempering behaviour of AISI 410 steel is studied within 673–923 K temperature range. • Temperature regime of maximum embrittlement is identified as 773–848 K. • Results show that type of

  5. Phages of lactic acid bacteria: The role of genetics in understanding phage-host interactions and their co-evolutionary processes

    Energy Technology Data Exchange (ETDEWEB)

    Mahony, Jennifer, E-mail: j.mahony@ucc.ie [Department of Microbiology, University College Cork, Western Road, Cork (Ireland); Ainsworth, Stuart; Stockdale, Stephen [Department of Microbiology, University College Cork, Western Road, Cork (Ireland); Sinderen, Douwe van, E-mail: d.vansinderen@ucc.ie [Department of Microbiology, University College Cork, Western Road, Cork (Ireland); Alimentary Pharmabiotic Centre, Biosciences Institute, University College Cork, Western Road, Cork (Ireland)

    2012-12-20

    Dairy fermentations are among the oldest food processing applications, aimed at preservation and shelf-life extension through the use of lactic acid bacteria (LAB) starter cultures, in particular strains of Lactococcus lactis, Streptococcus thermophilus, Lactobacillus spp. and Leuconostoc spp. Traditionally this was performed by continuous passaging of undefined cultures from a finished fermentation to initiate the next fermentation. More recently, consumer demands on consistent and desired flavours and textures of dairy products have led to a more defined approach to such processes. Dairy (starter) companies have responded to the need to define the nature and complexity of the starter culture mixes, and dairy fermentations are now frequently based on defined starter cultures of low complexity, where each starter component imparts specific technological properties that are desirable to the product. Both mixed and defined starter culture approaches create the perfect environment for the proliferation of (bacterio)phages capable of infecting these LAB. The repeated use of the same starter cultures in a single plant, coupled to the drive towards higher and consistent production levels, increases the risk and negative impact of phage infection. In this review we will discuss recent advances in tracking the adaptation of phages to the dairy industry, the advances in understanding LAB phage-host interactions, including evolutionary and genomic aspects.

  6. 55.2, a phage T4 ORFan gene, encodes an inhibitor of Escherichia coli topoisomerase I and increases phage fitness.

    Directory of Open Access Journals (Sweden)

    Yves Mattenberger

    Full Text Available Topoisomerases are enzymes that alter the topological properties of DNA. Phage T4 encodes its own topoisomerase but it can also utilize host-encoded topoisomerases. Here we characterized 55.2, a phage T4 predicted ORF of unknown function. High levels of expression of the cloned 55.2 gene are toxic in E. coli. This toxicity is suppressed either by increased topoisomerase I expression or by partial inactivation of the ATPase subunit of the DNA gyrase. Interestingly, very low-level expression of 55.2, which is non-lethal to wild type E. coli, prevents the growth of a deletion mutant of the topoisomerase I (topA gene. In vitro, gp55.2 binds DNA and blocks specifically the relaxation of negatively supercoiled DNA by topoisomerase I. In vivo, expression of gp55.2 at low non-toxic levels alters the steady state DNA supercoiling of a reporter plasmid. Although 55.2 is not an essential gene, competition experiments indicate that it is required for optimal phage growth. We propose that the role of gp55.2 is to subtly modulate host topoisomerase I activity during infection to insure optimal T4 phage yield.

  7. Correlation of conversion of Salmonella enterica serovar enteritidis phage type 1, 4, or 6 to phage type 7 with loss of lipopolysaccharide

    DEFF Research Database (Denmark)

    Baggesen, Dorte Lau; Wegener, Henrik Caspar; Madsen, M.

    1997-01-01

    Studies of pairs of Salmonella enterica serovar Enteritidis isolates from different poultry flocks showed that phage type (PT) 7 may be derived from PT 1, 4, and 6. The conversion appeared to be associated with loss of lipopolysaccharide. It is concluded that PT 7 may be of little value...... as an epidemiological marker of S. enterica serovar Enteritidis....

  8. Forecasting cyanobacteria dominance in Canadian temperate lakes.

    Science.gov (United States)

    Persaud, Anurani D; Paterson, Andrew M; Dillon, Peter J; Winter, Jennifer G; Palmer, Michelle; Somers, Keith M

    2015-03-15

    Predictive models based on broad scale, spatial surveys typically identify nutrients and climate as the most important predictors of cyanobacteria abundance; however these models generally have low predictive power because at smaller geographic scales numerous other factors may be equally or more important. At the lake level, for example, the ability to forecast cyanobacteria dominance is of tremendous value to lake managers as they can use such models to communicate exposure risks associated with recreational and drinking water use, and possible exposure to algal toxins, in advance of bloom occurrence. We used detailed algal, limnological and meteorological data from two temperate lakes in south-central Ontario, Canada to determine the factors that are closely linked to cyanobacteria dominance, and to develop easy to use models to forecast cyanobacteria biovolume. For Brandy Lake (BL), the strongest and most parsimonious model for forecasting % cyanobacteria biovolume (% CB) included water column stability, hypolimnetic TP, and % cyanobacteria biovolume two weeks prior. For Three Mile Lake (TML), the best model for forecasting % CB included water column stability, hypolimnetic TP concentration, and 7-d mean wind speed. The models for forecasting % CB in BL and TML are fundamentally different in their lag periods (BL = lag 1 model and TML = lag 2 model) and in some predictor variables despite the close proximity of the study lakes. We speculate that three main factors (nutrient concentrations, water transparency and lake morphometry) may have contributed to differences in the models developed, and may account for variation observed in models derived from large spatial surveys. Our results illustrate that while forecast models can be developed to determine when cyanobacteria will dominate within two temperate lakes, the models require detailed, lake-specific calibration to be effective as risk-management tools. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Phosphorous dynamics in a temperate intertidal estuary

    Science.gov (United States)

    Lillebø, A. I.; Neto, J. M.; Flindt, M. R.; Marques, J. C.; Pardal, M. A.

    2004-09-01

    Conservation and management of aquatic systems require detailed information of the processes that affect their functioning and development. The objectives of the present work were to describe the phosphorus dynamics during a complete tidal cycle and to quantify the relative contribution of the most common estuarine areas (e.g. seagrass beds, salt marshes, mud- and sand-flats without vegetation) to phosphorus net internal loading in a temperate intertidal estuary. Results show that phosphate efflux rates were higher during the first hours of tidal flood, and that phosphate concentrations were lowest at high tide. During tidal ebbing, ephemeral tide pools may cover a considerable percentage of the intertidal area. In these tide pools, water shallowness combined with enhanced temperatures stimulate the occurrence of high phosphate effluxes. The effluxes to the main water body during high tide contributed 57% of dissolved inorganic phosphorus and efflux during low tide contributed 43% to the net internal loading. Calculations of the phosphate net effluxes (kg P) indicate a strong contribution of the bare bottom mud-flats to the whole system internal phosphate loading, especially during the warmer periods. As a consequence of eutrophication, perennial benthic macrophytes are commonly replaced by fast-growing epiphytic macroalgae. Calculations showed that for a hypothetical intertidal estuary in a temperate region, management programs considering an eventual re-colonization of mud-flats by seagrasses or salt marsh plants may reduce the P-efflux by 13-16 kg ha -1. For example, in the small Mondego estuary, eutrophication has contributed to a reduction of the Zostera noltii meadows, leading to an increase in 190 kg of phosphorus net internal loading.

  10. Phenology of temperate trees in tropical climates

    Science.gov (United States)

    Borchert, Rolf; Robertson, Kevin; Schwartz, Mark D.; Williams-Linera, Guadalupe

    2005-09-01

    Several North American broad-leaved tree species range from the northern United States at ˜47°N to moist tropical montane forests in Mexico and Central America at 15-20°N. Along this gradient the average minimum temperatures of the coldest month (T Jan), which characterize annual variation in temperature, increase from -10 to 12°C and tree phenology changes from deciduous to leaf-exchanging or evergreen in the southern range with a year-long growing season. Between 30 and 45°N, the time of bud break is highly correlated with T Jan and bud break can be reliably predicted for the week in which mean minimum temperature rises to 7°C. Temperature-dependent deciduous phenology—and hence the validity of temperature-driven phenology models—terminates in southern North America near 30°N, where T Jan>7°C enables growth of tropical trees and cultivation of frost-sensitive citrus fruits. In tropical climates most temperate broad-leaved species exchange old for new leaves within a few weeks in January-February, i.e., their phenology becomes similar to that of tropical leaf-exchanging species. Leaf buds of the southern ecotypes of these temperate species are therefore not winter-dormant and have no chilling requirement. As in many tropical trees, bud break of Celtis, Quercus and Fagus growing in warm climates is induced in early spring by increasing daylength. In tropical climates vegetative phenology is determined mainly by leaf longevity, seasonal variation in water stress and day length. As water stress during the dry season varies widely with soil water storage, climate-driven models cannot predict tree phenology in the tropics and tropical tree phenology does not constitute a useful indicator of global warming.

  11. Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase.

    Science.gov (United States)

    Gasparian, Marine E; Bobik, Tatyana V; Kim, Yana V; Ponomarenko, Natalia A; Dolgikh, Dmitry A; Gabibov, Alexander G; Kirpichnikov, Mikhail P

    2013-11-01

    Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D4K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-β-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective Km values of 19 μM and 20 μM and kcat of 115 and 92 s(-1). Fusion proteins containing a D4K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  12. [Phage types recognized within Salmonella Enteritidis strains isolated in Poland in 1996-2007].

    Science.gov (United States)

    Dera-Tomaszewska, Bozena; Tokarska-Pietrzak, Ewa

    2012-01-01

    Phage typing carried out according to the well-defined schemes is still recommended as a standard, fast and inexpensive method for epidemiological investigations all over the world to control Salmonella infections. However, the method should be in the hands of well-trained staff. This means that it is generally limited to reference laboratories. The aim of this study was to perform an analysis of Salmonella Enteritidis phage types occurring in Poland in 1996-2007 basing on the strains received by the National Salmonella Centre for phage typing. The phage typing (according to the Lalko et al. scheme) carried out in this research work was associated with 750 Salmonella Enteritidis strains isolated in Poland from human and non-human sources, in the dying out period of over 25 years lasting second epidemic caused by this serovar. Types 1, 6 and 7 were the most often identified phage types. They were dominant among human as well as non-human (food, feeds, animals, environment and others) isolates. The great majority of them were of type 7. Type 1 Salmonella Enteritidis strains were isolated in the same number as type 6 strains. A little higher number of strains which presented phage type 3 was reported in comparison with the previous periods of time. It is worth to be noted that except the same, permanent phage types continuously existing in the environment (i.e., 1, 3, 6, 7), the new types start to appear. They can suggest an appearance of new sources of Salmonella Enteritidis infections, unknown in our country yet, which is very possible as a result of effective elimination of currently existing ones. The obtained results constitute the continuation of the previously done studies and provide the essential and unique data which allow to estimate the more completely epidemiological situation associated with occurrence of this pathogen in Poland.

  13. Biased selection of propagation-related TUPs from phage display peptide libraries.

    Science.gov (United States)

    Zade, Hesam Motaleb; Keshavarz, Reihaneh; Shekarabi, Hosna Sadat Zahed; Bakhshinejad, Babak

    2017-08-01

    Phage display is rapidly advancing as a screening strategy in drug discovery and drug delivery. Phage-encoded combinatorial peptide libraries can be screened through the affinity selection procedure of biopanning to find pharmaceutically relevant cell-specific ligands. However, the unwanted enrichment of target-unrelated peptides (TUPs) with no true affinity for the target presents an important barrier to the successful screening of phage display libraries. Propagation-related TUPs (Pr-TUPs) are an emerging but less-studied category of phage display-derived false-positive hits that are displayed on the surface of clones with faster propagation rates. Despite long regarded as an unbiased selection system, accumulating evidence suggests that biopanning may create biological bias toward selection of phage clones with certain displayed peptides. This bias can be dependent on or independent of the displayed sequence and may act as a major driving force for the isolation of fast-growing clones. Sequence-dependent bias is reflected by censorship or over-representation of some amino acids in the displayed peptide and sequence-independent bias is derived from either point mutations or rare recombination events occurring in the phage genome. It is of utmost interest to clean biopanning data by identifying and removing Pr-TUPs. Experimental and bioinformatic approaches can be exploited for Pr-TUP discovery. With no doubt, obtaining deeper insight into how Pr-TUPs emerge during biopanning and how they could be detected provides a basis for using cell-targeting peptides isolated from phage display screening in the development of disease-specific diagnostic and therapeutic platforms.

  14. Genome of a SAR116 bacteriophage shows the prevalence of this phage type in the oceans.

    Science.gov (United States)

    Kang, Ilnam; Oh, Hyun-Myung; Kang, Dongmin; Cho, Jang-Cheon

    2013-07-23

    The abundance, genetic diversity, and crucial ecological and evolutionary roles of marine phages have prompted a large number of metagenomic studies. However, obtaining a thorough understanding of marine phages has been hampered by the low number of phage isolates infecting major bacterial groups other than cyanophages and pelagiphages. Therefore, there is an urgent requirement for the isolation of phages that infect abundant marine bacterial groups. In this study, we isolated and characterized HMO-2011, a phage infecting a bacterium of the SAR116 clade, one of the most abundant marine bacterial lineages. HMO-2011, which infects "Candidatus Puniceispirillum marinum" strain IMCC1322, has an ~55-kb dsDNA genome that harbors many genes with novel features rarely found in cultured organisms, including genes encoding a DNA polymerase with a partial DnaJ central domain and an atypical methanesulfonate monooxygenase. Furthermore, homologs of nearly all HMO-2011 genes were predominantly found in marine metagenomes rather than cultured organisms, suggesting the novelty of HMO-2011 and the prevalence of this phage type in the oceans. A significant number of the viral metagenome sequences obtained from the ocean surface were best assigned to the HMO-2011 genome. The number of reads assigned to HMO-2011 accounted for 10.3%-25.3% of the total reads assigned to viruses in seven viromes from the Pacific and Indian Oceans, making the HMO-2011 genome the most or second-most frequently assigned viral genome. Given its ability to infect the abundant SAR116 clade and its widespread distribution, Puniceispirillum phage HMO-2011 could be an important resource for marine virus research.

  15. Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

    Directory of Open Access Journals (Sweden)

    Muyrers Joep PP

    2003-01-01

    Full Text Available Abstract Background The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined. Results Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα but only requires a phage annealing protein (RecT or Redβ. Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein. Conclusion Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.

