Bacteria, phages and septicemia.

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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.

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...... analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were...... examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates...

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

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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.

Stumbling across the same phage

DEFF Research Database (Denmark)

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

2017-01-01

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...... 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...

The Caulobacter crescentus phage phiCbK: genomics of a canonical phage

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Gill Jason J

2012-10-01

Full Text Available Abstract Background The bacterium Caulobacter crescentus is a popular model for the study of cell cycle regulation and senescence. The large prolate siphophage phiCbK has been an important tool in C. crescentus biology, and has been studied in its own right as a model for viral morphogenesis. Although a system of some interest, to date little genomic information is available on phiCbK or its relatives. Results Five novel phiCbK-like C. crescentus bacteriophages, CcrMagneto, CcrSwift, CcrKarma, CcrRogue and CcrColossus, were isolated from the environment. The genomes of phage phiCbK and these five environmental phage isolates were obtained by 454 pyrosequencing. The phiCbK-like phage genomes range in size from 205 kb encoding 318 proteins (phiCbK to 280 kb encoding 448 proteins (CcrColossus, and were found to contain nonpermuted terminal redundancies of 10 to 17 kb. A novel method of terminal ligation was developed to map genomic termini, which confirmed termini predicted by coverage analysis. This suggests that sequence coverage discontinuities may be useable as predictors of genomic termini in phage genomes. Genomic modules encoding virion morphogenesis, lysis and DNA replication proteins were identified. The phiCbK-like phages were also found to encode a number of intriguing proteins; all contain a clearly T7-like DNA polymerase, and five of the six encode a possible homolog of the C. crescentus cell cycle regulator GcrA, which may allow the phage to alter the host cell’s replicative state. The structural proteome of phage phiCbK was determined, identifying the portal, major and minor capsid proteins, the tail tape measure and possible tail fiber proteins. All six phage genomes are clearly related; phiCbK, CcrMagneto, CcrSwift, CcrKarma and CcrRogue form a group related at the DNA level, while CcrColossus is more diverged but retains significant similarity at the protein level. Conclusions Due to their lack of any apparent relationship to

Phage Therapy: Eco-Physiological Pharmacology

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Stephen T. Abedon

2014-01-01

Full Text Available Bacterial virus use as antibacterial agents, in the guise of what is commonly known as phage therapy, is an inherently physiological, ecological, and also pharmacological process. Physiologically we can consider metabolic properties of phage infections of bacteria and variation in those properties as a function of preexisting bacterial states. In addition, there are patient responses to pathogenesis, patient responses to phage infections of pathogens, and also patient responses to phage virions alone. Ecologically, we can consider phage propagation, densities, distribution (within bodies, impact on body-associated microbiota (as ecological communities, and modification of the functioning of body “ecosystems” more generally. These ecological and physiological components in many ways represent different perspectives on otherwise equivalent phenomena. Comparable to drugs, one also can view phages during phage therapy in pharmacological terms. The relatively unique status of phages within the context of phage therapy as essentially replicating antimicrobials can therefore result in a confluence of perspectives, many of which can be useful towards gaining a better mechanistic appreciation of phage therapy, as I consider here. Pharmacology more generally may be viewed as a discipline that lies at an interface between organism-associated phenomena, as considered by physiology, and environmental interactions as considered by ecology.

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

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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.

A Genotypic Analysis of Five P. aeruginosa Strains after Biofilm Infection by Phages Targeting Different Cell Surface Receptors

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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.

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

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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.

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......, studies of the genetic diversity and susceptibility patterns of F. psychrophilum strains and phages isolated in three geographically distinct areas (Chile, Denmark, and USA) showed that the strains and phages clustered into geographically distinct groups. However, cross-infectivity between Chilean phage......-phage. In the third paper, a detailed analysis of the resistance mechanisms in F. psychrophilum and six phage resistant mutants was done. The results revealed unique changes in the genomes in all the phage resistant strains and that some of these changes were related to cell surface properties which were suggested...

Antibody production in response to staphylococcal MS-1 phage cocktail in patients undergoing phage therapy

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Maciej Żaczek

2016-10-01

Full Text Available In this study, we investigated the humoral immune response (through the release of IgG, IgA, and IgM antiphage antibodies to a staphylococcal phage cocktail in patients undergoing experimental phage therapy at the Phage Therapy Unit, Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław, Poland. We also evaluated whether occurring antiphage antibodies had neutralizing properties towards applied phages (K rate. Among 20 examined patients receiving the MS-1 phage cocktail orally and/or locally, the majority did not show a noticeably higher level of antiphage antibodies in their sera during phage administration. Even in those individual cases with an increased immune response, mostly by induction of IgG and IgM, the presence of antiphage antibodies did not translate into unsatisfactory clinical results of phage therapy. On the other hand, a negative outcome of the treatment occurred in some patients who showed relatively weak production of antiphage antibodies before and during treatment. This may imply that possible induction of antiphage antibodies is not an obstacle to the implementation of phage therapy and support our assumption that the outcome of the phage treatment does not primarily depend on the appearance of antiphage antibodies in sera of patients during therapy. These conclusions are in line with our previous findings. The confirmation of this thesis is of great interest as regards the efficacy of phage therapy in humans.

Antibody Production in Response to Staphylococcal MS-1 Phage Cocktail in Patients Undergoing Phage Therapy.

Science.gov (United States)

Żaczek, Maciej; Łusiak-Szelachowska, Marzanna; Jończyk-Matysiak, Ewa; Weber-Dąbrowska, Beata; Międzybrodzki, Ryszard; Owczarek, Barbara; Kopciuch, Agnieszka; Fortuna, Wojciech; Rogóż, Paweł; Górski, Andrzej

2016-01-01

In this study, we investigated the humoral immune response (through the release of IgG, IgA, and IgM antiphage antibodies) to a staphylococcal phage cocktail in patients undergoing experimental phage therapy at the Phage Therapy Unit, Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław, Poland. We also evaluated whether occurring antiphage antibodies had neutralizing properties toward applied phages (K rate). Among 20 examined patients receiving the MS-1 phage cocktail orally and/or locally, the majority did not show a noticeably higher level of antiphage antibodies in their sera during phage administration. Even in those individual cases with an increased immune response, mostly by induction of IgG and IgM, the presence of antiphage antibodies did not translate into unsatisfactory clinical results of phage therapy. On the other hand, a negative outcome of the treatment occurred in some patients who showed relatively weak production of antiphage antibodies before and during treatment. This may imply that possible induction of antiphage antibodies is not an obstacle to the implementation of phage therapy and support our assumption that the outcome of the phage treatment does not primarily depend on the appearance of antiphage antibodies in sera of patients during therapy. These conclusions are in line with our previous findings. The confirmation of this thesis is of great interest as regards the efficacy of phage therapy in humans.

Complete genome sequence of Vibrio anguillarum phage CHOED successfully used for phage therapy in aquaculture

DEFF Research Database (Denmark)

Romero, Jaime; Higuera, Gastón; Gajardo, Felipe

2014-01-01

Vibrio anguillarum phage CHOED was isolated from Chilean mussels. It is a virulent phage showing effective inhibition of V. anguillarum. CHOED has potential in phage therapy, because it can protect fish from vibriosis in fish farms. Here, we announce the completely sequenced genome of V....... anguillarum phage CHOED....

Effects of aspirin on risk and severity of early recurrent stroke after transient ischaemic attack and ischaemic stroke : time-course analysis of randomised trials

NARCIS (Netherlands)

Rothwell, Peter M; Algra, Ale; Chen, Zhengming; Diener, Hans-Christoph; Norrving, Bo; Mehta, Ziyah

2016-01-01

BACKGROUND: Aspirin is recommended for secondary prevention after transient ischaemic attack (TIA) or ischaemic stroke on the basis of trials showing a 13% reduction in long-term risk of recurrent stroke. However, the risk of major stroke is very high for only the first few days after TIA and minor

Unusual case of recurrent SMART (stroke-like migraine attacks after radiation therapy syndrome

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Ramnath Santosh Ramanathan

2016-01-01

Full Text Available Stroke-like migraine attacks after radiation therapy (SMART syndrome is a rare delayed complication of cerebral radiation therapy. A 53-year-old female initially presented with headache, confusion and left homonymous hemianopia. Her medical history was notable for cerebellar hemangioblastoma, which was treated with radiation in 1987. Her initial brain MRI (magnetic resonance imaging revealed cortical enhancement in the right temporo-parieto-occipital region. She improved spontaneously in 2 weeks and follow-up scan at 4 weeks revealed no residual enhancement or encephalomalacia. She presented 6 weeks later with aphasia. Her MRI brain revealed similar contrast-enhancing cortical lesion but on the left side. Repeat CSF studies was again negative other than elevated protein. She was treated conservatively and recovered completely within a week. Before diagnosing SMART syndrome, it is important to rule out tumor recurrence, encephalitis, posterior reversible encephalopathy syndrome (PRES and stroke. Typically the condition is self-limiting, and gradually resolves.