  16. Transmission of Vibrio cholerae is antagonized by lytic phage and entry into the aquatic environment.

    Directory of Open Access Journals (Sweden)

    Eric J Nelson

    2008-10-01

    Full Text Available Cholera outbreaks are proposed to propagate in explosive cycles powered by hyperinfectious Vibrio cholerae and quenched by lytic vibriophage. However, studies to elucidate how these factors affect transmission are lacking because the field experiments are almost intractable. One reason for this is that V. cholerae loses the ability to culture upon transfer to pond water. This phenotype is called the active but non-culturable state (ABNC; an alternative term is viable but non-culturable because these cells maintain the capacity for metabolic activity. ABNC bacteria may serve as the environmental reservoir for outbreaks but rigorous animal studies to test this hypothesis have not been conducted. In this project, we wanted to determine the relevance of ABNC cells to transmission as well as the impact lytic phage have on V. cholerae as the bacteria enter the ABNC state. Rice-water stool that naturally harbored lytic phage or in vitro derived V. cholerae were incubated in a pond microcosm, and the culturability, infectious dose, and transcriptome were assayed over 24 h. The data show that the major contributors to infection are culturable V. cholerae and not ABNC cells. Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low. However, V. cholerae failed to colonize the small intestine after 24 h of incubation in pond water-the point when the phage and ABNC cell titers were highest. The transcriptional analysis traced the transformation into the non-infectious ABNC state and supports models for the adaptation to nutrient poor aquatic environments. Phage had an undetectable impact on this adaptation. Taken together, the rise of ABNC cells and lytic phage blocked transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment.

  17. Commensal E. coli Stx2 lysogens produce high levels of phages after spontaneous prophage induction

    Directory of Open Access Journals (Sweden)

    Hildegunn eIversen

    2015-02-01

    Full Text Available Enterohemorrhagic E. coli (EHEC is a food-borne pathogen that causes disease ranging from uncomplicated diarrhea to life-threatening hemolytic uremic syndrome (HUS and nervous system complications. Shiga toxin 2 (Stx2 is the major virulence factor of EHEC and is critical for development of HUS. The genes encoding Stx2 are carried by lambdoid bacteriophages and the toxin production is tightly linked to the production of phages during lytic cycle. It has previously been suggested that commensal E. coli could amplify the production of Stx2-phages and contribute to the severity of disease. In this study we examined the susceptibility of commensal E. coli strains to the Stx2-converting phage ϕ734, isolated from a highly virulent EHEC O103:H25 (NIPH-11060424. Among 38 commensal E. coli strains from healthy children below five years, 15 were lysogenized by the ϕ734 phage, whereas lytic infection was not observed. Three of the commensal E. coli ϕ734 lysogens were tested for stability, and appeared stable and retained the phage for at least 10 cultural passages. When induced to enter lytic cycle by H2O2 treatment, 8 out of 13 commensal lysogens produced more ϕ734 phages than NIPH-11060424. Strikingly, five of them even spontaneously (non-induced produced higher levels of phage than the H2O2 induced NIPH-11060424. An especially high frequency of HUS (60% was seen among children infected by NIPH-11060424 during the outbreak in 2006. Based on our findings, a high Stx2 production by commensal E. coli lysogens cannot be ruled out as a contributor to the high frequency of HUS during this outbreak.

  18. The population dynamics of bacteria, phage and RM Systems

    Science.gov (United States)

    Guet, Calin; Levin, Bruce; Pleska, Maros

    Viruses drive and mediate bacterial evolution as parasites and vectors of horizontal gene transfer, respectively. Temperate bacteriophages, defined by the ability to lysogenize a fraction of hosts and to transmit horizontally as well as vertically in the form of prophages, frequently carry genes that increase fitness or contribute to bacterial pathogenicity. Restriction-modification (RM) systems, which are widely diverse and ubiquitous among bacteria, can prevent infections leading to lysis, but their effect on lysogeny is not clear. We show that RM systems prevent lytic and lysogenic infections to the same extent and therefore represent a molecular barrier to prophage acquisition. Surprisingly, we find that this negative effect can be overcome and even reversed at the population level, as a consequence of dynamic interactions between viruses, hosts and RM systems. Thus the population dynamics of bacteria carrying RM systems impacts bacterial genome-wide evolution. .

  19. Stress relaxation in tempered glass caused by heat soak testing

    DEFF Research Database (Denmark)

    Schneider, Jens; Hilcken, Jonas; Aronen, Antti

    2016-01-01

    of commercial soda-lime-silica glass, it causes stress relaxation in tempered glass and the fracture pattern of the glass changes accordingly, especially thin glasses are affected. Based on the Tool-Narayanaswamy-Model, this paper comprises the theoretical background of the stress......Heat soak testing of tempered glass is a thermal process required after the tempering process itself to bring glasses of commercial soda-lime-silica-glass to failure that are contaminated with nickel sulphide inclusions, diameter 50 mm to 500 mm typically. Thus, the tests avoid a so...

  20. Bacteria between protists and phages: from antipredation strategies to the evolution of pathogenicity.

    Science.gov (United States)

    Brüssow, Harald

    2007-08-01

    Bacteriophages and protists are major causes of bacterial mortality. Genomics suggests that phages evolved well before eukaryotic protists. Bacteria were thus initially only confronted with phage predators. When protists evolved, bacteria were caught between two types of predators. One successful antigrazing strategy of bacteria was the elaboration of toxins that would kill the grazer. The released cell content would feed bystander bacteria. I suggest here that, to fight grazing protists, bacteria teamed up with those phage predators that concluded at least a temporary truce with them in the form of lysogeny. Lysogeny was perhaps initially a resource management strategy of phages that could not maintain infection chains. Subsequently, lysogeny might have evolved into a bacterium-prophage coalition attacking protists, which became a food source for them. When protists evolved into multicellular animals, the lysogenic bacteria tracked their evolving food source. This hypothesis could explain why a frequent scheme of bacterial pathogenicity is the survival in phagocytes, why a significant fraction of bacterial pathogens have prophage-encoded virulence genes, and why some virulence factors of animal pathogens are active against unicellular eukaryotes. Bacterial pathogenicity might thus be one playing option of the stone-scissor-paper game played between phages-bacteria-protists, with humans getting into the crossfire.

  1. Structural characterizations of phage antitoxin Dmd and its interactions with bacterial toxin RnlA.

    Science.gov (United States)

    Wei, Yong; Gao, Zengqiang; Zhang, Heng; Dong, Yuhui

    2016-04-15

    Toxin-antitoxin (TA) loci are widespread in bacteria plasmids and chromosomes, and target various cellular functions to regulate cell growth and death. A type II TA system RnlA-RnlB from Escherichia coli is associated with phage-resistance. After the infection of bacteriophage T4 with Dmd defection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs. Dmd can bind to RnlA directly and neutralize RnlA toxicity to allow phage reproduction. Dmd represent a heterogenous antitoxin of RnlA replacing antitoxin RnlB. Here, we reported two structures of Dmd from T4 phage and RB69 phage. Both Dmd structures are high similar with a compacted domain composed of a four-stranded anti-parallel β-sheet and an α-helix. Chromatography and SAXS suggest Dmd forms a dimer in solution consistent with that in crystal. Structure-based mutagenesis of Dmd reveals key residues involved in RnlA-binding. Possibility cavities in Dmd used for compounds design were modeled. Our structural study revealed the recognition and inhibition mechanism of RnlA by Dmd and providing a potential laboratory phage prevention target for drug design. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Identification of peptides that selectively bind to myoglobin by biopanning of phage displayed-peptide library.

    Science.gov (United States)

    Padmanaban, Guruprasath; Park, Hyekyung; Choi, Ji Suk; Cho, Yong-Woo; Kang, Woong Chol; Moon, Chan-Il; Kim, In-San; Lee, Byung-Heon

    2014-10-10

    Biopanning of phage displayed-peptide library was performed against myoglobin, a marker for the early assessment of acute myocardial infarction (AMI), to identify peptides that selectively bind to myoglobin. Using myoglobin-conjugated magnetic beads, phages that bound to myoglobin were collected and amplified for the next round of screening. A 148-fold enrichment of phage titer was observed after five rounds of screening relative to the first round. After phage binding ELISA, three phage clones were selected (3R1, 3R7 and 3R10) and the inserted peptides were chemically synthesized. The analysis of binding affinity showed that the 3R7 (CPSTLGASC) peptide had higher binding affinity (Kd=57 nM) than did the 3R1 (CNLSSSWIC) and 3R10 (CVPRLSAPC) peptide (Kd=125 nM and 293 nM, respectively). Cross binding activity to other proteins, such as bovine serum albumin, troponin I, and creatine kinase-MB, was minimal. In a peptide-antibody sandwich ELISA, the selected peptides efficiently captured myoglobin. Moreover, the concentrations of myoglobin in serum samples measured by a peptide-peptide sandwich assay were comparable to those measured by a commercial antibody-based kit. These results indicate that the identified peptides can be used for the detection of myoglobin and may be a cost effective alternative to antibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Inferring phage-bacteria infection networks from time-series data.

    Science.gov (United States)

    Jover, Luis F; Romberg, Justin; Weitz, Joshua S

    2016-11-01

    In communities with bacterial viruses (phage) and bacteria, the phage-bacteria infection network establishes which virus types infect which host types. The structure of the infection network is a key element in understanding community dynamics. Yet, this infection network is often difficult to ascertain. Introduced over 60 years ago, the plaque assay remains the gold standard for establishing who infects whom in a community. This culture-based approach does not scale to environmental samples with increased levels of phage and bacterial diversity, much of which is currently unculturable. Here, we propose an alternative method of inferring phage-bacteria infection networks. This method uses time-series data of fluctuating population densities to estimate the complete interaction network without having to test each phage-bacteria pair individually. We use in silico experiments to analyse the factors affecting the quality of network reconstruction and find robust regimes where accurate reconstructions are possible. In addition, we present a multi-experiment approach where time series from different experiments are combined to improve estimates of the infection network. This approach also mitigates against the possibility of evolutionary changes to relevant phenotypes during the time course of measurement.

  4. Encapsidation and transfer of phage DNA into host cells: from in vivo to single particles studies.

    Science.gov (United States)

    Ponchon, Luc; Mangenot, Stéphanie; Boulanger, Pascale; Letellier, Lucienne

    2005-08-05

    A remarkable property of bacteriophages is their capacity to encapsidate large amounts of DNA during morphogenesis and to maintain their genome in the capsid in a very stable form even under extreme conditions. Even as remarkable is the efficiency with which their genome is ejected from the phage particle and transferred into the host bacteria. Biophysical techniques have led to significant progresses in characterizing these mechanisms. The molecular motor of encapsidation of several phages as well as the organization of viral capsids have been described at atomic resolution. Cryo-electron microscopy and fluorescence microscopy have permitted to describe DNA ejection at the level of single phage particles. Theoretical models of encapsidation and ejection have been proposed that can be confronted to experimental data. This review will present the state of the art on the recent advances brought by biophysics in this field. Reference will be given to the work performed on double-stranded DNA phages and on one of its representative, phage T5, our working model.

  5. Real-time analysis of dual-display phage immobilization and autoantibody screening using quartz crystal microbalance with dissipation monitoring.

    Science.gov (United States)

    Rajaram, Kaushik; Losada-Pérez, Patricia; Vermeeren, Veronique; Hosseinkhani, Baharak; Wagner, Patrick; Somers, Veerle; Michiels, Luc

    2015-01-01

    Over the last three decades, phage display technology has been used for the display of target-specific biomarkers, peptides, antibodies, etc. Phage display-based assays are mostly limited to the phage ELISA, which is notorious for its high background signal and laborious methodology. These problems have been recently overcome by designing a dual-display phage with two different end functionalities, namely, streptavidin (STV)-binding protein at one end and a rheumatoid arthritis-specific autoantigenic target at the other end. Using this dual-display phage, a much higher sensitivity in screening specificities of autoantibodies in complex serum sample has been detected compared to single-display phage system on phage ELISA. Herein, we aimed to develop a novel, rapid, and sensitive dual-display phage to detect autoantibodies presence in serum samples using quartz crystal microbalance with dissipation monitoring as a sensing platform. The vertical functionalization of the phage over the STV-modified surfaces resulted in clear frequency and dissipation shifts revealing a well-defined viscoelastic signature. Screening for autoantibodies using antihuman IgG-modified surfaces and the dual-display phage with STV magnetic bead complexes allowed to isolate the target entities from complex mixtures and to achieve a large response as compared to negative control samples. This novel dual-display strategy can be a potential alternative to the time consuming phage ELISA protocols for the qualitative analysis of serum autoantibodies and can be taken as a departure point to ultimately achieve a point of care diagnostic system.

  6. Mur-LH, the Broad-Spectrum Endolysin of Lactobacillus helveticus Temperate Bacteriophage φ-0303

    Science.gov (United States)

    Deutsch, Stéphanie-Marie; Guezenec, Stéphane; Piot, Michel; Foster, Simon; Lortal, Sylvie

    2004-01-01

    φ-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycin C induction. In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a library of φ-0303 in Escherichia coli DH5α. The lytic activity was detected by its expression, using whole cells of the sensitive strain L. helveticus CNRZ 892 as the substrate. The lysin gene was within a 4.1-kb DNA fragment of φ-0303 containing six open reading frames (ORFs) and two truncated ORFs. No sequence homology with holin genes was found within the cloned fragment. An integrase-encoding gene was also present in the fragment, but it was transcribed in a direction opposite that of the lysin gene. The lysin-encoding lys gene was verified by PCR amplification from the total phage DNA and subcloned. The lys gene is a 1,122-bp sequence encoding a protein of 373 amino acids (Mur-LH), whose product had a deduced molecular mass of 40,207 Da. Comparisons with sequences in sequence databases showed homology with numerous endolysins of other bacteriophages. Mur-LH was expressed in E. coli BL21, and by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with L. helveticus CNRZ 892 as the substrate, the recombinant protein showed an apparent molecular mass of 40 kDa. The N-terminal sequence of the protein confirmed the start codon. Hydrolysis of cell walls of L. helveticus CNRZ 303 by the endolysin and biochemical analysis of the residues produced demonstrated that Mur-LH has N-acetylmuramidase activity. Last, the endolysin exhibited a broad spectrum of lytic activity, as it was active on different species, mainly thermophilic lactobacilli but also lactococci, pediococci, Bacillus subtilis, Brevibacterium linens, and Enterococcus faecium. PMID:14711630

  7. Stability and instability in the lysogenic state of phage lambda.

    Science.gov (United States)

    Little, John W; Michalowski, Christine B

    2010-11-01

    Complex gene regulatory circuits exhibit emergent properties that are difficult to predict from the behavior of the components. One such property is the stability of regulatory states. Here we analyze the stability of the lysogenic state of phage λ. In this state, the virus maintains a stable association with the host, and the lytic functions of the virus are repressed by the viral CI repressor. This state readily switches to the lytic pathway when the host SOS system is induced. A low level of SOS-dependent switching occurs without an overt stimulus. We found that the intrinsic rate of switching to the lytic pathway, measured in a host lacking the SOS response, was almost undetectably low, probably less than 10(-8)/generation. We surmise that this low rate has not been selected directly during evolution but results from optimizing the rate of switching in a wild-type host over the natural range of SOS-inducing conditions. We also analyzed a mutant, λprm240, in which the promoter controlling CI expression was weakened, rendering lysogens unstable. Strikingly, the intrinsic stability of λprm240 lysogens depended markedly on the growth conditions; lysogens grown in minimal medium were nearly stable but switched at high rates when grown in rich medium. These effects on stability likely reflect corresponding effects on the strength of the prm240 promoter, measured in an uncoupled assay system. Several derivatives of λprm240 with altered stabilities were characterized. This mutant and its derivatives afford a model system for further analysis of stability.