Unusual case of recurrent SMART (stroke-like migraine attacks after radiation therapy) syndrome.

Science.gov (United States)

Ramanathan, Ramnath Santosh; Sreedher, Gayathri; Malhotra, Konark; Guduru, Zain; Agarwal, Deeksha; Flaherty, Mary; Leichliter, Timothy; Rana, Sandeep

2016-01-01

Stroke-like migraine attacks after radiation therapy (SMART) syndrome is a rare delayed complication of cerebral radiation therapy. A 53-year-old female initially presented with headache, confusion and left homonymous hemianopia. Her medical history was notable for cerebellar hemangioblastoma, which was treated with radiation in 1987. Her initial brain MRI (magnetic resonance imaging) revealed cortical enhancement in the right temporo-parieto-occipital region. She improved spontaneously in 2 weeks and follow-up scan at 4 weeks revealed no residual enhancement or encephalomalacia. She presented 6 weeks later with aphasia. Her MRI brain revealed similar contrast-enhancing cortical lesion but on the left side. Repeat CSF studies was again negative other than elevated protein. She was treated conservatively and recovered completely within a week. Before diagnosing SMART syndrome, it is important to rule out tumor recurrence, encephalitis, posterior reversible encephalopathy syndrome (PRES) and stroke. Typically the condition is self-limiting, and gradually resolves.

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.

Current insights into phage biodiversity and biogeography.

Science.gov (United States)

Thurber, Rebecca Vega

2009-10-01

Phages exert tremendous ecological and evolutionary forces directly on their bacterial hosts. Phage induced cell lysis also indirectly contributes to organic and inorganic nutrient recycling. Phage abundance, diversity, and distribution are therefore important parameters in ecosystem function. The assumption that phage consortia are ubiquitous and homogenous across habitats (everything is everywhere) is currently being re-evaluated. New studies on phage biogeography have found that some phages are globally distributed while others are unique and perhaps endemic to specific environments. Furthermore, advances in technology have allowed scientists to conduct experiments aimed at analyzing phage consortia over temporal scales, and surprisingly have found reoccurring patterns. This review discusses currents in the field of phage ecology with particular focus on efforts to characterize phage diversity and biogeography across various spatial and temporal scales.

Recurrent ophthalmoplegia in childhood: diagnostic and etiologic considerations

Energy Technology Data Exchange (ETDEWEB)

Oestergaard, J.R.; Moeller, H.U.; Christensen, T.

1996-06-01

The authors present two patients with recurrent painful ophthalmoplegia starting in early childhood. Clinically, both patients fulfilled the criteria for ophthalmologic migraine. In one case, magnetic resonance investigations were performed following the second attack, between the third and fourth and during the fourth attack. The left third cranial nerve was significantly thickened in its course from the brainstem through the prepontine cistern to the cavernous sinus during the attacks and moderately thickened between the attacks. In the second case, magnetic resonance imaging was performed during the 14th attack, when the oculomotor nerve dysfunction was almost permanent, and the imaging demonstrated a swollen oculomotor nerve. Whether these finding are pathognomonic of ophthalmoplegic migraine awaits further reports using magnetic resonance imaging in infants showing recurrent painful ophthalmoplegia of eraly onset. 17 refs.

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.

HostPhinder: A Phage Host Prediction Tool

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Julia Villarroel

2016-05-01

Full Text Available The current dramatic increase of antibiotic resistant bacteria has revitalised the interest in bacteriophages as alternative antibacterial treatment. Meanwhile, the development of bioinformatics methods for analysing genomic data places high-throughput approaches for phage characterization within reach. Here, we present HostPhinder, a tool aimed at predicting the bacterial host of phages by examining the phage genome sequence. Using a reference database of 2196 phages with known hosts, HostPhinder predicts the host species of a query phage as the host of the most genomically similar reference phages. As a measure of genomic similarity the number of co-occurring k-mers (DNA sequences of length k is used. Using an independent evaluation set, HostPhinder was able to correctly predict host genus and species for 81% and 74% of the phages respectively, giving predictions for more phages than BLAST 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 bacterial hosts. HostPhinder is available as an interactive web service [1] and as a stand alone download from the Docker registry [2].

Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

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Catarina Moreirinha

2018-03-01

Full Text Available The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells.

Assembling filamentous phage occlude pIV channels.

Science.gov (United States)

Marciano, D K; Russel, M; Simon, S M

2001-07-31

Filamentous phage f1 is exported from its Escherichia coli host without killing the bacterial cell. Phage-encoded protein pIV, which is required for phage assembly and secretion, forms large highly conductive channels in the outer membrane of E. coli. It has been proposed that the phage are extruded across the bacterial outer membrane through pIV channels. To test this prediction, we developed an in vivo assay by using a mutant pIV that functions in phage export but whose channel opens in the absence of phage extrusion. In E. coli lacking its native maltooligosacharride transporter LamB, this pIV variant allowed oligosaccharide transport across the outer membrane. This entry of oligosaccharide was decreased by phage production and still further decreased by production of phage that cannot be released from the cell surface. Thus, exiting phage block the pIV-dependent entry of oligosaccharide, suggesting that phage occupy the lumen of pIV channels. This study provides the first evidence, to our knowledge, for viral exit through a large aqueous channel.

Phage inactivation by triplet acetone

International Nuclear Information System (INIS)

Gomes, R.A.

1985-01-01

The exposure of lambda phage to triplet acetone is studied. The triplet acetone is obtained from aerobic oxidation of isobutanal catalysed by peroxidase. A decrease of lambda phage ability to infect Escherichia coli is reported, perhaps, partially due to the possible production of lesions in the phage genome. (M.A.C.) [pt

Characterization of Two Virulent Phages of Lactobacillus plantarum

Science.gov (United States)

Briggiler Marcó, Mariángeles; Garneau, Josiane E.; Tremblay, Denise; Quiberoni, Andrea

2012-01-01

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria. PMID:23042172

Characterizing Phage Genomes for Therapeutic Applications

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Casandra W. Philipson

2018-04-01

Full Text Available Multi-drug resistance is increasing at alarming rates. The efficacy of phage therapy, treating bacterial infections with bacteriophages alone or in combination with traditional antibiotics, has been demonstrated in emergency cases in the United States and in other countries, however remains to be approved for wide-spread use in the US. One limiting factor is a lack of guidelines for assessing the genomic safety of phage candidates. We present the phage characterization workflow used by our team to generate data for submitting phages to the Federal Drug Administration (FDA for authorized use. Essential analysis checkpoints and warnings are detailed for obtaining high-quality genomes, excluding undesirable candidates, rigorously assessing a phage genome for safety and evaluating sequencing contamination. This workflow has been developed in accordance with community standards for high-throughput sequencing of viral genomes as well as principles for ideal phages used for therapy. The feasibility and utility of the pipeline is demonstrated on two new phage genomes that meet all safety criteria. We propose these guidelines as a minimum standard for phages being submitted to the FDA for review as investigational new drug candidates.

Characterizing Phage Genomes for Therapeutic Applications.