  8. Drilling in tempered glass – modelling and experiments

    DEFF Research Database (Denmark)

    Nielsen, Jens Henrik

    The present paper reports experimentally and numerically obtained results for the process of drilling in tempered glass. The experimental results are drilling depths on the edge in 19mm tempered glass with a known residual stress state measured by a scattered light polariscope. The experiments have...... been modelled using a state-of-the-art model and compared with satisfying result to the performed experiments. The numerical model has been used for a parametric study, investigating the redistribution of residual stresses during the process of drilling. This is done for investigating the possibility...... of applying forces in such holes and thereby being able to mechanically assemble tempered glass without the need of drilling holes before the tempering process. The paper is the result of currently ongoing research and the results should be treated as so....

  9. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  10. The Lytic SA Phage Demonstrate Bactericidal Activity against Mastitis Causing Staphylococcus aureus

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    Hamza Ameer

    2016-01-01

    Full Text Available Staphylococcus aureus is the major causative agent of mastitis among dairy animals as it causes intramammary gland infection. Due to antibiotic resistance and contamination of antibiotics in the milk of diseased animals; alternative therapeutic agents are required to cure mastitis. Lytic bacteriophages and their gene products can be potential therapeutic agents against bacteria as they are host specific and less harmful than antibiotics. In this study, Staphylococcus aureus were isolated from milk samples of the infected animals and identified biochemically. SA phage was isolated from sewage water showing lytic activity against Staphylococcus aureus isolates. The highest lytic activity of bacteriophages was observed at 37°C and pH 7, and the most suitable storage condition was at 4°C. SA phage efficiently reduced bacterial growth in the bacterial reduction assay. The characterization and bacterial growth reduction activity of the bacteriophages against Staphylococcus aureus signifies their underlying potential of phage therapy against mastitis.

  11. Relative entropy differences in bacterial chromosomes, plasmids, phages and genomic islands

    DEFF Research Database (Denmark)

    Bohlin, Jon; van Passel, Mark W. J.; Snipen, Lars

    2012-01-01

    Background: We sought to assess whether the concept of relative entropy (information capacity), could aid our understanding of the process of horizontal gene transfer in microbes. We analyzed the differences in information capacity between prokaryotic chromosomes, genomic islands (GI), phages...... with the strongest association being in phages. Relative entropy was also found to be lower in the obligate intracellular Mycobacterium leprae than in the related M. tuberculosis when measured on a shared set of highly conserved genes. Conclusions: We argue that relative entropy differences reflect how plasmids......, phages and GIs interact with microbial host chromosomes and that all these biological entities are, or have been, subjected to different selective pressures. The rate at which amelioration of horizontally acquired DNA occurs within the chromosome is likely to account for the small differences between...

  12. Phagekines: Screening Binding Properties and Biological Activity of Functional Cytokines Displayed on Phages.

    Science.gov (United States)

    Rojas, Gertrudis; Carmenate, Tania

    2018-01-01

    The current chapter focuses on the use of filamentous phages to display, modify, and characterize cytokines, which are proteins belonging to a versatile group of essential mediators involved in cell-cell communication. Cytokines exhibit a considerable diversity, both in functions and in structural features underlying their biological effects. A broad variety of cytokines have been successfully displayed on phages, allowing the high-throughput study of their binding properties and biological activities and the discovery of novel therapeutics through directed evolution. The technical singularities and some potential applications of cytokine phage display are illustrated here with the case of Interleukin-2, a prototypic member of the four-alpha-helix bundle cytokine family playing a pivotal role in the immune response and having a long history of therapeutic use.

  13. The factors affecting effectiveness of treatment in phages therapy, mini review

    Directory of Open Access Journals (Sweden)

    Mai Huong eCHATAIN-LY

    2014-02-01

    Full Text Available In recent years, the use of lytic bacteriophages as antimicrobial agents controlling pathogenic bacteria has appeared as a promising new alternative strategy in the face of growing antibiotic resistance which has caused problems in many fields including medicine, veterinary medicine and aquaculture. The use of bacteriophages has numerous advantages over traditional antimicrobials. The effectiveness of phage applications in fighting against pathogenic bacteria depends on several factors such as the bacteriophages/target bacteria ratio, the mode and moment of treatment, environmental conditions (pH, temperature ..., the neutralization of phage and accessibility to target bacteria, amongst others. This report presents these factors and the challenges involved in developing phage therapy applications

  14. Development of a renal collecting duct homing peptide using phage display

    DEFF Research Database (Denmark)

    Svenningsen, Per; Peti-Peterdi, Janos

    Homing peptides are useful for in vivo labeling and nonviral gene transfer to selective tissues and cell types. The aim of this project was to develop a renal collecting duct homing peptide. Using phage display, we identified a phage expressing a cyclic 7 amino acid peptide, which was internalized...... developed a peptide, which can be used to label the collecting duct cells in mice and rats; however, further development is needed in order to use the peptide as a vector for nonviral gene transfer in vivo....... in a collecting duct cell line. Moreover, the phage was internalized in the collecting duct cells after i.v. injection in mice. To test if the peptide could be used for nonviral gene transfer, we synthesized the identified peptide fused to a protamine fragment. The fusion peptide was able to bind plasmid GFP c...

  15. Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd

    NARCIS (Netherlands)

    Verhaert, R.M D; van Duin, J; Quax, Wim

    1999-01-01

    The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (alpha subunit-internal peptide-beta subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the

  16. Identification and Characterization of the Phage Gene sav, Involved in Sensitivity to the Lactococcal Abortive Infection Mechanism AbiV

    DEFF Research Database (Denmark)

    Haaber, Jakob Brandt Borup; Rousseau, G. M.; Hammer, Karin

    2009-01-01

    Lactococcus lactis phage mutants that are insensitive to the recently characterized abortive infection mechanism AbiV were isolated and analyzed in an effort to elucidate factors involved in the sensitivity to AbiV. Whole-genome sequencing of the phage mutants p2.1 and p2.2 revealed mutations...

  17. Salmonellosis in garden birds in Scotland, 1995 to 2008: geographic region, Salmonella enterica phage type and bird species.

    Science.gov (United States)

    Pennycott, T W; Mather, H A; Bennett, G; Foster, G

    2010-04-03

    Salmonellosis was diagnosed in garden birds from 198 incidents in Scotland between September 1995 and August 2008. Salmonellosis was essentially a disease of finches in the north of Scotland, but in the south of Scotland it was also a problem in house sparrows. Almost all of the incidents were caused by Salmonella Typhimurium phage types 40 or 56/variant, but regional variation in phage types was observed. In the north of Scotland, one phage type (DT 40) predominated, but in the south of Scotland two phage types were commonly isolated (DTs 40 and 56/variant, with the latter the more common of the two phage types). This regional difference was statistically significant for salmonellosis in greenfinches, chaffinches and 'other garden birds', but not for house sparrows. Different temporal patterns for different species of bird and different phage types were also observed within regions. These findings suggest that the epidemiology of salmonellosis in garden birds varies depending on the phage type of Salmonella and the species of garden bird, with additional regional differences depending on the wild bird populations and the phage types of Salmonella in circulation. An awareness of these differences will help when formulating guidelines aimed at reducing the impact of salmonellosis in garden birds.

  18. THE CHARACTERIZATION OF DANISH ISOLATES OF SALMONELLA-ENTERICA SEROVAR ENTERITIDIS BY PHAGE TYPING AND PLASMID PROFILING - 1980-1990

    DEFF Research Database (Denmark)

    Brown, D. J.; Baggesen, Dorte Lau; Hansen, H. B.

    1994-01-01

    flocks, quarantined imported poultry, environmental samples from hatchery units, and from bovines. Phage type (PT) 1 was found to be the most common type among isolates of poultry origin (57.6%) followed by PT4 (28.8%). Isolates belonging to PT8 were found exclusively in imported birds. Phage typing...

  19. A Novel Application of Synthetic Biology and Directed Evolution to Engineer Phage-based Antibiotics

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Meiye [Sandia National Lab. (SNL-CA), Livermore, CA (United States)

    2014-09-01

    The emergence of multiple drug resistant bacteria poses threats to human health, agriculture and food safety. Annually over 100,000 deaths and up to $20 billion loss to the U.S. economy are attributed to multiple drug resistant bacteria. With only four new chemical antibiotics in the drug development pipeline, we are in dire need of new solutions to address the emerging threat of multiple drug resistance. We propose a paradigm-changing approach to address the multi-drug resistant bacteria problem by utilizing Synthetic Biology (SynBio) methodologies to create and evolve “designer” bacteriophages or phages – viruses that specifically infect bacteria – to infect and kill newly emerging pathogenic bacterial strains WITHOUT the need for chemical antibiotics. A major advantage of using phage to combat pathogenic bacteria is that phages can co-evolve with their bacterial host, and Sandia can be the first in the world to establish an industrial scale Synthetic Biology pipeline for phage directed evolution for safe, targeted, customizable solution to bacterial drug resistance. Since there is no existing phage directed evolution effort within or outside of Sandia, this proposal is suitable as a high-risk LDRD effort to create the first pipeline for such an endeavor. The high potential reward nature of this proposal will be the immediate impact in decontamination and restoration of surfaces and infrastructure, with longer term impact in human or animal therapeutics. The synthetic biology and screening approaches will lead to fundamental knowledge of phage/bacteria co-evolution, making Sandia a world leader in directed evolution of bacteriophages.

  20. Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model.

    Science.gov (United States)

    Beeton, M L; Alves, D R; Enright, M C; Jenkins, A T A

    2015-08-01

    The Galleria mellonella infection model was used to assess the in vivo efficacy of phage therapy against laboratory and clinical strains of Pseudomonas aeruginosa. In a first series of experiments, Galleria were infected with the laboratory strain P. aeruginosa PAO1 and were treated with varying multiplicity of infection (MOI) of phages either 2h post-infection (treatment) or 2h pre-infection (prevention) via injection into the haemolymph. To address the kinetics of infection, larvae were bled over a period of 24h for quantification of bacteria and phages. Survival rates at 24h when infected with 10 cells/larvae were greater in the prevention versus treatment model (47% vs. 40%, MOI=10; 47% vs. 20%, MOI=1; and 33% vs. 7%, MOI=0.1). This pattern held true when 100 cells/larvae were used (87% vs. 20%, MOI=10; 53% vs. 13%, MOI=1; 67% vs. 7%, MOI=0.1). By 24h post-infection, phages kept bacterial cell numbers in the haemolymph 1000-fold lower than in the non-treated group. In a second series of experiments using clinical strains to further validate the prevention model, phages protected Galleria when infected with both a bacteraemia (0% vs. 85%) and a cystic fibrosis (80% vs. 100%) isolate. Therefore, this study validates the use of G. mellonella as a simple, robust and cost-effective model for initial in vivo examination of P. aeruginosa-targeted phage therapy, which may be applied to other pathogens with similarly low infective doses. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  1. Diversity of the antibody response to tetanus toxoid: comparison of hybridoma library to phage display library.

    Directory of Open Access Journals (Sweden)

    Mahsa Sorouri

    Full Text Available Monoclonal antibodies are important tools in research and since the 1990s have been an important therapeutic class targeting a wide variety of diseases. Earlier methods of mAb production relied exclusively on the lengthy process of making hybridomas. The advent of phage display technology introduced an alternative approach for mAb production. A potential concern with this approach is its complete dependence on an in vitro selection process, which may result in selection of V(H-V(L pairs normally eliminated during the in vivo selection process. The diversity of V(H-V(L pairs selected from phage display libraries relative to an endogenous response is unknown. To address these questions, we constructed a panel of hybridomas and a phage display library using the spleen of a single tetanus toxoid-immunized mouse and compared the diversity of the immune response generated using each technique. Surprisingly, the tetanus toxoid-specific antibodies produced by the hybridoma library exhibited a higher degree of V(H-V(L genetic diversity than their phage display-derived counterparts. Furthermore, the overlap among the V-genes from each library was very limited. Consistent with the notion that accumulation of many small DNA changes lead to increased antigen specificity and affinity, the phage clones displayed substantial micro-heterogeneity. Contrary to previous reports, we found that antigen specificity against tetanus toxoid is encoded by both V(κ and V(H genes. Finally, the phage-derived tetanus-specific clones had a lower binding affinity than the hybridomas, a phenomenon thought to be the result of random pairing of the V-genes.