Science.gov (United States)

Philipson, Casandra W; Voegtly, Logan J; Lueder, Matthew R; Long, Kyle A; Rice, Gregory K; Frey, Kenneth G; Biswas, Biswajit; Cer, Regina Z; Hamilton, Theron; Bishop-Lilly, Kimberly A

2018-04-10

Multi-drug resistance is increasing at alarming rates. The efficacy of phage therapy, treating bacterial infections with bacteriophages alone or in combination with traditional antibiotics, has been demonstrated in emergency cases in the United States and in other countries, however remains to be approved for wide-spread use in the US. One limiting factor is a lack of guidelines for assessing the genomic safety of phage candidates. We present the phage characterization workflow used by our team to generate data for submitting phages to the Federal Drug Administration (FDA) for authorized use. Essential analysis checkpoints and warnings are detailed for obtaining high-quality genomes, excluding undesirable candidates, rigorously assessing a phage genome for safety and evaluating sequencing contamination. This workflow has been developed in accordance with community standards for high-throughput sequencing of viral genomes as well as principles for ideal phages used for therapy. The feasibility and utility of the pipeline is demonstrated on two new phage genomes that meet all safety criteria. We propose these guidelines as a minimum standard for phages being submitted to the FDA for review as investigational new drug candidates.

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

DEFF Research Database (Denmark)

Tan, Demeng

The disease vibriosis is caused by the bacterial pathogen Vibrio anguillarum and results in large losses in aquaculture both in Denmark and around the world. Antibiotics have been widely used in antimicrobial prophylaxis and treatment of vibriosis. Recently, numerous multidrug-resistant strains...... of V. anguillarum have been isolated, indicating that antibiotic use has to be restricted and alternatives have to be developed. Lytic phages have been demonstrated to play an essential role in preventing bacterial infection. However, phages are also known to play a critical role in the evolution...... 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...

Basics of Antibody Phage Display Technology.

Science.gov (United States)

Ledsgaard, Line; Kilstrup, Mogens; Karatt-Vellatt, Aneesh; McCafferty, John; Laustsen, Andreas H

2018-06-09

Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments. Moreover, the pros and cons of different antigen display methods and the use of naïve versus immunized phage display antibody libraries is discussed, and selected examples from the field of antivenom research are highlighted. This review thus provides in-depth knowledge on the principles and use of phage display technology with a special focus on discovery of antibodies that target animal toxins.

Discovery of Selective Nanobodies against α-elapitoxin Dpp2c from Black Mamba through Phage Display Screening

DEFF Research Database (Denmark)

Milbo, Christina; Laustsen, Andreas Hougaard; Lohse, Brian

Feared for its highly neurotoxic venom and rapid attack technique, the Black mamba (Dendroaspis polylepis) is Africa’s largest venomous snake. The clinical manifestations of a bitefrom D. polylepis include flaccid paralysis leading to respiratory failure and death due to postsynaptic blockade of ......-neurotoxins. Here, we report the discovery of selective nanobodies targeting α-elapitoxin Dpp2c from D. polylepis through phage display screening....

[Peptide phage display in biotechnology and biomedicine].

Science.gov (United States)

Kuzmicheva, G A; Belyavskaya, V A

2016-07-01

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

MVP: a microbe-phage interaction database.

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Gao, Na L; Zhang, Chengwei; Zhang, Zhanbing; Hu, Songnian; Lercher, Martin J; Zhao, Xing-Ming; Bork, Peer; Liu, Zhi; Chen, Wei-Hua

2018-01-04

Phages invade microbes, accomplish host lysis and are of vital importance in shaping the community structure of environmental microbiota. More importantly, most phages have very specific hosts; they are thus ideal tools to manipulate environmental microbiota at species-resolution. The main purpose of MVP (Microbe Versus Phage) is to provide a comprehensive catalog of phage-microbe interactions and assist users to select phage(s) that can target (and potentially to manipulate) specific microbes of interest. We first collected 50 782 viral sequences from various sources and clustered them into 33 097 unique viral clusters based on sequence similarity. We then identified 26 572 interactions between 18 608 viral clusters and 9245 prokaryotes (i.e. bacteria and archaea); we established these interactions based on 30 321 evidence entries that we collected from published datasets, public databases and re-analysis of genomic and metagenomic sequences. Based on these interactions, we calculated the host range for each of the phage clusters and accordingly grouped them into subgroups such as 'species-', 'genus-' and 'family-' specific phage clusters. MVP is equipped with a modern, responsive and intuitive interface, and is freely available at: http://mvp.medgenius.info. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Control of Pierce's Disease by Phage.

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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.

Phase variable expression of a single phage receptor in Campylobacter jejuni NCTC12662 influences sensitivity toward several diverse CPS-dependent phages

DEFF Research Database (Denmark)

Gencay, Yilmaz Emre; Sørensen, Martine C.H.; Wenzel, Cory Q.

2018-01-01

Campylobacter jejuni NCTC12662 is sensitive to infection by many Campylobacter bacteriophages. Here we used this strain to investigate the molecular mechanism behind phage resistance development when exposed to a single phage and demonstrate how phase variable expression of one surface component...... influences phage sensitivity against many diverse C. jejuni phages. When C. jejuni NCTC12662 was exposed to phage F207 overnight, 25% of the bacterial cells were able to grow on a lawn of phage F207, suggesting that resistance develops at a high frequency. One resistant variant, 12662R, was further...... characterized and shown to be an adsorption mutant. Plaque assays using our large phage collection showed that seven out of 36 diverse capsular polysaccharide (CPS)-dependent phages could not infect 12662R, whereas the remaining phages formed plaques on 12662R with reduced efficiencies. Analysis of the CPS...

Phages in the Human Body.

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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.

Current taxonomy of phages infecting lactic acid bacteria

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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.

Formulation, stabilisation and encapsulation of bacteriophage for phage therapy.

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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

2017-11-01

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

Vibrio vulnificus phage PV94 is closely related to temperate phages of V. cholerae and other Vibrio species.

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Mark Pryshliak

Full Text Available BACKGROUND: Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with V. cholerae. Thus far, only two phages (SSP002 and VvAW1 infecting V. vulnificus have been genetically characterized. These phages were isolated from the environment and are not related to Vibrio cholerae phages. The lack of information on temperate V. vulnificus phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other Vibrio species. RESULTS: In this study the temperate phage PV94 was isolated from a V. vulnificus biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5'-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to V. cholerae kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in V. furnissii NCTC 11218. CONCLUSION: We present the first genomic sequence of a temperate phage isolated from a human V. vulnificus isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various Vibrio species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus Vibrio, by which V. vulnificus might acquire virulence-associated genes from other species.

Phages of life - the path to pharma.

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Forde, Amanda; Hill, Colin

2018-02-01

Bacteriophage (phage) therapy has encountered both enthusiasm and scepticism in the past century. New antimicrobial strategies against lethal pathogens are now a top priority for the World Health Organization, and although compassionate use of phages recently met with significant success, regulated clinical interventions seem unlikely in the near future. The hundredth anniversary of their discovery seems an appropriate time for a revival of phage therapy, particularly as the dilemma of antibiotic resistance grows. Phages are ubiquitous in the environment, on our food and in and on our bodies. Their influence on human health is currently being evaluated, and in this mini-review, we examine data from recent metagenomic studies that propose a role for phages in the structure of the microbiome and in health and disease. We assess evidence for phages as vehicles for gene transfer in the context of antibiotic resistance and discuss challenges and opportunities along the critical path from phage discovery to a patient-focused pharmaceutical intervention. © 2017 The British Pharmacological Society.

Heat tolerance of dairy lactococcal c2 phages

DEFF Research Database (Denmark)

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

2011-01-01

-order kinetics with correlation coefficients of 0.96–0.99. D70-values of 12 s and 16.6 min were calculated for the most sensitive and resistant phage, respectively. Release of phage DNA from capsids, and disintegration of phage heads and tails were among the first morphological changes observed for moderately...... thermal inactivated lysates (15% phage inactivation) of the heat tolerant phage P635....

The Staphylococci Phages Family: An Overview

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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.

Development of a phage typing system for Staphylococcus hyicus

DEFF Research Database (Denmark)

Wegener, Henrik Caspar

1993-01-01

Bacteriophages were released by 98% of 100 Staphylococcus hyicus strains studied after treatment with mitomycin C. Twenty-three phages with different lytic spectra were included in a phage typing system and used f or typing S. hyicus. On a test-set of 100 epidemiologically unrelated S. hyicus...... 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...... 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...

[Isolation and characterization of siphovirus phages infecting bovine Streptococcus agalactiae].