  2. Phage display-derived binders able to distinguish Listeria monocytogenes from other Listeria species.

    Science.gov (United States)

    Morton, Josephine; Karoonuthaisiri, Nitsara; Charlermroj, Ratthaphol; Stewart, Linda D; Elliott, Christopher T; Grant, Irene R

    2013-01-01

    The objective of this study was to produce phage display-derived binders with the ability to distinguish Listeria monocytogenes from other Listeria spp., which may have potential utility to enhance detection of Listeria monocytogenes. To obtain binders with the desired binding specificity a series of surface and solution phage-display biopannings were performed. Initially, three rounds of surface biopanning against gamma-irradiated L. monocytogenes serovar 4b cells were performed followed by an additional surface biopanning round against L. monocytogenes 4b which included prior subtraction biopanning against gamma-irradiated L. innocua cells. In an attempt to further enhance binder specificity for L. monocytogenes 4b two rounds of solution biopanning were performed, both rounds included initial subtraction solution biopanning against L. innocua. Subsequent evaluations were performed on the phage clones by phage binding ELISA. All phage clones tested from the second round of solution biopanning had higher specificity for L. monocytogenes 4b than for L. innocua and three other foodborne pathogens (Salmonella spp., Escherichia coli and Campylobacter jejuni). Further evaluation with five other Listeria spp. revealed that one phage clone in particular, expressing peptide GRIADLPPLKPN, was highly specific for L. monocytogenes with at least 43-fold more binding capability to L. monocytogenes 4b than to any other Listeria sp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity. L. monocytogenes-specific binders were obtained which could have potential application in novel detection tests for L. monocytogenes, benefiting both the food and medical industries.

  3. Phage display-derived binders able to distinguish Listeria monocytogenes from other Listeria species.

    Directory of Open Access Journals (Sweden)

    Josephine Morton

    Full Text Available The objective of this study was to produce phage display-derived binders with the ability to distinguish Listeria monocytogenes from other Listeria spp., which may have potential utility to enhance detection of Listeria monocytogenes. To obtain binders with the desired binding specificity a series of surface and solution phage-display biopannings were performed. Initially, three rounds of surface biopanning against gamma-irradiated L. monocytogenes serovar 4b cells were performed followed by an additional surface biopanning round against L. monocytogenes 4b which included prior subtraction biopanning against gamma-irradiated L. innocua cells. In an attempt to further enhance binder specificity for L. monocytogenes 4b two rounds of solution biopanning were performed, both rounds included initial subtraction solution biopanning against L. innocua. Subsequent evaluations were performed on the phage clones by phage binding ELISA. All phage clones tested from the second round of solution biopanning had higher specificity for L. monocytogenes 4b than for L. innocua and three other foodborne pathogens (Salmonella spp., Escherichia coli and Campylobacter jejuni. Further evaluation with five other Listeria spp. revealed that one phage clone in particular, expressing peptide GRIADLPPLKPN, was highly specific for L. monocytogenes with at least 43-fold more binding capability to L. monocytogenes 4b than to any other Listeria sp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity. L. monocytogenes-specific binders were obtained which could have potential application in novel detection tests for L. monocytogenes, benefiting both the food and medical industries.

  4. Phage as a molecular recognition element in biosensors immobilized by physical adsorption.

    Science.gov (United States)

    Nanduri, Viswaprakash; Sorokulova, Iryna B; Samoylov, Alexandre M; Simonian, Aleksandr L; Petrenko, Valery A; Vodyanoy, Vitaly

    2007-01-15

    Biosensors based on landscape phages immobilized by physical adsorption on the surface of a quartz crystal microbalance was used for detection of beta-galactosidase from Escherichia coli. The sensor had a detection limit of a few nanomoles and a response time of a approximately 100 s over the range of 0.003-210 nM. The binding dose-response curve had a typical sigmoid shape and the signal was saturated at the beta-galactosidase concentration of about 200 nM. A marked selectivity for beta-galactosidase over BSA was observed in mixed solutions even when the concentration of BSA exceeded the concentration of beta-galactosidase by a factor of approximately 2000. The apparent value of the dissociation constant (K(d)) of the interaction of free phage with beta-galactosidase (9.1+/-0.9 pM) was smaller compared with the one calculated for the bound phage (1.7+/-0.5 nM). The binding was specific with three binding sites needed to bind a single molecule of beta-galactosidase. The K(d) obtained from the enzyme-linked immunosorbent assay (ELISA) for the phage and the monoclonal anti-beta-galactosidase antibodies were 21+/-2 and 26+/-2 nM, respectively. Although the method of physical adsorption is simpler and more economical in comparison with Langmuir-Blodgett and molecular assembling methods the performances of the sensors made by these technologies compare well. This work provides evidence that phage can be used as a recognition element in biosensors using physical adsorption method for immobilization of phage on the sensor surface.

  5. The Agricultural Antibiotic Carbadox Induces Phage-mediated Gene Transfer in Salmonella

    Directory of Open Access Journals (Sweden)

    Bradley L. Bearson

    2014-02-01

    Full Text Available Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the U.S. during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness

  6. Evolution of parasitism and mutualism between filamentous phage M13 and Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jason W. Shapiro

    2016-05-01

    Full Text Available Background. How host-symbiont interactions coevolve between mutualism and parasitism depends on the ecology of the system and on the genetic and physiological constraints of the organisms involved. Theory often predicts that greater reliance on horizontal transmission favors increased costs of infection and may result in more virulent parasites or less beneficial mutualists. We set out to understand transitions between parasitism and mutualism by evolving the filamentous bacteriophage M13 and its host Escherichia coli. Results. The effect of phage M13 on bacterial fitness depends on the growth environment, and initial assays revealed that infected bacteria reproduce faster and to higher density than uninfected bacteria in 96-well microplates. These data suggested that M13 is, in fact, a facultative mutualist of E. coli. We then allowed E. coli and M13 to evolve in replicated environments, which varied in the relative opportunity for horizontal and vertical transmission of phage in order to assess the evolutionary stability of this mutualism. After 20 experimental passages, infected bacteria from treatments with both vertical and horizontal transmission of phage had evolved the fastest growth rates. At the same time, phage from these treatments no longer benefited the ancestral bacteria. Conclusions. These data suggest a positive correlation between the positive effects of M13 on E. coli hosts from the same culture and the negative effects of the same phage toward the ancestral bacterial genotype. The results also expose flaws in applying concepts from the virulence-transmission tradeoff hypothesis to mutualism evolution. We discuss the data in the context of more recent theory on how horizontal transmission affects mutualisms and explore how these effects influence phages encoding virulence factors in pathogenic bacteria.

  7. Error and efficiency of simulated tempering simulations.

    Science.gov (United States)

    Rosta, Edina; Hummer, Gerhard

    2010-01-21

    We derive simple analytical expressions for the error and computational efficiency of simulated tempering (ST) simulations. The theory applies to the important case of systems whose dynamics at long times is dominated by the slow interconversion between two metastable states. An extension to the multistate case is described. We show that the relative gain in efficiency of ST simulations over regular molecular dynamics (MD) or Monte Carlo (MC) simulations is given by the ratio of their reactive fluxes, i.e., the number of transitions between the two states summed over all ST temperatures divided by the number of transitions at the single temperature of the MD or MC simulation. This relation for the efficiency is derived for the limit in which changes in the ST temperature are fast compared to the two-state transitions. In this limit, ST is most efficient. Our expression for the maximum efficiency gain of ST simulations is essentially identical to the corresponding expression derived by us for replica exchange MD and MC simulations [E. Rosta and G. Hummer, J. Chem. Phys. 131, 165102 (2009)] on a different route. We find quantitative agreement between predicted and observed efficiency gains in a test against ST and replica exchange MC simulations of a two-dimensional Ising model. Based on the efficiency formula, we provide recommendations for the optimal choice of ST simulation parameters, in particular, the range and number of temperatures, and the frequency of attempted temperature changes.

  8. The tempered polymerization of human neuroserpin.

    Directory of Open Access Journals (Sweden)

    Rosina Noto

    Full Text Available Neuroserpin, a member of the serpin protein superfamily, is an inhibitor of proteolytic activity that is involved in pathologies such as ischemia, Alzheimer's disease, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB. The latter belongs to a class of conformational diseases, known as serpinopathies, which are related to the aberrant polymerization of serpin mutants. Neuroserpin is known to polymerize, even in its wild type form, under thermal stress. Here, we study the mechanism of neuroserpin polymerization over a wide range of temperatures by different techniques. Our experiments show how the onset of polymerization is dependent on the formation of an intermediate monomeric conformer, which then associates with a native monomer to yield a dimeric species. After the formation of small polymers, the aggregation proceeds via monomer addition as well as polymer-polymer association. No further secondary mechanism takes place up to very high temperatures, thus resulting in the formation of neuroserpin linear polymeric chains. Most interesting, the overall aggregation is tuned by the co-occurrence of monomer inactivation (i.e. the formation of latent neuroserpin and by a mechanism of fragmentation. The polymerization kinetics exhibit a unique modulation of the average mass and size of polymers, which might suggest synchronization among the different processes involved. Thus, fragmentation would control and temper the aggregation process, instead of enhancing it, as typically observed (e.g. for amyloid fibrillation.

  9. Crenarchaeota and Euryarchaeota in temperate estuarine sediments.

    Science.gov (United States)

    Abreu, C; Jurgens, G; De Marco, P; Saano, A; Bordalo, A A

    2001-05-01

    Application of molecular techniques to ecological studies has unveiled a wide diversity of micro-organisms in natural communities, previously unknown to microbial ecologists. New lineages of Archaea were retrieved from several non-extreme environments, showing that these micro-organisms are present in a large variety of ecosystems. The aim was therefore to assess the presence and diversity of Archaea in the sediments of the river Douro estuary (Portugal), relating the results obtained to ecological data. Total DNA was extracted from sediment samples obtained from an estuary deprived of vegetation, amplified by PCR and the resulting DNA fragments cloned. The archaeal origin of the cloned inserts was checked by Southern blot, dot blot or colony blot hybridization. Recombinant plasmids were further analysed by restriction with AvaII and selected for sequencing. Phylogenetic analyses of 14 sequences revealed the presence of members of the domain Archaea. Most of the sequences could be assigned to the kingdom Crenarchaeota. Most of these sequences were closely related to those obtained from non-extreme Crenarchaeota members previously retrieved from diverse ecosystems, such as freshwater and marine environments. The presence of archaeal 16S rDNA sequences in temperate estuarine sediments emerges as a valuable contribution to the understanding of the complexity of the ecosystem.

  10. Influence of tempering temperature on mechanical properties of cast steels

    Directory of Open Access Journals (Sweden)

    G. Golański

    2008-12-01

    Full Text Available The paper presents results of research on the influence of tempering temperature on structure and mechanical properties of bainite hardened cast steel: G21CrMoV4 – 6 (L21HMF and G17CrMoV5 – 10 (L17HMF. Investigated cast steels were taken out from internal frames of steam turbines serviced for long time at elevated temperatures. Tempering of the investigated cast steel was carried out within the temperature range of 690 ÷ 730 C (G21CrMoV4 – 6 and 700 ÷ 740 C (G17CrMoV5 – 10. After tempering the cast steels were characterized by a structure of tempered lower bainite with numerous precipitations of carbides. Performed research of mechanical properties has shown that high temperatures of tempering of bainitic structure do not cause decrease of mechanical properties beneath the required minimum.oo It has also been proved that high-temperature tempering (>720 oC ensures high impact energy at the 20% decrease of mechanical properties.

  11. Dung beetle communities: a neotropical-north temperate comparison.

    Science.gov (United States)

    Radtke, Meghan G; Fonseca, Cláudio R V da; Williamson, G Bruce

    2010-01-01

    Dung beetle communities have been compared across north temperate latitudes. Tropical dung beetle communities appear to be more diverse based on studies using different methodologies. Here, we present results from a standardized sampling protocol used to compare dung beetle communities across five neotropical forests in Brazil and Ecuador and two warm, north temperate forests in Mississippi and Louisiana. Species richness in the tropical forests was three to seven times higher than the temperate forests, as would be expected by studies of other taxa across tropical and temperate latitudes. Average body size in the temperate forests was larger than the tropical forests, as predicted by Bergmann's rule. Dung beetle abundance and volume per trap-day were generally higher in Ecuador than Brazil, and higher in Mississippi than Louisiana, but there were no tropical-temperate differences. Species rank-abundance curves were similar within countries and between countries. Rank-volume distributions indicated a smaller range of beetle body sizes in Ecuador versus Brazil or the USA. Community similarity was high within countries and low between countries. Community differences between Brazil and Ecuador sites may be explained by differences in productivity based on geological age of the soils.

  12. Changing patterns among the subgroups of strains of Staphylococcus aureus of phage group II in Danish hospitals from 1961-91

    DEFF Research Database (Denmark)

    Eriksen, N H; Hartzen, S H; Bangsborg, Jette Marie

    1994-01-01

    During the period 1961-91 a total of 567,635 strains of Staphylococcus aureus from hospitalized patients in Denmark have been characterized according to their antibiotic resistance, site of isolation and phage type. Strains of phage group II (typed by the phages 3A, 3C, 55 and 71) have been analy...

  13. Re-engineering of the PAM1 phage display monoclonal antibody to produce a soluble, versatile anti-homogalacturonan scFv

    DEFF Research Database (Denmark)

    Manfield, I. W.; Bernal Giraldo, Adriana Jimena; Møller, I.

    2006-01-01

    Antibody phage display is an increasingly important alternative method for the production of monoclonal antibodies (mAbs) and involves the expression of antibody fragments (scFvs) at the surface of bacteriophage particles. We have previously used this technique to generate a phage mAb (PAM1phage)...

  14. A Partially Purified Acinetobacter baumannii Phage Preparation Exhibits no Cytotoxicity in 3T3 Mouse Fibroblast Cells.

    Science.gov (United States)

    Henein, Alexandra E; Hanlon, Geoffrey W; Cooper, Callum J; Denyer, Stephen P; Maillard, Jean-Yves

    2016-01-01

    A surge in the level and scale of antibiotic resistance has prompted renewed interest in the application of bacteriophages to treat bacterial infections. However, concerns still exist over their efficacy and safety. Acinetobacter baumannii phage BS46, a member of the family Myoviridae, has previously been shown to be effective in murine models. The cytotoxic effect of this phage was evaluated in mouse fibroblast 3T3 cells using four different assays: trypan blue; staining with Hoechst and propidium iodide; lactate dehydrogenase release; and the MTS assay. The addition of phage concentrations up to 2 × 10(9) pfu/mL showed little to no impact on the viability of 3T3 cells after 24 h exposure using the different assays. This study demonstrates that phage BS46 is non-cytotoxic to 3T3 cells using four different assays and that appropriate quality assurance protocols for phage therapeutics are required.

  15. Phage-host interactions analysis of newly characterized Oenococcus oeni bacteriophages: Implications for malolactic fermentation in wine.