Science.gov (United States)

Bai, Qinqin; Yang, Yongchun; Lu, Chengping

2016-02-04

To isolate and identify Streptococcus agalactiae phages and screen candidate phages to control infection caused by bovine S. agalactiae. We used two methods for isolation of S. agalactiae phages, namely (1) isolation of phages from milk and environmental samples, and (2) isolation of phages via induction of lysogens with Mitomycin C. Double-layer agar culture method was used to purify phages. Then the newly obtained phages, with S. agalactiae phage JX01 isolated from mastitis milk, were comparatively analyzed in the following aspects: morphology of phages by transmission electron microscopy, host range of phages to 55 S. agalactiae strains and other Streptococcus strains, phages DNA using EcoR I, Xba I, Pst I and Sal I, the optical multiplicity of infection, absorption curve and one step growth curve, and the stability of phages at different storage conditions. The comparative analysis of the 3 novel phages LYGO9, HZ04 and pA11 (induced from S. agalctiae bovine clinical isolate HAJL2011070601) with JX01 showed that the 4 phages were classified as the member of Siphovirdae family. EcoR I, Sal I, Xba I and Pst I separately digested the 4 phages DNA provided 4, 3, 3 and 2 profiles, respectively. This suggested that they were different strains. All the 4 phages specifically infected bovine S. agalactiae isolates. LYGO9, pA11, JX01 and HZ04 could lyse 12, 13, 20 and 23 of 42 tested bovine S. agalctiae isolates, respectively. This clearly indicated that these 4 phages are closely related. The 3 new phages which specifically lyse bovine S. agalactiae isolates are siphovirus phages. Phage LYGO9 was shown having a short latent period and a larger burst size.

Phage-Bacterial Dynamics with Spatial Structure: Self Organization around Phage Sinks Can Promote Increased Cell Densities.

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Bull, James J; Christensen, Kelly A; Scott, Carly; Jack, Benjamin R; Crandall, Cameron J; Krone, Stephen M

2018-01-29

Bacteria growing on surfaces appear to be profoundly more resistant to control by lytic bacteriophages than do the same cells grown in liquid. Here, we use simulation models to investigate whether spatial structure per se can account for this increased cell density in the presence of phages. A measure is derived for comparing cell densities between growth in spatially structured environments versus well mixed environments (known as mass action). Maintenance of sensitive cells requires some form of phage death; we invoke death mechanisms that are spatially fixed, as if produced by cells. Spatially structured phage death provides cells with a means of protection that can boost cell densities an order of magnitude above that attained under mass action, although the effect is sometimes in the opposite direction. Phage and bacteria self organize into separate refuges, and spatial structure operates so that the phage progeny from a single burst do not have independent fates (as they do with mass action). Phage incur a high loss when invading protected areas that have high cell densities, resulting in greater protection for the cells. By the same metric, mass action dynamics either show no sustained bacterial elevation or oscillate between states of low and high cell densities and an elevated average. The elevated cell densities observed in models with spatial structure do not approach the empirically observed increased density of cells in structured environments with phages (which can be many orders of magnitude), so the empirical phenomenon likely requires additional mechanisms than those analyzed here.

Phage Therapy -- Everything Old Is New again

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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.

Isolation and Characterization of Phages Infecting Bacillus subtilis

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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.

Pseudomonas predators: understanding and exploiting phage-host interactions.

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De Smet, Jeroen; Hendrix, Hanne; Blasdel, Bob G; Danis-Wlodarczyk, Katarzyna; Lavigne, Rob

2017-09-01

Species in the genus Pseudomonas thrive in a diverse set of ecological niches and include crucial pathogens, such as the human pathogen Pseudomonas aeruginosa and the plant pathogen Pseudomonas syringae. The bacteriophages that infect Pseudomonas spp. mirror the widespread and diverse nature of their hosts. Therefore, Pseudomonas spp. and their phages are an ideal system to study the molecular mechanisms that govern virus-host interactions. Furthermore, phages are principal catalysts of host evolution and diversity, which directly affects the ecological roles of environmental and pathogenic Pseudomonas spp. Understanding these interactions not only provides novel insights into phage biology but also advances the development of phage therapy, phage-derived antimicrobial strategies and innovative biotechnological tools that may be derived from phage-bacteria interactions.

Phage typing of Staphylococcus saprophyticus.

Science.gov (United States)

Torres Pereira, A.; Melo Cristino, J. A.

1991-01-01

This study included 502 staphylococcus strains; Staphylococcus saprophyticus (297 strains) S. cohnii (47), S. xylosus (10), S. epidermidis (67) and S. aureus (81). Mitomycin C induction was performed on 100 isolates of S. saprophyticus and all induced strains were reacted with each other. Twenty-six strains proved to be lysogenic. Phages were propagated and titrated. With 12 of the phages there were three frequent associations, named lytic groups A, B and C, which included 75% of all typable strains. Typability of the system was 45% and reproducibility was between 94.2% and 100%. Phages did not lyse S. aureus and S. epidermidis strains, but they lysed S. saprophyticus and only rare strains of other novobiocin resistant species. Effective S. saprophyticus typing serves ecological purposes and tracing the origin of urinary strains from the skin or mucous membranes. Phage typing in association with plasmid profiling previously described, are anticipated as complementary methods with strong discriminatory power for differentiating among S. saprophyticus strains. PMID:1752305

Recurrent transient ischaemic attack and early risk of stroke: data from the PROMAPA study.

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Purroy, Francisco; Jiménez Caballero, Pedro Enrique; Gorospe, Arantza; Torres, María José; Alvarez-Sabin, José; Santamarina, Estevo; Martínez-Sánchez, Patricia; Cánovas, David; Freijo, María José; Egido, Jose Antonio; Ramírez-Moreno, Jose M; Alonso-Arias, Arantza; Rodríguez-Campello, Ana; Casado, Ignacio; Delgado-Mederos, Raquel; Martí-Fàbregas, Joan; Fuentes, Blanca; Silva, Yolanda; Quesada, Helena; Cardona, Pere; Morales, Ana; de la Ossa, Natalia Pérez; García-Pastor, Antonio; Arenillas, Juan F; Segura, Tomas; Jiménez, Carmen; Masjuán, Jaime

2013-06-01

Many guidelines recommend urgent intervention for patients with two or more transient ischaemic attacks (TIAs) within 7 days (multiple TIAs) to reduce the early risk of stroke. To determine whether all patients with multiple TIAs have the same high early risk of stroke. Between April 2008 and December 2009, we included 1255 consecutive patients with a TIA from 30 Spanish stroke centres (PROMAPA study). We prospectively recorded clinical characteristics. We also determined the short-term risk of stroke (at 7 and 90 days). Aetiology was categorised using the TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification. Clinical variables and extracranial vascular imaging were available and assessed in 1137/1255 (90.6%) patients. 7-Day and 90-day stroke risk were 2.6% and 3.8%, respectively. Large-artery atherosclerosis (LAA) was confirmed in 190 (16.7%) patients. Multiple TIAs were seen in 274 (24.1%) patients. Duration <1 h (OR=2.97, 95% CI 2.20 to 4.01, p<0.001), LAA (OR=1.92, 95% CI 1.35 to 2.72, p<0.001) and motor weakness (OR=1.37, 95% CI 1.03 to 1.81, p=0.031) were independent predictors of multiple TIAs. The subsequent risk of stroke in these patients at 7 and 90 days was significantly higher than the risk after a single TIA (5.9% vs 1.5%, p<0.001 and 6.8% vs 3.0%, respectively). In the logistic regression model, among patients with multiple TIAs, no variables remained as independent predictors of stroke recurrence. According to our results, multiple TIAs within 7 days are associated with a greater subsequent risk of stroke than after a single TIA. Nevertheless, we found no independent predictor of stroke recurrence among these patients.

Twelve previously unknown phage genera are ubiquitous in global oceans.

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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.

Phages of Listeria offer novel tools for diagnostics and biocontrol

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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.

[Analysis of the relevant factors for recurrent sudden sensorineural hearing loss].