    Science.gov (United States)

    Costantini, Antonella; Doria, Francesca; Saiz, Juan-Carlos; Garcia-Moruno, Emilia

    2017-04-04

    Nowadays, only few phages infecting Oenococcus oeni, the principal lactic acid bacteria (LAB) species responsible for malolactic fermentation (MLF) in wine, have been characterized. In the present study, to better understanding the factors affecting the lytic activity of Oenococcus phages, fifteen O. oeni bacteriophages have been studied in detail, both with molecular and microbiological methods. No correlations were found between genome sizes, type of integrase genes, or morphology and the lytic activity of the 15 tested phages. Interestingly, though phage attack in a wine at the end of alcoholic fermentation seems not to be a problem, it can indeed represent a risk factor for MLF when the alcohol content is low, feature that may be a key point for choosing the appropriate time for malolactic starter inoculation. Additionally, it was observed that some phages genomes bear 2 or 3 types of integrase genes, which point to horizontal gene transfer between O. oeni bacteriophages. Copyright © 2017. Published by Elsevier B.V.

  16. Phage and bacteria support mutual diversity in a narrowing staircase of coexistence

    DEFF Research Database (Denmark)

    Härter, Jan Olaf Mirko; Mitarai, Namiko; Sneppen, Kim

    2014-01-01

    The competitive exclusion principle states that phage diversity M should not exceed bacterial diversity N. By analyzing the steady-state solutions of multistrain equations, we find a new constraint: the diversity N of bacteria living on the same resources is constrained to be M or M+1 in terms of...... arms race will typically favor high growth rate, but a phage that infects two bacterial strains differently can occasionally eliminate the fastest growing bacteria. This context-dependent fitness allows abrupt resetting of the 'Red-Queen's race' and constrains the local diversity....

  17. Stability of Staphylococcus aureus phage ISP after freeze-drying (lyophilization).

    Science.gov (United States)

    Merabishvili, Maia; Vervaet, Chris; Pirnay, Jean-Paul; De Vos, Daniel; Verbeken, Gilbert; Mast, Jan; Chanishvili, Nino; Vaneechoutte, Mario

    2013-01-01

    Staphylococcus aureus phage ISP was lyophilized, using an Amsco-Finn Aqua GT4 freeze dryer, in the presence of six different stabilizers at different concentrations. Stability of the lyophilized phage at 4 °C was monitored up to 37 months and compared to stability in Luria Bertani broth and physiological saline at 4 °C. Sucrose and trehalose were shown to be the best stabilizing additives, causing a decrease of only 1 log immediately after the lyophilization procedure and showing high stability during a 27 month storage period.

  18. War is peace--dispatches from the bacterial and phage killing fields.

    Science.gov (United States)

    Comeau, André M; Krisch, Henry M

    2005-08-01

    Large-scale sequence analyses of phage and bacteria have provided new insights into the diverse and multifaceted interactions of these genomes. Such interactions are important because they determine the partitioning of a large fraction of global biomass. Furthermore, the struggle between phage and bacteria has had a significant impact on the evolution of the biosphere. This competition for resources has created an enormous pool of genetic diversity. Eons of horizontal genetic transfer have permitted the entire biosphere to directly benefit from a bargain-basement source of evolutionary innovation.

  19. Peptide substrate identification for yeast Hsp40 Ydj1 by screening the phage display library

    Directory of Open Access Journals (Sweden)

    Li Jingzhi

    2004-01-01

    Full Text Available We have identified a peptide substrate for molecular chaperone Hsp40 Ydj1 by utilizing the combination of phage display library screening and isothemol titration calirimetry (ITC. The initial peptide substrate screening for Hsp40 Ydj1 has been carried out by utilizing a 7-mer phage display library. The peptide sequences from the bio-panning were synthesized and object to the direct affinity measurement for Hsp40 Ydj1 by isothemol titration calirimetry studies. The peptide which has the measurable affinity with Ydj1 shows enriched hydrophobic residues in the middle of the substrate fragment. The peptide substrate specificity for molecular chaperone Hsp40 has been analyzed.

  20. Isolation and characterization of ZZ1, a novel lytic phage that infects Acinetobacter baumannii clinical isolates

    Directory of Open Access Journals (Sweden)

    Jin Jing

    2012-07-01

    Full Text Available Abstract Background Acinetobacter baumannii, a significant nosocomial pathogen, has evolved resistance to almost all conventional antimicrobial drugs. Bacteriophage therapy is a potential alternative treatment for multidrug-resistant bacterial infections. In this study, one lytic bacteriophage, ZZ1, which infects A. baumannii and has a broad host range, was selected for characterization. Results Phage ZZ1 and 3 of its natural hosts, A. baumanni clinical isolates AB09V, AB0902, and AB0901, are described in this study. The 3 strains have different sensitivities to ZZ1, but they have the same sensitivity to antibiotics. They are resistant to almost all of the antibiotics tested, except for polymyxin. Several aspects of the life cycle of ZZ1 were investigated using the sensitive strain AB09V under optimal growth conditions. ZZ1 is highly infectious with a short latent period (9 min and a large burst size (200 PFU/cell. It exhibited the most powerful antibacterial activity at temperatures ranging from 35°C to 39°C. Moreover, when ZZ1 alone was incubated at different pHs and different temperatures, the phage was stable over a wide pH range (4 to 9 and at extreme temperatures (between 50°C and 60°C. ZZ1 possesses a 100-nm icosahedral head containing double-stranded DNA with a total length of 166,682 bp and a 120-nm long contractile tail. Morphologically, it could be classified as a member of the Myoviridae family and the Caudovirales order. Bioinformatic analysis of the phage whole genome sequence further suggested that ZZ1 was more likely to be a new member of the Myoviridae phages. Most of the predicted ORFs of the phage were similar to the predicted ORFs from other Acinetobacter phages. Conclusion The phage ZZ1 has a relatively broad lytic spectrum, high pH stability, strong heat resistance, and efficient antibacterial potential at body temperature. These characteristics greatly increase the utility of this phage as an antibacterial agent

  1. Isolation and characterization of ZZ1, a novel lytic phage that infects Acinetobacter baumannii clinical isolates.

    Science.gov (United States)

    Jin, Jing; Li, Zhen-Jiang; Wang, Shu-Wei; Wang, Shan-Mei; Huang, De-Hai; Li, Ya-Hui; Ma, Yun-Yun; Wang, Jin; Liu, Fang; Chen, Xiang-Dong; Li, Guang-Xing; Wang, Xiao-Ting; Wang, Zhong-Quan; Zhao, Guo-Qiang

    2012-07-28

    Acinetobacter baumannii, a significant nosocomial pathogen, has evolved resistance to almost all conventional antimicrobial drugs. Bacteriophage therapy is a potential alternative treatment for multidrug-resistant bacterial infections. In this study, one lytic bacteriophage, ZZ1, which infects A. baumannii and has a broad host range, was selected for characterization. Phage ZZ1 and 3 of its natural hosts, A. baumanni clinical isolates AB09V, AB0902, and AB0901, are described in this study. The 3 strains have different sensitivities to ZZ1, but they have the same sensitivity to antibiotics. They are resistant to almost all of the antibiotics tested, except for polymyxin. Several aspects of the life cycle of ZZ1 were investigated using the sensitive strain AB09V under optimal growth conditions. ZZ1 is highly infectious with a short latent period (9 min) and a large burst size (200 PFU/cell). It exhibited the most powerful antibacterial activity at temperatures ranging from 35°C to 39°C. Moreover, when ZZ1 alone was incubated at different pHs and different temperatures, the phage was stable over a wide pH range (4 to 9) and at extreme temperatures (between 50°C and 60°C). ZZ1 possesses a 100-nm icosahedral head containing double-stranded DNA with a total length of 166,682 bp and a 120-nm long contractile tail. Morphologically, it could be classified as a member of the Myoviridae family and the Caudovirales order. Bioinformatic analysis of the phage whole genome sequence further suggested that ZZ1 was more likely to be a new member of the Myoviridae phages. Most of the predicted ORFs of the phage were similar to the predicted ORFs from other Acinetobacter phages. The phage ZZ1 has a relatively broad lytic spectrum, high pH stability, strong heat resistance, and efficient antibacterial potential at body temperature. These characteristics greatly increase the utility of this phage as an antibacterial agent; thus, it should be further investigated.

  2. [Study on phage display technology and target protein screening for active ingredients of traditional Chinese medicines].

    Science.gov (United States)

    Zhang, Fang; Qiu, Zheng

    2013-07-01

    Phage display technology refers to a high-throughput in vitro screening technology for extracting required peptides/ proteins from colonies with mass mutants. Due to its high efficiency, practicability and convenience, it has been widely applied in pharmaceutical research and development, as well as target protein screening for active ingredients of traditional Chinese medicines. Target protein is the binding site of drug molecules in vivo, and good targets are the basis of excellent pharmaceuticals. This article summarizes the advance in studies on the phage display technology and its application in targeted protein screening for active ingredients of Chinese materia medica.

  3. Stability of Staphylococcus aureus phage ISP after freeze-drying (lyophilization.

    Directory of Open Access Journals (Sweden)

    Maia Merabishvili

    Full Text Available Staphylococcus aureus phage ISP was lyophilized, using an Amsco-Finn Aqua GT4 freeze dryer, in the presence of six different stabilizers at different concentrations. Stability of the lyophilized phage at 4 °C was monitored up to 37 months and compared to stability in Luria Bertani broth and physiological saline at 4 °C. Sucrose and trehalose were shown to be the best stabilizing additives, causing a decrease of only 1 log immediately after the lyophilization procedure and showing high stability during a 27 month storage period.

  4. Competition between conjugation and M13 phage infection in Escherichia coli in the absence of selection pressure: a kinetic study.

    Science.gov (United States)

    Wan, Zhenmao; Goddard, Noel L

    2012-10-01

    Inter- and intraspecies horizontal gene transfer enabled by bacterial secretion systems is a powerful mechanism for bacterial genome plasticity. The type IV secretion system of Escherichia coli, encoded by the F plasmid, enables cell-to-cell contact and subsequent DNA transfer known as conjugation. Conjugation is compromised by phage infection that specifically targets the secretion machinery. Hence, the use of phages to regulate the spread of genes, such as acquired antibiotic resistance or as general biosanitation agents, has gained interest. To predict the potential efficacy, the competition kinetics must first be understood. Using quantitative PCR to enumerate genomic loci in a resource-limited batch culture, we quantify the infection kinetics of the nonlytic phage M13 and its impact on conjugation in the absence of selection pressure (isogenic set). Modeling the resulting experimental data reveals the cellular growth rate to be reduced to 60% upon phage infection. We also find a maximum phage infection rate of 3×10(-11) mL phage(-1) min(-1) which is only 1 order of magnitude slower than the maximum conjugation rate (3×10(-10) mL cell(-1) min(-1)), suggesting phages must be in significant abundance to be effective antagonists to horizontal gene transfer. In the regime where the number of susceptible cells (F(+)) and phages are equal upon initial infection, we observe the spread of the conjugative plasmid throughout the cell population despite phage infection, but only at 10% of the uninfected rate. This has interesting evolutionary implications, as even in the absence of selection pressure, cells maintain the ability to conjugate despite phage vulnerability and the associated growth consequences.

  5. Phage-associated mutator phenotype in group A streptococcus.

    Science.gov (United States)

    Scott, Julie; Thompson-Mayberry, Prestina; Lahmamsi, Stephanie; King, Catherine J; McShan, W Michael

    2008-10-01

    Defects in DNA mismatch repair (MMR) occur frequently in natural populations of pathogenic and commensal bacteria, resulting in a mutator phenotype. We identified a unique genetic element in Streptococcus pyogenes strain SF370 that controls MMR via a dynamic process of prophage excision and reintegration in response to growth. In S. pyogenes, mutS and mutL are organized on a polycistronic mRNA under control of a common promoter. Prophage SF370.4 is integrated between the two genes, blocking expression of the downstream gene (mutL) and resulting in a mutator phenotype. However, in rapidly growing cells the prophage excises and replicates as an episome, allowing mutL to be expressed. Excision of prophage SF370.4 and expression of MutL mRNA occur simultaneously during early logarithmic growth when cell densities are low; this brief window of MutL gene expression ends as the cell density increases. However, detectable amounts of MutL protein remain in the cell until the onset of stationary phase. Thus, MMR in S. pyogenes SF370 is functional in exponentially growing cells but defective when resources are limiting. The presence of a prophage integrated into the 5' end of mutL correlates with a mutator phenotype (10(-7) to 10(-8) mutation/generation, an approximately a 100-fold increase in the rate of spontaneous mutation compared with prophage-free strains [10(-9) to 10(-10) mutation/generation]). Such genetic elements may be common in S. pyogenes since 6 of 13 completed genomes have related prophages, and a survey of 100 strains found that about 20% of them are positive for phages occupying the SF370.4 attP site. The dynamic control of a major DNA repair system by a bacteriophage is a novel method for achieving the mutator phenotype and may allow the organism to respond rapidly to a changing environment while minimizing the risks associated with long-term hypermutability.

  6. Phage-encoded colanic acid-degrading enzyme permits lytic phage infection of a capsule-forming resistant mutant Escherichia coli strain.

    Science.gov (United States)

    Kim, Min Soo; Kim, Young Deuk; Hong, Sung Sik; Park, Kwangseo; Ko, Kwan Soo; Myung, Heejoon

    2015-02-01

    In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Concentration of MS2 phage in river water by a combined ferric colloid adsorption and foam separation-based method, with MS2 phage leaching from ferric colloid.