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Liang, H; Zhong, S X

2016-09-07

Objective: To investigate the possible factors related to recurrence and prognosis of sudden sensorineural hearing loss(SSNHL). Methods: Four hundred and ninety-five patients with unilateral sudden sensorineural hearing loss between January 2013 to April 2014 were analyzed retrospectively(34 patients lost to follow-up with a dropout rate of 6.87%). Twenty of the 495 patients were diagnosed as recurrent SSNHL and treated again in the same hospital. The data of the patients were summarized to analyze the related factors which might influence the recurrence and prognosis of SSNHL. Results: In the 20 patients with recurrent SSNHL, 19 had the second attack in same ear as the first attack, and the other one had in both ears. There were seven male patients, and thirteen female patients. Patients ranged in age from 24 to 77years, with a median age of 39.5 years. Types of hearing loss: low frequency in eight patients, high frequency in two patients, flat frequency in eight patients, total deafness in two patients, the types of the second attack in 17 patients were same as the first attack, only one patient was changed from total deafness to flat frequency, one case was changed from flat frequency to high frequency, one case changed from flat frequency to total deafness. The intervals between of the first attack time and the second attack time were 1-36 months with the median time of 3.5 months. After systemic oral and (or) transtympanic steroid treatment, recovered in three cases, effective in three cases and 14 cases invalid, the cure rate was 15%, and the total effective rate was 30%. There were statistically significant differences in the recovery rate(χ 2 =8.640, P vertigo and/or dizziness, with a history more than seven days, with hypertension or diabetes mellitus, and with a type of hearing loss except low frequency type, the treatment effect was invalid. The patients with hearing loss at low frequency had the best outcomes. The total effective rates were significant

A century of the phage: past, present and future.

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Salmond, George P C; Fineran, Peter C

2015-12-01

Viruses that infect bacteria (bacteriophages; also known as phages) were discovered 100 years ago. Since then, phage research has transformed fundamental and translational biosciences. For example, phages were crucial in establishing the central dogma of molecular biology - information is sequentially passed from DNA to RNA to proteins - and they have been shown to have major roles in ecosystems, and help drive bacterial evolution and virulence. Furthermore, phage research has provided many techniques and reagents that underpin modern biology - from sequencing and genome engineering to the recent discovery and exploitation of CRISPR-Cas phage resistance systems. In this Timeline, we discuss a century of phage research and its impact on basic and applied biology.

Efficient identification of phosphatidylserine-binding proteins by ORF phage display

International Nuclear Information System (INIS)

Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela; Fan, Xianqun; Li, Wei

2009-01-01

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in ∼300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.

The genomes and comparative genomics of Lactobacillus delbrueckii phages.

Science.gov (United States)

Riipinen, Katja-Anneli; Forsman, Päivi; Alatossava, Tapani

2011-07-01

Lactobacillus delbrueckii phages are a great source of genetic diversity. Here, the genome sequences of Lb. delbrueckii phages LL-Ku, c5 and JCL1032 were analyzed in detail, and the genetic diversity of Lb. delbrueckii phages belonging to different taxonomic groups was explored. The lytic isometric group b phages LL-Ku (31,080 bp) and c5 (31,841 bp) showed a minimum nucleotide sequence identity of 90% over about three-fourths of their genomes. The genomic locations of their lysis modules were unique, and the genomes featured several putative overlapping transcription units of genes. LL-Ku and c5 virions displayed peptidoglycan hydrolytic activity associated with a ~36-kDa protein similar in size to the endolysin. Unexpectedly, the 49,433-bp genome of the prolate phage JCL1032 (temperate, group c) revealed a conserved gene order within its structural genes. Lb. delbrueckii phages representing groups a (a phage LL-H), b and c possessed only limited protein sequence homology. Genomic comparison of LL-Ku and c5 suggested that diversification of Lb. delbrueckii phages is mainly due to insertions, deletions and recombination. For the first time, the complete genome sequences of group b and c Lb. delbrueckii phages are reported.

Restoring logic and data to phage-cures for infectious disease

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Philip Serwer

2017-08-01

Full Text Available Antibiotic therapy for infectious disease is being compromised by emergence of multi-drug-resistant bacterial strains, often called superbugs. A response is to use a cocktail of several bacteria-infecting viruses (bacteriophages or phages to supplement antibiotic therapy. Use of such cocktails is called phage therapy, which has the advantage of response to bacterial resistance that is rapid and not exhaustible. A procedure of well-established success is to make cocktails from stockpiles of stored environmental phages. New phages are added to stockpiles when phage therapy becomes thwarted. The scientific subtext includes optimizing the following aspects: (1 procedure for rapidly detecting, purifying, storing and characterizing phages for optimization of phage cocktails, (2 use of directed evolution in the presence of bacteriostatic compounds to obtain phages that can be most efficiently used for therapy in the presence of these compounds, (3 phage genome sequencing technology and informatics to improve the characterization of phages, and (4 database technology to make optimal use of all relevant information and to rapidly retrieve phages for cocktails that will vary with the infection(s involved. The use of phage stockpiles has an established record, including a recent major human-therapy success by the US Navy. However, I conclude that most research is not along this track and, therefore, is not likely to lead to real world success. I find that a strong case exists for action to rectify this situation.

Morphological evidence for phages in Xylella fastidiosa

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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.

Supersize me: Cronobacter sakazakii phage GAP32

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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.

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.

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.

Phage therapy reduces Campylobacter jejuni colonization in broilers

NARCIS (Netherlands)

Wagenaar, J.A.; Bergen, van M.A.P.; Mueller, M.A.; Wassenaar, T.M.; Carlton, R.M.

2005-01-01

The effect of phage therapy in the control of Campylobacter jejuni colonization in young broilers, either as a preventive or a therapeutic measure, was tested. A prevention group was infected with C. jejuni at day 4 of a 10-day phage treatment. A therapeutic group was phage treated for 6 days,

Molecular Characterization of Three Lactobacillus delbrueckii subsp. bulgaricus Phages

Science.gov (United States)

Casey, Eoghan; Mahony, Jennifer; O'Connell-Motherway, Mary; Bottacini, Francesca; Cornelissen, Anneleen; Neve, Horst; Heller, Knut J.; Noben, Jean-Paul; Dal Bello, Fabio

2014-01-01

In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for one phage, transcriptional analysis by Northern hybridization, reverse transcription-PCR, and primer extension. The three obtained phage genomes display high levels of sequence identity to each other and to genomes of the so-called group b L. delbrueckii phages c5, LL-Ku, and phiLdb, where some of the observed differences are believed to be responsible for host range variations. PMID:25002431

Characterisation of a novel enterobacteria phage, CAjan, isolated from rat faeces.

Science.gov (United States)

Carstens, Alexander B; Kot, Witold; Lametsch, Rene; Neve, Horst; Hansen, Lars H

2016-08-01

In this study, we describe the isolation and characterisation of the novel enterobacteria phage CAjan. This phage belongs to the order Caudovirales and the family Siphoviridae. The phage possesses a linear, double-stranded DNA genome consisting of 59,670 bp with a G+C content of 44.7 % and 91 predicted open reading frames (ORFs). Putative functions were assigned to 39 of the ORFs (37.4 %). The phage structural genes were furthermore functionally characterised by LC MS/MS. CAjan, together with Escherichia phage Seurat and Escherichia phage slur01, represent a novel and genetically distinct clade of Siphoviridae phages that could be considered to constitute a new phage genus. Despite limited sequence similarity, the phages in this group share a number of other common features, including genome structure and the presence of queuosine biosynthesis genes.

Advanced Analysis to Distinguish between Physical Decrease and Inactivation of Viable Phages in Aerosol by Quantitating Phage-Specific Particles.