    Science.gov (United States)

    Suzuki, Yoshihiro; Kobayashi, Takumi; Nishiyama, Masateru; Kono, Tomoya

    2016-08-01

    The concentration of MS2 phage as a model RNA virus in river water using a combined ferric colloid adsorption and foam separation-based method was examined. The MS2 phage concentrations were determined by the plaque-forming unit (PFU) method and reverse transcription quantitative PCR (RT-qPCR) analysis. When ferric colloid adsorption was performed prior to foam separation, MS2 phage was effectively removed from river water and concentrated in the generated foam within 7 min. The removal efficiency was >99% at the optimum iron and casein concentrations of 5 mg-Fe/L and 10 mg/L, respectively. Furthermore, based on the analysis of the collected ferric colloid dissolved using deferoxamine, the MS2 concentration in the colloid-dissolved solution was 190-fold higher than that found in raw water according to RT-qPCR analysis. This is a novel method for concentrating RNA viruses to facilitate their detection in river water using coagulation and foam separation combined with chelate dissolution of ferric flocs. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  8. Landscape phenology of Wisconsin's temperate mixed forest

    Science.gov (United States)

    Liang, Liang

    This dissertation covers an intensive study of the landscape phenology of Wisconsin's temperate mixed forest. It endeavors to connect conventional plant phenology study back to its ecological complexity (from gardens/trees to the forest) and to compare field-observed phenology with remotely sensed phenology for regional to global monitoring/forecasting applications (from the forest to biomes). A new research perspective: landscape phenology (LP) is proposed in this dissertation. LP is defined as an approach to seasonal vegetation dynamics that integrates spatial patterns and temporal processes within heterogeneous environment across multiple scales. High density in situ observations, remote sensing data, and spatio-temporal analysis are employed for understanding patterns and processes within the complexity of seasonal landscape dynamics. In particular, bi-daily spring forest phenologies of multiple tree/shrub species and understory plants were observed using field protocols or digital photography; high-frequency micrometeorological measurements were used in tandem with LiDAR-based microtopography/canopy heights as well as soil condition data, to characterize microenvironments; and high-resolution, multi-temporal satellite images were employed to facilitate plant community delineation and landscape scaling. A hierarchical upscaling approach is introduced, aiming to integrate in situ phenological observations with the remotely sensed phenological measures. Primary results from this work include: a detailed account of spatio-temporal variations of spring plant phenology and their environmental drivers within a typical seasonal forest; thermal time (accumulated growing degree hours) driven linear phenological models for six forest species; a landscape-level phenological progression regime driven by antecedent weather fluctuations; a conceptual landscape phenology model that assigns phenological behaviors to levels of population, community, and ecosystem patch; and a

  9. Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries.

    Science.gov (United States)

    Huang, Renhua; Fang, Pete; Kay, Brian K

    2012-09-01

    Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded two- to sevenfold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99-100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound two- to fourfold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell

  11. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies

    Science.gov (United States)

    Mu, Xihui; Tong, Zhaoyang; Huang, Qibin; Liu, Bing; Liu, Zhiwei; Hao, Lanqun; Dong, Hua; Zhang, Jinping; Gao, Chuan

    2016-01-01

    Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL) technique, Staphylococcus protein A (SPA) functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb) as magnetic capturing probes, and Ru(bpy)32+-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD) was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy)32+-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy)32+-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility. PMID:26927130

  12. Replicating phages in the epidermal mucosa of the eel (Anguilla anguilla

    Directory of Open Access Journals (Sweden)

    Miguel eCarda Diéguez

    2015-01-01

    Full Text Available In this work, we used the eel (Anguilla anguilla as an animal model to test the hypothesis of Barr et al. about the putative role of the epidermal mucosa as a phage enrichment layer. To this end, we analyzed the microbial content of the skin mucus of wild and farmed eels by using a metagenomic approach. We found a great abundance of replicating phages (concatemers in all the samples. They were assembled in four complete genomes of three Myovirus and one Podovirus. We also found evidences that ΦKZ and Podovirus phages could be part of the resident microbiota associated to the eel mucosal surface and persist on them over the time. Moreover, the viral abundance estimated by epiflorescent counts and by metagenomic recruitment from eel mucosa was higher than that of the surrounding water. Taken together, our results support the hypothesis that claims a possible role of phages in the animal mucus as agents controlling bacterial populations, including pathogenic species, providing a kind of innate immunity.

  13. Genome sequence of the Bacteroides fragilis phage ATCC 51477-B1.

    Science.gov (United States)

    Hawkins, Shawn A; Layton, Alice C; Ripp, Steven; Williams, Dan; Sayler, Gary S

    2008-08-18

    The genome of a fecal pollution indicator phage, Bacteroides fragilis ATCC 51477-B1, was sequenced and consisted of 44,929 bases with a G+C content of 38.7%. Forty-six putative open reading frames were identified and genes were organized into functional clusters for host specificity, lysis, replication and regulation, and packaging and structural proteins.

  14. Complete Genome Sequence of Bacillus cereus Group Phage TsarBomba

    OpenAIRE

    Erill, Ivan; Caruso, Steven M.

    2015-01-01

    The Bacillus cereus group bacteriophage TsarBomba, a double-stranded DNA Myoviridae, was isolated from soil collected in Saratov, Russia. TsarBomba was found to be similar to Bacillus phages BCP78 and BCU4, and to have a wide host range among Bacillus cereus group species.

  15. [Research on utilization of phage display technology to screen chloramine ketone simulated surface point].

    Science.gov (United States)

    Xiong, Yonghua; Chen, Xuelan; Liu, Xiaoxing

    2010-09-01

    Utilize phage display technology to screen chloramine ketone simulated surface point, providing the basis for establishing drug-free ELISA test system. Use the anti chloramine ketone monoclonal antibody as the ligand, and screen the Ph.D. -7(TM) phage display peptide library to select the chloramine ketone simulated surface point. After 4 rounds of screening, select the phase, which can combine the chloramines ketone monoclonal antibody with different degrees, and further, determine the positive clone with competitive inhibition of the hydrochloric chloramine ketone by the indirect competitive ELISA method. It is discovered that all the 10 phages can combine the chloramine ketone monoclonal antibody with different degrees. Use the indirect competitive ELISA method to determine, the phenomenon that 5 positive clones, among them, have competitive inhibition with the hydrochloric chloramine ketone exists. The sequencing results of those 5 positive clones show that 4 different amino acid sequenced are obtained. Tentatively judge these 4 phages display the chloramine ketone simulated antigen surface points.

  16. Inactivation of Phage MS2 by Iron-Aided Titanium Dioxide Photocatalysis

    OpenAIRE

    Sjogren, Jon C.; Sierka, Raymond A.

    1994-01-01

    Near-UV photocatalytic disinfection was accomplished in aqueous titanium dioxide suspensions. A level of inactivation of phage MS2 of 90% increased to 99.9% after 2 μM ferrous sulfate was added. Hydroxyl radical oxidation, with Fenton reaction enhancement, is suspected to be primarily responsible for the viral degradation observed.

  17. Real-time imaging of DNA ejection from single phage particles.

    Science.gov (United States)

    Mangenot, Stéphanie; Hochrein, Marion; Rädler, Joachim; Letellier, Lucienne

    2005-03-08

    Infection by tailed dsDNA phages is initiated by release of the viral DNA from the capsid and its polarized injection into the host. The driving force for the genome transport remains poorly defined. Among many hypothesis [1], it has been proposed that the internal pressure built up during packaging of the DNA in the capsid is responsible for its injection [2-4]. Whether the energy stored during packaging is sufficient to cause full DNA ejection or only to initiate the process was tested on phage T5 whose DNA (121,400 bp) can be released in vitro by mere interaction of the phage with its E. coli membrane receptor FhuA [5-7]. We present a fluorescence microscopy study investigating in real time the dynamics of DNA ejection from single T5 phages adsorbed onto a microfluidic cell. The ejected DNA was fluorescently stained, and its length was measured at different stages of the ejection after being stretched in a hydrodynamic flow. We conclude that DNA release is not an all-or-none process but occurs in a stepwise fashion and at a rate reaching 75,000 bp/sec. The relevance of this stepwise ejection to the in vivo DNA transfer is discussed.

  18. Insights of Phage-Host Interaction in Hypersaline Ecosystem through Metagenomics Analyses

    NARCIS (Netherlands)

    Motlagh, Amir Mohaghegh; Bhattacharjee, Ananda S; Coutinho, Felipe H; Dutilh, Bas E; Casjens, Sherwood R; Goel, Ramesh K

    2017-01-01

    Bacteriophages, as the most abundant biological entities on Earth, place significant predation pressure on their hosts. This pressure plays a critical role in the evolution, diversity, and abundance of bacteria. In addition, phages modulate the genetic diversity of prokaryotic communities through

  19. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo

    NARCIS (Netherlands)

    Dolk, E.; Vaart, M. van der; Lutje Hulsik, D.; Vriend, G.; Haard, H. de; Spinelli, S.; Cambillau, C.; Frenken, L.; Verrips, T.

    As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama

  20. Intravitreal injection of a chimeric phage endolysin Ply187 protects mice from Staphylococcus aureus endophthalmitis

    Science.gov (United States)

    Objectives: The treatment of endophthalmitis is becoming very challenging due to the emergence of multidrug-resistant bacteria. Hence, the development of novel therapeutic alternatives for ocular use is essential. Here, we evaluated the therapeutic potential of Ply187AN-KSH3b, a chimeric phage endol...

  1. Intravitreal injection of the chimeric phage endolysin Ply187 protects mice from Staphylococcus aureus endophthalmitis.

    Science.gov (United States)

    Singh, Pawan Kumar; Donovan, David M; Kumar, Ashok

    2014-08-01

    The treatment of endophthalmitis is becoming very challenging due to the emergence of multidrug-resistant bacteria. Hence, the development of novel therapeutic alternatives for ocular use is essential. Here, we evaluated the therapeutic potential of Ply187AN-KSH3b, a chimeric phage endolysin derived from the Ply187 prophage, in a mouse model of Staphylococcus aureus endophthalmitis. Our data showed that the chimeric Ply187 endolysin exhibited strong antimicrobial activity against both methicillin-sensitive S. aureus and methicillin-resistant S. aureus (MRSA) strains, as evidenced by MIC determinations, reductions in turbidity, and disruption of biofilms. Moreover, exposure of S. aureus to Ply187 for up to 10 generations did not lead to resistance development. The intravitreal injection of chimeric Ply187 (at 6 or 12 h postinfection) significantly improved the outcome of endophthalmitis, preserved retinal structural integrity, and maintained visual function as assessed by electroretinogram analysis. Furthermore, phage lysin treatment significantly reduced the bacterial burden and the levels of inflammatory cytokines and neutrophil infiltration in the eyes. These results indicate that the intravitreal administration of a phage lytic enzyme attenuates the development of bacterial endophthalmitis in mice. To the best of our knowledge, this is the first study demonstrating the therapeutic use of phage-based antimicrobials in ocular infections. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Use of a Regression Model to Study Host-Genomic Determinants of Phage Susceptibility in MRSA

    DEFF Research Database (Denmark)

    Zschach, Henrike; Larsen, Mette V; Hasman, Henrik

    2018-01-01

    Staphylococcus aureus is a major agent of nosocomial infections. Especially in methicillin-resistant strains, conventional treatment options are limited and expensive, which has fueled a growing interest in phage therapy approaches. We have tested the susceptibility of 207 clinical S. aureus...

  3. Contributions of supercoiling to Tn3 resolvase and phage Mu Gin site-specific recombination

    NARCIS (Netherlands)

    K.R. Benjamin (Kirsten); M.T.B. Abola; R. Kanaar (Roland); N.R. Cozzarelli (Nicholas)

    1996-01-01

    textabstractMembers of the resolvase/invertase family of site-specific recombinases require supercoiled substrates containing two recombination sites. To dissect the roles of supercoiling in recombination by the Tn3 and γδ resolvases and the phage Mu Gin invertase, we used substrates that provided

  4. Genome Sequence of Jumbo Phage vB_AbaM_ME3 of Acinetobacter baumanni.

    Science.gov (United States)

    Buttimer, Colin; O'Sullivan, Lisa; Elbreki, Mohamed; Neve, Horst; McAuliffe, Olivia; Ross, R Paul; Hill, Colin; O'Mahony, Jim; Coffey, Aidan

    2016-08-25

    Bacteriophage (phage) vB_AbaM_ME3 was previously isolated from wastewater effluent using the propagating host Acinetobacter baumannii DSM 30007. The full genome was sequenced, revealing it to be the largest Acinetobacter bacteriophage sequenced to date with a size of 234,900 bp and containing 326 open reading frames (ORFs). Copyright © 2016 Buttimer et al.

  5. Genome sequence of jumbo phage vB_AbaM_ME3 of Acinetobacter baumanni

    OpenAIRE

    Buttimer, Colin; O'Sullivan, Lisa; Elbreki, Mohamed; Neve, Horst; McAuliffe, Olivia; Ross, R. Paul; Hill, Colin; O'Mahony, Jim; Coffey, Aidan

    2016-01-01

    Bacteriophage (phage) vB_AbaM_ME3 was previously isolated from wastewater effluent using the propagating host Acinetobacter baumannii DSM 30007. The full genome was sequenced, revealing it to be the largest Acinetobacter bacteriophage sequenced to date with a size of 234,900?bp and containing 326 open reading frames (ORFs).

  6. Characterization of Campylobacter phages including analysis of host range by selected Campylobacter Penner serotypes

    DEFF Research Database (Denmark)

    Hansen, Vinni; Rosenquist, Hanne; Baggesen, Dorte Lau

    2007-01-01

    (PFGE) and restriction endonuclease analysis (REA) were used to characterize the phage genomes. Three categories of bacteriophages were observed. I: a genome size of similar to 194 kb and refractory to digestion with HhaI; II: a genome size of similar to 140 kb and digestible by HhaI; and III: a genome...

  7. Identification of Novel Single Chain Fragment Variable Antibodies Against TNF-α Using Phage Display Technology.

    Science.gov (United States)

    Alizadeh, Ali Akbar; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

    2015-12-01

    Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Because of central role of TNF-α in pathogenesis of inflammatory diseases, in the current study, we aimed to identify novel scFv antibodies against TNF-α using phage display technology. Using libraries composed of phagemid displaying scFv antibodies, four rounds of biopanning against TNF-α were carried out, which led to identification of scFvs capable of binding to TNF-α. The scFv antibody with appropriate binding affinity towards TNF-α, was amplified and used in ELISA experiment. Titration of phage achieved from different rounds of biopanning showed an enrichment of specific anti-TNF-α phages during biopanning process. Using ELISA experiment, a binding constant (Kd) of 1.11 ± 0.32 nM was determined for the phage displaying J48 scFv antibody. The findings in the current work revealed that the identified novel scFv antibody displayed at the N-terminal of minor coat proteins of phagemid binds TNF-α with suitable affinity. However, the soluble form of the antibody is needed to be produced and evaluated in more details regarding its binding properties to TNF-α.

  8. The in-feed antibiotic carbadox induces phage gene transcription in the swine gut microbiome

    Science.gov (United States)

    Carbadox is a quinoxaline-di-N-oxide antibiotic fed to over 40 percent of young pigs in the U.S. and has been shown to induce phage DNA transduction in vitro; however, the effects of carbadox on swine microbiome functions are poorly understood. We investigated the in vivo effects of carbadox on swin...

  9. Display technology on filamentous phage in the search for anti-infective biological agents

    Directory of Open Access Journals (Sweden)

    Nelson Santiago Vispo

    2016-01-01

    Full Text Available Introduction: The causes of antibiotic resistance are complex. The phage display technology has been used mainly to produce monoclonal antibodies (MAbs and peptides directed against cancer or inflammatory disease targets. Today, this technology is recognized as a powerful tool for selecting novel peptides and antibodies that can bind to a wide range of antigens, ranging from whole cells to proteins and lipid targets. In this review, we highlight research that exploits the phage display technology to discover new drugs against infectious diseases, with a focus on antimicrobial peptides and antibodies. Methods: Basic and recent literature review was made, mainly focused on general aspects of phage display technology and the application in the search of new peptides or antibodies of pharmaceutical use to combat the infectious diseases transmitted by bacteria and virus. Results: Updated information on the selected topics is shown, with a guiding and practical approach aimed at researchers in the field of molecular biology to continue deepening the technology with special emphasis in the applications that have been developed in Cuba. Conclusions: Advances in methods of screening, manufacturing, and humanization technologies show that phage display technology can significantly contribute in the fight against clinically important pathogens.

  10. A century of phage research: bacteriophages and the shaping of modern biology.

    Science.gov (United States)

    Keen, Eric C

    2015-01-01

    2015 marks the centennial of the discovery of bacteriophages, viruses that infect bacteria. Phages have been central to some of biology's most meaningful advances over the past hundred years (shown here); they greatly influence the workings of the biosphere, and are poised to play expanded roles in biomedicine, biotechnology, and ecology. © 2015 WILEY Periodicals, Inc.

  11. Effects of salts on internal DNA pressure and mechanical properties of phage capsids

    NARCIS (Netherlands)

    Evilevitch, Alex; Roos, Wouter H.; Ivanovska, Irena L.; Jeembaeva, Meerim; Jönsson, Bengt; Wuite, Gijs J.L.

    2011-01-01

    Based on atomic force microscopy nanoindentation measurements of phage λ, we previously proposed a minimal model describing the effect of water hydrating DNA that strengthens viral capsids against external deformation at wild-type DNA packing density. Here, we report proof of this model by testing

  12. Complete nucleotide sequence of a new filamentous phage, Xf109, which integrates its genome into the chromosomal DNA of Xanthomonas oryzae.

    Science.gov (United States)

    Yeh, Ting Y

    2017-02-01

    Unlike Ff-like coliphages, certain filamentous Inoviridae phages integrate their genomes into the host chromosome and enter a prophage state in their infectious cycle. This lysogenic life cycle was first reported for Xanthomonas citri Cf phage. However, except for the X. citri phages Cf and XacF1, complete genome sequence information about lysogenic Xanthomonas phages is not available to date. A proviral sequence of Xf109 phage was identified in the genome of Xanthomonas oryzae, the rice bacterial blight pathogen, and revived as infectious virions to lysogenize its host de novo. The genome of Xf109 phage is 7190 nucleotides in size and contains 12 predicted open reading frames in an organization similar to that of the Cf phage genome. Seven of the Xf109 proteins show significant sequence similarity to Cf and XacF1 phage proteins, while its ORF4 shares 92 % identity with the major coat protein of X. phage oryzae Xf. Integration of Xf109 phage DNA into the host genome is site-specific, and the attP/attB sequence contains the dif core sequence 5'-TATACATTATGCGAA-3', which is identical to that of Cf, XacF1, and Xanthomonas campestris phage ϕLf. To my knowledge, this is the first complete genome sequence of a filamentous bacteriophage that infects X. oryzae.

  13. Effectiveness of a Lytic Phage SRG1 against Vancomycin-Resistant Enterococcus faecalis in Compost and Soil

    Directory of Open Access Journals (Sweden)

    Sidra Rahmat Ullah

    2017-01-01

    Full Text Available Nosocomial infections caused by vancomycin-resistant Enterococcus have become a major problem. Bacteriophage therapy is proposed as a potential alternative therapy. Bacteriophages are viruses that infect bacteria and are ubiquitous in nature. Lytic bacteriophage was isolated from sewage water that infects VREF, the causative agent of endocarditis, bacteraemia, and urinary tract infections (UTIs. The phage produced clear plaques with unique clear morphology and well-defined boundaries. TEM results of phage revealed it to be 108±0.2 nm long and 90±0.5 nm wide. The characterization of bacteriophage revealed that infection process of phage was calcium and magnesium dependent and phage titers were highest under optimum conditions for VREF, with an optimal temperature range of 37–50°C. The maximum growth was observed at 37°C, hence having 100% viability. The latent period for phage was small with a burst size of 512 viral particles per bacterial cell. The phage was tested against various clinical strains and results proved it to be host specific. It can be used as a potential therapeutic agent for VREF infections. The phage efficiently eradicated VREF inoculated in cattle compost, poultry compost, and a soil sample which makes it a potential agent for clearing compost and soil sample.

  14. Assessment of the effects of various UV sources on inactivation and photoproduct induction in phage T7 dosimeter.

    Science.gov (United States)

    Fekete, A; Vink, A A; Gaspar, S; Berces, A; Modos, K; Ronto, G; Roza, L

    1998-10-01

    The correlation between the biologically effective dose (BED) of a phage T7 biological dosimeter and the induction of cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts ((6-4)PD) in the phage DNA was determined using seven various UV sources. The BED is the inactivation rate of phage T7 expressed in HT7 units. The CPD and (6-4)PD were determined by lesion-specific monoclonal antibodies in an immunodot-blot assay. The various lamps induced these lesions at different rates; the relative induction ratios of CPD to (6-4)PD increased with increasing effective wavelength of irradiation source. The amount of total adducts per phage was compared to the BED of phage T7 dosimeter, representing the average number of UV lesions in phage. For UVC (200-280 nm radiation) and unfiltered TL01 the number of total adducts approximates the reading; however, UV sources having longer effective wavelengths produced fewer CPD and (6-4)PD. A possible explanation is that although the most relevant lesions by UVC are the CPD and (6-4)PD, at longer wavelengths other photoproducts can contribute to the lethal damage of phages. The results emphasize the need to study the biological effects of solar radiation because the lesions responsible for the lethal effect may be different from those produced by various UV sources.

  15. Clinical Antibiotic-resistant Acinetobacter baumannii Strains with Higher Susceptibility to Environmental Phages than Antibiotic-sensitive Strains.

    Science.gov (United States)

    Chen, Li-Kuang; Kuo, Shu-Chen; Chang, Kai-Chih; Cheng, Chieh-Chen; Yu, Pei-Ying; Chang, Chih-Hui; Chen, Tren-Yi; Tseng, Chun-Chieh

    2017-07-24

    Antibiotic-resistant Acinetobacter baumannii is associated with nosocomial infections worldwide. Here, we used clinically isolated A. baumannii strains as models to demonstrate whether antibiotic resistance is correlated with an increased susceptibility to bacteriophages. In this study, 24 active phages capable of infecting A. baumannii were isolated from various environments, and the susceptibilities of both antibiotic-sensitive and antibiotic-resistant strains of A. baumannii to different phages were compared. In our study, a total of 403 clinically isolated A. baumannii strains were identified. On average, the phage infection percentage of the antibiotic-resistant A. baumannii strains was 84% (from 81-86%), whereas the infection percentage in the antibiotic-sensitive A. baumannii strains was only 56.5% (from 49-64%). In addition, the risk of phage infection for A. baumannii was significantly increased in the strains that were resistant to at least four antibiotics and exhibited a dose-dependent response (p-trend baumannii isolates, 75.6% were phage typeable. The results of phage typing might also reveal the antibiotic-resistant profiles of clinical A. baumannii strains. In conclusion, phage susceptibility represents an evolutionary trade-off in A. baumannii strains that show adaptations for antibiotic resistance, particularly in medical environments that have high antibiotic use.

  16. Phage Types and Genotypes of Shiga Toxin-Producing Escherichia coli O157:H7 Isolates from Humans and Animals in Spain: Identification and Characterization of Two Predominating Phage Types (PT2 and PT8)

    Science.gov (United States)

    Mora, Azucena; Blanco, Miguel; Blanco, Jesús E.; Alonso, M. Pilar; Dhabi, Ghizlane; Thomson-Carter, Fiona; Usera, Miguel A.; Bartolomé, Rosa; Prats, Guillermo; Blanco, Jorge

    2004-01-01

    Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused

  17. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

    Directory of Open Access Journals (Sweden)

    Shigeyuki Yamaguchi

    Full Text Available The production of cells capable of expressing gene(s of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

  18. A Genotypic Analysis of Five P. aeruginosa Strains after Biofilm Infection by Phages Targeting Different Cell Surface Receptors

    Directory of Open Access Journals (Sweden)

    Diana P. Pires

    2017-06-01

    Full Text Available Antibiotic resistance constitutes one of the most serious threats to the global public health and urgently requires new and effective solutions. Bacteriophages are bacterial viruses increasingly recognized as being good alternatives to traditional antibiotic therapies. In this study, the efficacy of phages, targeting different cell receptors, against Pseudomonas aeruginosa PAO1 biofilm and planktonic cell cultures was evaluated over the course of 48 h. Although significant reductions in the number of viable cells were achieved for both cases, the high level of adaptability of the bacteria in response to the selective pressure caused by phage treatment resulted in the emergence of phage-resistant variants. To further investigate the genetic makeup of phage-resistant variants isolated from biofilm infection experiments, some of these bacteria were selected for phenotypic and genotypic characterization. Whole genome sequencing was performed on five phage-resistant variants and all of them carried mutations affecting the galU gene as well as one of pil genes. The sequencing analysis further revealed that three of the P. aeruginosa PAO1 variants carry large deletions (>200 kbp in their genomes. Complementation of the galU mutants with wild-type galU in trans restored LPS expression on the bacterial cell surface of these bacterial strains and rendered the complemented strains to be sensitive to phages. This provides unequivocal evidence that inactivation of galU function was associated with resistance to the phages that uses LPS as primary receptors. Overall, this work demonstrates that P. aeruginosa biofilms can survive phage attack and develop phage-resistant variants exhibiting defective LPS production and loss of type IV pili that are well adapted to the biofilm mode of growth.

  19. Removal of phages and viral pathogens in a full-scale MBR: Implications for wastewater reuse and potable water.

    Science.gov (United States)

    Purnell, Sarah; Ebdon, James; Buck, Austen; Tupper, Martyn; Taylor, Huw

    2016-09-01

    The aim of this study was to demonstrate how seasonal variability in the removal efficacy of enteric viral pathogens from an MBR-based water recycling system might affect risks to human health if the treated product were to be used for the augmentation of potable water supplies. Samples were taken over a twelve month period (March 2014-February 2015), from nine locations throughout a water recycling plant situated in East London and tested for faecal indicator bacteria (thermotolerant coliforms, intestinal enterococci n = 108), phages (somatic coliphage, F-specific RNA phage and Bacteroides phage (GB-124) n = 108), pathogenic viruses (adenovirus, hepatitis A, norovirus GI/GII n = 48) and a range of physico-chemical parameters (suspended solids, DO, BOD, COD). Thermotolerant coliforms and intestinal enterococci were removed effectively by the water recycling plant throughout the study period. Significant mean log reductions of 3.9-5.6 were also observed for all three phage groups monitored. Concentrations of bacteria and phages did not vary significantly according to season (P < 0.05; Kruskal-Wallis), though recorded levels of norovirus (GI) were significantly higher during autumn/winter months (P = 0.027; Kruskal-Wallis). Log reduction values for norovirus and adenovirus following MBR treatment were 2.3 and 4.4, respectively. However, both adenovirus and norovirus were detected at low levels (2000 and 3240 gene copies/L, respectively) post chlorination in single samples. Whilst phage concentrations did correlate with viral pathogens, the results of this study suggest that phages may not be suitable surrogates, as viral pathogen concentrations varied to a greater degree seasonally than did the phage indicators and were detected on a number of occasions on which phages were not detected (false negative sample results). Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Characterization and Complete Genome Sequences of Three N4-Like Roseobacter Phages Isolated from the South China Sea.

    Science.gov (United States)

    Li, Baolian; Zhang, Si; Long, Lijuan; Huang, Sijun

    2016-09-01

    Three bacteriophages (RD-1410W1-01, RD-1410Ws-07, and DS-1410Ws-06) were isolated from the surface water of Sanya Bay, northern South China Sea, on two marine bacteria type strains of the Roseobacter lineage. These phages have an isometric head and a short tail, morphologically belonging to the Podoviridae family. Two of these phages can infect four of seven marine roseobacter strains tested and the other one can infect three of them, showing relatively broader host ranges compared to known N4-like roseophages. One-step growth curves showed that these phages have similar short latent periods (1-2 h) but highly variable burst sizes (27-341 pfu cell(-1)). Their complete genomes show high level of similarities to known N4-like roseophages in terms of genome size, G + C content, gene content, and arrangement. The morphological and genomic features of these phages indicate that they belong to the N4likevirus genus. Moreover, comparative genomic analysis based on 43 N4-like phages (10 roseobacter phages and 33 phages infecting other lineages of bacteria) revealed a core genome of 18 genes shared by all the 43 phages and 38 genes shared by all the ten roseophages. The 38 core genes of N4-like roseophages nearly make up 70 % of each genome in length. Phylogenetic analysis based on the concatenated core gene products showed that our phage isolates represent two new phyletic branches, suggesting the broad genetic diversity of marine N4-like roseophages remains.

  1. Transcription Profiling of Bacillus subtilis Cells Infected with AR9, a Giant Phage Encoding Two Multisubunit RNA Polymerases.

    Science.gov (United States)

    Lavysh, Daria; Sokolova, Maria; Slashcheva, Marina; Förstner, Konrad U; Severinov, Konstantin

    2017-02-14

    Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5' ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases.IMPORTANCE Phages regulate the timing of the expression of their own genes to coordinate processes in the infected cell and maximize the release of viral progeny. Phages also alter the levels of host transcripts. Here we present the results of a temporal analysis of the host and viral transcriptomes of Bacillus subtilis infected with a giant phage, AR9. We identify viral promoters recognized by two virus-encoded RNA polymerases that are a unique feature of the phiKZ-related group of phages to which AR9 belongs. Our results set the stage for future analyses of highly unusual RNA polymerases encoded by AR9 and other phiKZ-related phages. Copyright © 2017 Lavysh et al.