Science.gov (United States)

Shimasaki, Noriko; Nojima, Yasuhiro; Sakakibara, Masaya; Kikuno, Ritsuko; Iizuka, Chiori; Okaue, Akira; Okuda, Shunji; Shinohara, Katsuaki

2018-01-01

　Recent studies have investigated the efficacy of air-cleaning products against pathogens in the air. A standard method to evaluate the reduction in airborne viruses caused by an air cleaner has been established using a safe bacteriophage instead of pathogenic viruses; the reduction in airborne viruses is determined by counting the number of viable airborne phages by culture, after operating the air cleaner. The reduction in the number of viable airborne phages could be because of "physical decrease" or "inactivation". Therefore, to understand the mechanism of reduction correctly, an analysis is required to distinguish between physical decrease and inactivation. The purpose of this study was to design an analysis to distinguish between the physical decrease and inactivation of viable phi-X174 phages in aerosols. We established a suitable polymerase chain reaction (PCR) system by selecting an appropriate primer-probe set for PCR and validating the sensitivity, linearity, and specificity of the primer-probe set to robustly quantify phi-X174-specific airborne particles. Using this quantitative PCR system and culture assay, we performed a behavior analysis of the phage aerosol in a small chamber (1 m 3 ) at different levels of humidity, as humidity is known to affect the number of viable airborne phages. The results revealed that the reduction in the number of viable airborne phages was caused not only by physical decrease but also by inactivation under particular levels of humidity. Our study could provide an advanced analysis to differentiate between the physical decrease and inactivation of viable airborne phages.

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...

Molecular characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages.

Science.gov (United States)

Casey, Eoghan; Mahony, Jennifer; O'Connell-Motherway, Mary; Bottacini, Francesca; Cornelissen, Anneleen; Neve, Horst; Heller, Knut J; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2014-09-01

In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for one phage, transcriptional analysis by Northern hybridization, reverse transcription-PCR, and primer extension. The three obtained phage genomes display high levels of sequence identity to each other and to genomes of the so-called group b L. delbrueckii phages c5, LL-Ku, and phiLdb, where some of the observed differences are believed to be responsible for host range variations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

In Vivo Imaging of Molecularly Targeted Phage

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Kimberly A. Kelly

2006-12-01

Full Text Available Rapid identification of in vivo affinity ligands would have far-reaching applications for imaging specific molecular targets, in vivo systems imaging, and medical use. We have developed a high-throughput method for identifying and optimizing ligands to map and image biologic targets of interest in vivo. We directly labeled viable phage clones with far-red fluorochromes and comparatively imaged them in vivo by multichannel fluorescence ratio imaging. Using Secreted Protein Acidic and Rich in Cysteine (osteonectin and vascular cell adhesion molecule-1 as model targets, we show that: 1 fluorescently labeled phage retains target specificity on labeling; 2 in vivo distribution can be quantitated (detection thresholds of ~ 300 phage/mm3 tissue throughout the entire depth of the tumor using fluorescent tomographic imaging; and 3 fluorescently labeled phage itself can serve as a replenishable molecular imaging agent. The described method should find widespread application in the rapid in vivo discovery and validation of affinity ligands and, importantly, in the use of fluorochrome-labeled phage clones as in vivo imaging agents.

How to Name and Classify Your Phage: An Informal Guide

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Evelien Adriaenssens

2017-04-01

Full Text Available With this informal guide, we try to assist both new and experienced phage researchers through two important stages that follow phage discovery; that is, naming and classification. Providing an appropriate name for a bacteriophage is not as trivial as it sounds, and the effects might be long-lasting in databases and in official taxon names. Phage classification is the responsibility of the Bacterial and Archaeal Viruses Subcommittee (BAVS of the International Committee on the Taxonomy of Viruses (ICTV. While the BAVS aims at providing a holistic approach to phage taxonomy, for individual researchers who have isolated and sequenced a new phage, this can be a little overwhelming. We are now providing these researchers with an informal guide to phage naming and classification, taking a “bottom-up” approach from the phage isolate level.

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

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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

Recurrent Guillain-Barre Syndrome- A Case Report

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Dilesha WL

2015-05-01

Full Text Available Guillain-Barre syndrome (GBS manifests as acute flaccid weakness of limbs, and is considered a monophasic illness. But recurrences have been reported. Published case studies suggest that 1- 5% of patients who had GBS will have recurrent attacks. We describe a 66-year-old lady who presented with acute onset descending, symmetrical, areflexic, flaccid quadriparesis that progressed to respiratory failure. She had a history of GBS five years back of which she had made a complete recovery with no residual weakness. The diagnosis of recurrent GBS was made.

Mutagenesis of lambda phage by tif-expression or host-irradiation functions is largely independent of damage in the phage DNA

International Nuclear Information System (INIS)

Von Wright, A.; Bridges, B.A.

1980-01-01

The survival and mutagenesis of UV-irradiated phage lambda, as well as bacterial mutagenesis, are enhanced in tif mutants of Escherichia coli when these strains are grown at 43 0 C (Castellazzi et al., 1972). This was interpreted on the basis of a hypothesis (the SOS hypothesis) according to which the UV-inducible phenomena connected with reactivation and mutagenesis of UV-irradiated bacteriophages (Weigle, 1953; Radman, 1975) are constitutively expressed in tif-bacteria at high temperature (Witkin, 1974). In unpublished experiments with phage T3 we found that the survival of UV-irradiated phage is also better at 43 0 C than at 32 0 C in tif + cells and this made us reexamine the significance and nature of tif expression and examine its effects on both unirradiated and UV-irradiated phage lambda. Our results indicate that tif-induced mutagenesis and possibly reactivation of UV-irradiated phage lambda should be reinterpreted. (orig./AJ)

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.

Changing clinical profile of acute rheumatic fever and rheumatic recurrence

International Nuclear Information System (INIS)

Sheikh, A.M.; Sadiq, M.; Rehman, A.U.

2016-01-01

Background: Clinical profile of acute rheumatic fever and rheumatic recurrence seems to have changed in countries where rheumatic fever is still endemic. The objectives of this study were to compare clinical profile and outcome of patients suffering initial and recurrent episodes of acute rheumatic fever in children. Methods: This prospective study was conducted in two tertiary care hospitals from January to June 2011. The diagnosis was based on the modified Jones criteria. Sixty children were included in the study, 15 having first episode of rheumatic fever and 45 with rheumatic recurrence. The severity of carditis was assessed by Clinical and echocardiography means. Results: Carditis was the commonest presentation in both first (80 percentage) and recurrent attacks (100 percentage). Arthritis was seen in 60 percentage of children with first episode and in 26.7 percentage with recurrence. The frequency of subcutaneous nodules, invariably associated with carditis, was very high (33.3 percentage in the first and 48.3 percentage in recurrent episodes). Carditis was generally mild during first episode (53.3 percentage) and severe with rheumatic recurrence (55.6 percentage). There was no death in either group. One patient with severe mitral regurgitation and rheumatic recurrence underwent mitral valve repair for intractable heart failure. Conclusion: Clinical profile of rheumatic recurrence and acute rheumatic fever has changed. Rheumatic recurrence is associated with severe carditis. Carditis is more common than arthritis even in the first attack. Sub-cutaneous nodules are a frequent finding invariably associated with carditis. (author)

Recurrent painful ophthalmoplegic neuropathy; A case report

OpenAIRE

Semra Saygi; Tulun Savas; ilknur Erol

2014-01-01

Recurrent painful ophthalmoplegic neuropathy, typically seen as a serious childhood migraine attack which is followed by ptosis and diplopia due to oculomotor nerve palsy. This is regarded as a form of migraine in the previous classifications but according to the latest classification of the International Headache Society has been recognized as cranial neuralgia. Due to the poor pathological and radiological findings of oculomotor nerve during attack, it is difficult to make differential diag...

Perception of Recurrent Stroke Risk among Black, White and Hispanic Ischemic Stroke and Transient Ischemic Attack Survivors: The SWIFT Study

Science.gov (United States)

Boden-Albala, Bernadette; Carman, Heather; Moran, Megan; Doyle, Margaret; Paik, Myunghee C.

2011-01-01

Objectives Risk modification through behavior change is critical for primary and secondary stroke prevention. Theories of health behavior identify perceived risk as an important component to facilitate behavior change; however, little is known about perceived risk of vascular events among stroke survivors. Methods The SWIFT (Stroke Warning Information and Faster Treatment) study includes a prospective population-based ethnically diverse cohort of ischemic stroke and transient ischemic attack survivors. We investigate the baseline relationship between demographics, health beliefs, and knowledge on risk perception. Regression models examined predictors of inaccurate perception. Results Only 20% accurately estimated risk, 10% of the participants underestimated risk, and 70% of the 817 study participants significantly overestimated their risk for a recurrent stroke. The mean perceived likelihood of recurrent ischemic stroke in the next 10 years was 51 ± 7%. We found no significant differences by race-ethnicity with regard to accurate estimation of risk. Inaccurate estimation of risk was associated with attitudes and beliefs [worry (p risk factors. Conclusion This paper provides a unique perspective on how factors such as belief systems influence risk perception in a diverse population at high stroke risk. There is a need for future research on how risk perception can inform primary and secondary stroke prevention. Copyright © 2011 S. Karger AG, Basel PMID:21894045

Statistical optimization for enhanced yields of probiotic Bacillus coagulans and its phage resistant mutants followed by kinetic modelling of the process.

Science.gov (United States)

Pandey, Kavita R; Joshi, Chetan; Vakil, Babu V

2016-01-01

Probiotics are microorganisms which when administered in adequate amounts confer health benefits to the host. A leading pharmaceutical company producing Bacillus coagulans as a probiotic was facing the problem of recurring phage attacks. Two mutants viz. B. co PIII and B. co MIII that were isolated as phage resistant mutants after UV irradiation and MMS treatment of phage sensitive B. coagulans parental culture were characterized at functional and molecular level and were noted to have undergone interesting genetic changes. The non-specific genetic alterations induced by mutagenesis can also lead to alterations in cell performance. Hence, in the current study the parental strain and the two mutants were selected for shake flask optimization. Plackett-Burman design was used to select the significant culture variables affecting biomass production. Evolutionary operation method was applied for further optimization. The study showed wide variations in the nutritional requirements of phage resistant mutants, post exposure to mutagens. An increment of 150, 134 and 152 % was observed in the biomass productions of B. coagulans (parental type) and mutants B.co PIII and B.co MIII respectively, compared to the yield from one-factor-at-a-time technique. Using Logistic and modified Leudeking-Piret equations, biomass accumulation and substrate utilization efficiency of the bioprocess were determined. The experimental data was in agreement with the results predicted by statistical analysis and modelling. The developed model may be useful for controlling the growth and substrate consumption kinetics in large scale fermentation using B. coagulans .

Breakthrough attacks in patients with hereditary angioedema receiving long-term prophylaxis are responsive to icatibant

DEFF Research Database (Denmark)

Aberer, Werner; Maurer, Marcus; Bouillet, Laurence

2017-01-01

BACKGROUND: Patients with hereditary angioedema (HAE) due to C1-inhibitor deficiency (C1-INH-HAE) experience recurrent attacks of cutaneous or submucosal edema that may be frequent and severe; prophylactic treatments can be prescribed to prevent attacks. However, despite the use of long-term prop...

Identifying Risk of Future Asthma Attacks Using UK Medical Record Data : A Respiratory Effectiveness Group Initiative

NARCIS (Netherlands)

Blakey, John D.; Price, David B.; Pizzichini, Emilio; Popov, Todor A.; Dimitrov, Borislav D.; Postma, Dirkje S.; Josephs, Lynn K.; Kaplan, Alan; Papi, Alberto; Kerkhof, Marjan; Hillyer, Elizabeth V.; Chisholm, Alison; Thomas, Mike

BACKGROUND: Asthma attacks are common, serious, and costly. Individual factors associated with attacks, such as poor symptom control, are not robust predictors. OBJECTIVE: We investigated whether the rich data available in UK electronic medical records could identify patients at risk of recurrent

Marine phages as excellent tracers for reactive colloidal transport in porous media

Science.gov (United States)

Ghanem, Nawras; Chatzinotas, Antonis; Harms, Hauke; Wick, Lukas Y.

2016-04-01

Question: Here we evaluate marine phages as specific markers of hydrological flow and reactive transport of colloidal particles in the Earth's critical zone (CZ). Marine phages and their bacterial hosts are naturally absent in the CZ, and can be detected with extremely high sensitivity. In the framework of the DFG Collaborative Research Center AquaDiva, we asked the following questions: (1) Are marine phages useful specific markers of hydrological flow and reactive transport in porous media? and (2) Which phage properties are relevant drivers for the transport of marine phages in porous media? Methods: Seven marine phages from different families (as well two commonly used terrestrial phages) were selected based on their morphology, size and physico-chemical surface properties (surface charge and hydrophobicity). Phage properties were assessed by electron microscopy, dynamic light scattering and water contact angle analysis (CA). Sand-filled laboratory percolation columns were used to study transport. The breakthrough curves of the phages were analyzed using the clean bed filtration theory and the XDLVO theory of colloid stability, respectively. Phages were quantified by a modified high- throughput plaque assay and a culture-independent particle counting method approach. Results: Our data show that most marine tested phages exhibited highly variable transport rates and deposition efficiency, yet generally high colloidal stability and viability. We find that size, morphology and hydrophobicity are key factors shaping the transport efficiency of phages. Differing deposition efficiencies of the phages were also supported by calculated XDLVO interaction energy profile. Conclusion: Marine phages have a high potential for the use as sensitive tracers in terrestrial habitats with their surface properties playing a crucial role for their transport. Marine phages however, exhibit differences in their deposition efficiency depending on their morphology, hydrophobicity and

An improved plating assay for determination of phage titer

African Journals Online (AJOL)

RACHEL

antibiotics to control bacterial infections in swine (Thacker,. 2014). Phage therapy is re-valued by researchers to combat the growing menace of antibiotic-resistant infections (Torres-Barceló and Hochberg, 2016). Determination of phage titer in a sample is a key step in the study of the phage involved. It is very important to.

Phage lytic enzymes: a history.

Science.gov (United States)

Trudil, David

2015-02-01

There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery

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Teerapong Yata

2014-01-01

Full Text Available Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage, viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

Ultraviolet inactivation and photoreactivation of the cholera phage 'Kappa'

International Nuclear Information System (INIS)

Samad, S.A.; Bhattacharyya, S.C.; Chatterjee, S.N.

1987-01-01

The lysogenic cholera phage, 'Kappa' is some ten to twenty folds more resistant to UV (254 nm) than are most of the T. phages of E. coli, or the cholera phage PL 163/10, or the host V. cholerae strain H218 Sm r , the 37% (D 37 ) and 10% (D 10 ) survival doses being 255.8 J/m 2 and 633.6 J/m 2 respectively. The UV-irradiated 'Kappa' phages could be photoreactivated in the host V. cholerae strain H218 Sm r to a maximum extent of 40%. The removal of the number of lethal hits per phage by the survival-enhancement treatment (photoreactivation) with time followed an exponential relation, the constant probability of removal of lethal hit per unit time being 2.8x10 -2 min -1 . The UV-irradiated phages could also be Weigle reactivated in the host strain of H218 Sm r by a small degree, the maximum reactivation factor (ratio of survivals in UV-irradiated and non-irradiated hosts) being 1.50. (orig.)

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.

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli

International Nuclear Information System (INIS)

Wood, R.D.; Hutchinson, F.

1984-01-01

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr + host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one or two mutant phage per mutant burst. From the results of a series of experiments with various mutant host cells, a major pathway of non-targeted mutagenesis by ultraviolet light was proposed which acts in addition to ''SOS induction''. This pathway involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage. (author)

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

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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.

Filamentous phages of Ralstonia solanacearum: double-edged swords for pathogenic bacteria.

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Yamada, Takashi

2013-01-01

Some phages from genus Inovirus use host or bacteriophage-encoded site-specific integrases or recombinases establish a prophage state. During integration or excision, a superinfective form can be produced. The three states (free, prophage, and superinfective) of such phages exert different effects on host bacterial phenotypes. In Ralstonia solanacearum, the causative agent of bacterial wilt disease of crops, the bacterial virulence can be positively or negatively affected by filamentous phages, depending on their state. The presence or absence of a repressor gene in the phage genome may be responsible for the host phenotypic differences (virulent or avirulent) caused by phage infection. This strategy of virulence control may be widespread among filamentous phages that infect pathogenic bacteria of plants.

Complete genome sequences of three Campylobacter jejuni phage-propagating strains

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Bacteriophage therapy has the potential to reduce Campylobacter jejuni numbers in livestock, but requires a detailed understanding of phage-host interactions. Some C. jejuni strains are readily infected by certain phages, and are thus designated as phage-propagating strains. Here we report the compl...

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.