  2. PHYSICO CHEMICAL CHARACTERIZATION OF A T5-LIKE SALMONELLA PHAGE ΦSP-3

    Directory of Open Access Journals (Sweden)

    Jeena Augustine

    2014-10-01

    Full Text Available Antimicrobial resistance is a problem faced by humanity for quite some time now. Search for an intelligent alterative to antibiotics led the scientific community to join hands with an age old foe of pathogenic bacteria - bacteriophages. The present study elaborates the potential of a previously isolated phage with biocontrol capability, to survive and adsorb in various physicochemical environments . ΦSP-3, a Salmonella specific lytic phage capable of infecting host in nutrient deprived states with an added advantage of high host specificity and absence of virulence genes as previously reported was chosen for the study. Salmonella Enteritidis was used as host. Basic growth parameters like time taken for phage adsorption, optimal multiplicity of infection (MOI and one step growth curve, were determined, followed by physicochemical characterization. Various parameters studied include temperature, pH, salinity and presence of sugars and CaCl2. 25 minutes of exposure time was required for 100% adsorption and optimal MOI was calculated as one. The latent period and the rise period was 30 minutes each with a subsequent burst size of 60 phages per bacterium. ΦSP-3 could survive upto 70ºC. 1M NaCl was optimal for phage viability while 0.25M favored maximal adsorption. The optimum pH for ΦSP-3 viability and adsorption was 8. Among the sugars,Arabinose was most influential in inactivating ΦSP-3. 100% adsorption was achieved at both 37ºC and at 40ºC. 10mM of CaCl2 to be optimum for ΦSP-3. Thus the present study substantiates the candidature of ΦSP-3 as a sturdy biocontrol agent capable of thriving well in diverse physicochemical environments.

  3. Genome-wide characterization of vibrio phage ϕpp2 with unique arrangements of the mob-like genes

    Directory of Open Access Journals (Sweden)

    Lin Ying-Rong

    2012-06-01

    Full Text Available Abstract Background Vibrio parahaemolyticus is associated with gastroenteritis, wound infections, and septicemia in human and animals. Phages can control the population of the pathogen. So far, the only one reported genome among giant vibriophages is KVP40: 244,835 bp with 26% coding regions that have T4 homologs. Putative homing endonucleases (HE were found in Vibrio phage KVP40 bearing one segD and Vibrio cholerae phage ICP1 carrying one mobC/E and one segG. Results A newly isolated Vibrio phage ϕpp2, which was specific to the hosts of V. parahaemolyticus and V. alginolyticus, featured a long nonenveloped head of ~90 × 150 nm and tail of ~110 nm. The phage can survive at 50°C for more than one hour. The genome of the phage ϕpp2 was sequenced to be 246,421 bp, which is 1587 bp larger than KVP40. 383 protein-encoding genes (PEGs and 30 tRNAs were found in the phage ϕpp2. Between the genomes of ϕpp2 and KVP40, 254 genes including 29 PEGs for viral structure were of high similarity, whereas 17 PEGs of KVP40 and 21 PEGs of ϕpp2 were unmatched. In both genomes, the capsid and tail genes have been identified, as well as the extensive representation of the DNA replication, recombination, and repair enzymes. In addition to the three giant indels of 1098, 1143 and 3330 nt, ϕpp2 possessed unique proteins involved in potassium channel, gp2 (DNA end protector, tRNA nucleotidyltransferase, and mob-type HEs, which were not reported in KVP40. The ϕpp2 PEG274, with strong promoters and translational initiation, was identified to be a mobE type, flanked by NrdA and NrdB/C homologs. Coincidently, several pairs of HE-flanking homologs with empty center were found in the phages of Vibrio phages ϕpp2 and KVP40, as well as in Aeromonas phages (Aeh1 and Ae65, and cyanophage P-SSM2. Conclusions Vibrio phage ϕpp2 was characterized by morphology, growth, and genomics with three giant indels and different types of HEs. The gene analysis on the required

  4. Pseudomonas aeruginosa adaptation to lungs of cystic fibrosis patients leads to lowered resistance to phage and protist enemies

    DEFF Research Database (Denmark)

    Friman, Ville-Petri; Ghoul, Melanie; Molin, Søren

    2013-01-01

    ) patients affects its survival in the presence of natural phage (14/1, ΦKZ, PNM and PT7) and protist (Tetrahymena thermophila and Acanthamoebae polyphaga) enemies. We found that most of the bacteria isolated from relatively recently intermittently colonised patients (1-25 months), were innately phage......-resistant and highly toxic for protists. In contrast, bacteria isolated from long time chronically infected patients (2-23 years), were less efficient in both resisting phages and killing protists. Moreover, chronic isolates showed reduced killing of wax moth larvae (Galleria mellonella) probably due to weaker...... will likely slow down the emergence of beneficial resistance mutations....

  5. Hail resistance of solar collectors with tempered glass covers

    Science.gov (United States)

    Lof, G. O. G.; French, R. R.

    1980-01-01

    The resistance of solar collectors glazed with 3 mm of tempered glass to hailstones of up to 10 cm in diameter and 0.5 kg in weight which fell on Fort Collins, Colorado is discussed. Of the ten solar heating systems directly in the hailpath with tempered glass covers, seven were undamaged, two lost one collector panel each, and a 700-panel collector had seven broken covers, amounting to a total breakage of nine panels out of 956, approximately 1%. In addition, one system with nontempered glass covers suffered two glass punctures in a 26-panel collector. It is concluded that the risk of hail damage to commercial solar collectors glazed with 3-mm tempered glass is negligibly small, and greatly exceeded by the risk of hail damage to the roofs of buildings and automobiles or to fiberglass-reinforced polyester sheets used as collector glazings.

  6. Tempered stable distributions stochastic models for multiscale processes

    CERN Document Server

    Grabchak, Michael

    2015-01-01

    This brief is concerned with tempered stable distributions and their associated Levy processes. It is a good text for researchers interested in learning about tempered stable distributions.  A tempered stable distribution is one which takes a stable distribution and modifies its tails to make them lighter. The motivation for this class comes from the fact that infinite variance stable distributions appear to provide a good fit to data in a variety of situations, but the extremely heavy tails of these models are not realistic for most real world applications. The idea of using distributions that modify the tails of stable models to make them lighter seems to have originated in the influential paper of Mantegna and Stanley (1994). Since then, these distributions have been extended and generalized in a variety of ways. They have been applied to a wide variety of areas including mathematical finance, biostatistics,computer science, and physics.

  7. Diversity and abundance of photosynthetic sponges in temperate Western Australia

    Directory of Open Access Journals (Sweden)

    Brümmer Franz

    2009-02-01

    Full Text Available Abstract Background Photosynthetic sponges are important components of reef ecosystems around the world, but are poorly understood. It is often assumed that temperate regions have low diversity and abundance of photosynthetic sponges, but to date no studies have investigated this question. The aim of this study was to compare the percentages of photosynthetic sponges in temperate Western Australia (WA with previously published data on tropical regions, and to determine the abundance and diversity of these associations in a range of temperate environments. Results We sampled sponges on 5 m belt transects to determine the percentage of photosynthetic sponges and identified at least one representative of each group of symbionts using 16S rDNA sequencing together with microscopy techniques. Our results demonstrate that photosynthetic sponges are abundant in temperate WA, with an average of 63% of sponge individuals hosting high levels of photosynthetic symbionts and 11% with low to medium levels. These percentages of photosynthetic sponges are comparable to those found on tropical reefs and may have important implications for ecosystem function on temperate reefs in other areas of the world. A diverse range of symbionts sometimes occurred within a small geographic area, including the three "big" cyanobacterial clades, Oscillatoria spongeliae, "Candidatus Synechococcus spongiarum" and Synechocystis species, and it appears that these clades all occur in a wide range of sponges. Additionally, spongin-permeating red algae occurred in at least 7 sponge species. This study provides the first investigation of the molecular phylogeny of rhodophyte symbionts in sponges. Conclusion Photosynthetic sponges are abundant and diverse in temperate WA, with comparable percentages of photosynthetic to non-photosynthetic sponges to tropical zones. It appears that there are three common generalist clades of cyanobacterial symbionts of sponges which occur in a wide

  8. Influence of tempering temperature on mechanical properties of cast steels

    OpenAIRE

    G. Golański

    2008-01-01

    The paper presents results of research on the influence of tempering temperature on structure and mechanical properties of bainite hardened cast steel: G21CrMoV4 – 6 (L21HMF) and G17CrMoV5 – 10 (L17HMF). Investigated cast steels were taken out from internal frames of steam turbines serviced for long time at elevated temperatures. Tempering of the investigated cast steel was carried out within the temperature range of 690 ÷ 730 C (G21CrMoV4 – 6) and 700 ÷ 740 C (G17CrMoV5 – 10). After temperin...

  9. Heterogeneity in Induction Level, Infection Ability, and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

    Science.gov (United States)

    Bonanno, Ludivine; Petit, Marie-Agnès; Loukiadis, Estelle; Michel, Valérie

    2016-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event. PMID:26826235

  10. SWFSC/MMTD/CCE: Leatherback Use of Temperate Habitat (LUTH) 2008

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Leatherback Use of Temperate Habitat (LUTH) survey is an ecosystem assessment of temperate foraging habitats of endangered leatherback turtles off the coast of...

  11. Phage Biocontrol of Enteropathogenic and Shiga Toxin-Producing Escherichia coli in Meat Products

    Directory of Open Access Journals (Sweden)

    David Damian Tomat

    2013-06-01

    Full Text Available Ten bacteriophages were isolated from faeces and their lytic effects assayed on 103 pathogenic and non-pathogenic Enterobacteriaceae. Two phages (DT1 and DT6 were selected, based on their host ranges, and their lytic effects on previously characterized virulent strains of bacteria inoculated on pieces of beef were determined. We evaluated the reductions of viable cells (VC of Escherichia coli O157:H7 and non-O157 Shiga toxigenic E. coli (STEC strains on meat after exposure to DT6 at 5 and 24 °C for 3, 6 and 24 h and the effect of both phages against an enteropathogenic E. coli (EPEC strain. Significant VC reductions, compared to controls without phages, at both temperatures were observed, with the greatest decrease taking place within the first hours of the assays. Reductions were also influenced by phage concentration, being the highest concentrations, 1.7x1010 plaque forming units (PFU/mL for DT1 and 1.4x1010 PFU/mL for DT6, the most effective. When EPEC and STEC (O157:H7 strains were tested, we obtained VC reductions of 0.67 log (p=0.01 and 0.77 log (p=0.01 after 3 h incubation and 0.80 log (p=0.01 and 1.15 log (p=0.001 after 6h. In contrast, all the nonpathogenic strains of E. coli as well as the other enterobacteria tested were resistant. In addition, phage cocktail was evaluated on two strains and further biocontrol reductions were observed. However, E. coli bacteriophage insensitive mutants (BIMs emerged in meat assays. BIMs isolated from meat along with those isolated by using the secondary culture method were tested to evaluate resistance phenotype stability and reversion. They presented low emergence frequencies (6.5x10-7 - 1.8x10-6 and variable stability and reversion. Results indicate that isolated phages were stable on storage, negative for all the virulence factors assayed, highly specific for virotypes of E. coli and could be useful in reducing STEC and EPEC VC in meat products.

  12. Enterococcus phages as potential tool for identifying sewage inputs in the Great Lakes region

    Science.gov (United States)

    Vijayavel, K.; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Ebdon, J.; Taylor, H.; Kashian, D.R.

    2014-01-01

    Bacteriophages are viruses living in bacteria that can be used as a tool to detect fecal contamination in surface waters around the world. However, the lack of a universal host strain makes them unsuitable for tracking fecal sources. We evaluated the suitability of two newly isolated Enterococcus host strains (ENT-49 and ENT-55) capable for identifying sewage contamination in impacted waters by targeting phages specific to these hosts. Both host strains were isolated from wastewater samples and identified as E. faecium by 16S rRNA gene sequencing. Occurrence of Enterococcus phages was evaluated in sewage samples (n = 15) from five wastewater treatment plants and in fecal samples from twenty-two species of wild and domesticated animals (individual samples; n = 22). Levels of Enterococcus phages, F + coliphages, Escherichia coli and enterococci were examined from four rivers, four beaches, and three harbors. Enterococcus phages enumeration was at similar levels (Mean = 6.72 Log PFU/100 mL) to F + coliphages in all wastewater samples, but were absent from all non-human fecal sources tested. The phages infecting Enterococcus spp. and F + coliphages were not detected in the river samples (detection threshold < 10 PFU/100 mL), but were present in the beach and harbor samples (range = 1.83 to 2.86 Log PFU/100 mL). Slightly higher concentrations (range = 3.22 to 3.69 Log MPN/100 mL) of E. coli and enterococci when compared to F + coliphages and Enterococcus phages, were observed in the river, beach and harbor samples. Our findings suggest that the bacteriophages associated with these particular Enterococcus host strains offer potentially sensitive and human-source specific indicators of enteric pathogen risk.

  13. Klebsiella Phage ΦK64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types.

    Science.gov (United States)

    Pan, Yi-Jiun; Lin, Tzu-Lung; Chen, Ching-Ching; Tsai, Yun-Ting; Cheng, Yi-Hsiang; Chen, Yi-Yin; Hsieh, Pei-Fang; Lin, Yi-Tsung; Wang, Jin-Town

    2017-03-15

    The genome of the multihost bacteriophage ΦK64-1, capable of infecting Klebsiella capsular types K1, K11, K21, K25, K30, K35, K64, and K69, as well as new capsular types KN4 and KN5, was analyzed and revealed that 11 genes ( S1-1 , S1-2 , S1-3 , S2-1 , S2-2 , S2-3 , S2-4 , S2-5 , S2-6 , S2-7 , and S2-8 ) encode proteins with amino acid sequence similarity to tail fibers/spikes or lyases. S2-5 previously was shown to encode a K64 capsule depolymerase (K64dep). Specific capsule-degrading activities of an additional eight putative capsule depolymerases (S2-4 against K1, S1-1 against K11, S1-3 against K21, S2-2 against K25, S2-6 against K30/K69, S2-3 against K35, S1-2 against KN4, and S2-1 against KN5) was demonstrated by expression and purification of the recombinant proteins. Consistent with the capsular type-specific depolymerization activity of these gene products, phage mutants of S1-2 , S2-2 , S2-3 , or S2-6 lost infectivity for KN4, K25, K35, or K30/K69, respectively, indicating that capsule depolymerase is crucial for infecting specific hosts. In conclusion, we identified nine functional capsule depolymerase-encoding genes in a bacteriophage and correlated activities of the gene products to all ten hosts of this phage, providing an example of type-specific host infection mechanisms in a multihost bacteriophage. IMPORT