Killing cancer cells by targeted drug-carrying phage nanomedicines

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Bar, Hagit; Yacoby, Iftach; Benhar, Itai

2008-01-01

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. PMID:18387177

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Wood, R.D.; Hutchinson, F. (Yale Univ., New Haven, CT (USA). Dept. of Molecular Biophysics and Biochemistry)

1984-03-05

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr/sup +/ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one or two mutant phage per mutant burst. From the results of a series of experiments with various mutant host cells, a major pathway of non-targeted mutagenesis by ultraviolet light was proposed which acts in addition to ''SOS induction''. This pathway involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.

Chemical Strategies for the Covalent Modification of Filamentous Phage

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Matthew B Francis

2014-12-01

Full Text Available Historically filamentous bacteriophage have been known to be the workhorse of phage display due to their ability to link genotype to phenotype. More recently, the filamentous phage scaffold has proved to be powerful outside the realms of phage display technology in fields such as molecular imaging, cancer research and materials and vaccine development. The ability of the virion to serve as a platform for a variety of applications heavily relies on the functionalization of the phage coat proteins with a wide variety of functionalities. Genetic modification of the coat proteins has been the most widely used strategy for functionalizing the virion; however complementary chemical modification strategies can help to diversify the range of materials that can be developed. This review emphasizes the recent advances that have been made in the chemical modification of filamentous phage as well as some of the challenges that are involved functionalizing the virion.

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

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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

Ligand-directed profiling of organelles with internalizing phage libraries

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Dobroff, Andrey S.; Rangel, Roberto; Guzman-Roja, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Bologa, Cristian G.; Oprea, Tudor I.; Brinker, C. Jeffrey; Pasqualini, Renata; Arap, Wadih

2015-01-01

Phage display is a resourceful tool to, in an unbiased manner, discover and characterize functional protein-protein interactions, to create vaccines, and to engineer peptides, antibodies, and other proteins as targeted diagnostic and/or therapeutic agents. Recently, our group has developed a new class of internalizing phage (iPhage) for ligand-directed targeting of organelles and/or to identify molecular pathways within live cells. This unique technology is suitable for applications ranging from fundamental cell biology to drug development. Here we describe the method for generating and screening the iPhage display system, and explain how to select and validate candidate internalizing homing peptide. PMID:25640897

Evolution of phage display technology: from discovery to application.

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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.

Complete Genome Sequence of Pseudomonas aeruginosa Phage AAT-1.

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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.

Nongenetic individuality in the host-phage interaction.

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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.

DNA damage and mutagenesis of lambda phage induced by gamma-rays

International Nuclear Information System (INIS)

Bertram, Heidi

1988-01-01

Lambda phage DNA was gamma irradiated in aqueous solution and strand breakage determined. Twice as much minor structural damage per lethal hit was found in this DNA compared with DNA from irradiated phage suspensions. The in vitro irradiated DNA was repackaged into infectious particles. Induction of mutations in the cI or cII cistron was scored using SOS-induced host cells. In vitro prepared particles were found to have second-order kinetics for mutagenesis induced by gamma rays indicating two pre-mutational events were necessary to produce a mutation, but bacteria-free phage suspensions ('lys-phage') showed single hit kinetics for mutagenesis after irradiation. Increase in the mutation rate in the phage particles was mainly due to minor lesions, i.e. ssb, als and unidentified base damage. In lys-phage, mutagenesis might be enhanced by clustered DNA damage - configuration not existing in pack-phage. Loss of infectivity was analysed in comparison with structural damage. All lesions contributed to biological inactivation. Minor lesions were tolerated by lambda phage to a limited extent. Major lesions (e.g. dsb) contributed most to infectivity loss and were considered lethal events. (U.K.)

Changes of the Specific Infectivity of Tracer Phages during Transport in Porous Media.

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Ghanem, Nawras; Trost, Manuel; Sánchez Fontanet, Laura; Harms, Hauke; Chatzinotas, Antonis; Wick, Lukas Y

2018-03-20

Phages (i.e., viruses infecting bacteria) are considered to be good indicators and tracers for fecal pollution, hydraulic flow, or colloidal transport in the subsurface. They are typically quantified as total virus particles (VLP) or plaque forming units (PFU) of infectious phages. As transport may lead to phage deactivation, VLP quantification can overestimate the number of infectious phages. In contrast, PFU counts may underestimate the transport of total virus particles. Using PFU and tunable resistive pulse sensing-based counting for active and total phages, respectively, we quantified the effect of transport through laboratory percolation columns on the specific infectivity (SI). The SI is defined by the ratio of total VLP to PFU and is a measure for the minimum particle numbers needed to create a single infection. Transport of three marine tracer phages and the coli-phage (T4) was described by colloidal filtration theory. We found that apparent collision efficiencies of active and total phages differed. Depending on the phage properties (e.g., morphology or hydrophobicity), passage through a porous medium led to either an increasing or decreasing SI of effluent phages. Our data suggest that both phage mass recovery and the SI should be considered in quantitative phage tracer experiments.

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.

The Human Gut Phage Community and Its Implications for Health and Disease.

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Manrique, Pilar; Dills, Michael; Young, Mark J

2017-06-08

In this review, we assess our current understanding of the role of bacteriophages infecting the human gut bacterial community in health and disease. In general, bacteriophages contribute to the structure of their microbial communities by driving host and viral diversification, bacterial evolution, and by expanding the functional diversity of ecosystems. Gut bacteriophages are an ensemble of unique and shared phages in individuals, which encompass temperate phages found predominately as prophage in gut bacteria (prophage reservoir) and lytic phages. In healthy individuals, only a small fraction of the prophage reservoir is activated and found as extracellular phages. Phage community dysbiosis is characterized by a shift in the activated prophage community or an increase of lytic phages, and has been correlated with disease, suggesting that a proper balance between lysis and lysogeny is needed to maintain health. Consequently, the concept of microbial dysbiosis might be extended to the phage component of the microbiome as well. Understanding the dynamics and mechanisms to restore balance after dysbiosis is an active area of research. The use of phage transplants to re-establish health suggests that phages can be used as disease treatment. Such advances represent milestones in our understanding of gut phages in human health and should fuel research on their role in health and disease.

Recurrent Acute Tonsillitis, The 'Core' Issue

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Saikat Samaddar

2016-04-01

Full Text Available Introduction: Today bacteriological and patho-anatomical considerations too are taken into account while treating a case of tonsillitis. Past decade has seen the rise of resistance amongst the common pathogens, as well as rise in the number of unusual offenders. Determination of the true offending organism and prescribing an antibiotic as per the sensitivity pattern is of utmost importance.  Materials and Method: A prospective longitudinal study was conducted in a tertiary care hospital in Kolkata. The study population consisted of patients presenting with recurrent attacks of acute tonsillitis. Determination of throat swab micro flora, ASO titre and core tissue microflora was done and correlated statistically. Result: There was poor correlation between throat swab and core tissue microflora. Positive predictive value of throat swab was 10%. Pseudomonas is the predominant flora harbouring tonsillar core in our study population. Amoxicillin the most commonly prescribed antibiotic stands out to be the most resistant one. No statistical significance could be reached comparing streptococcal tonsillitis with ASO titre. Discussion: The real pathology within the tonsil core is not always reflected in routine throat swab and bacteriology of recurrent tonsillitis may differ in different regions. ASO titre estimation adds up to the economic burden if rheumatic fever is not suspected. Judicious use of antibiotics based on true sensitivity pattern is encouraged. In the era of antibiotics Tonsillectomy still holds an important position in the management of recurrent attacks. Conclusion: Pseudomonas was found to be the most common pathogen in recurrent acute tonsillitis. Core tissue study remains the Gold standard in identifying the pathogenic organism. Oral third generation Cephalosporin was the most efficacious antibiotic for recurrent tonsillitis in our study population.

Recurrent laughter-induced syncope.

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Gaitatzis, Athanasios; Petzold, Axel

2012-07-01

Syncope is a common presenting complaint in Neurology clinics or Emergency departments, but its causes are sometimes difficult to diagnose. Apart from vasovagal attacks, other benign, neurally mediated syncopes include "situational" syncopes, which occur after urination, coughing, swallowing, or defecation. A healthy 42-year-old male patient presented to the neurology clinic with a long history of faints triggered by spontaneous laughter, especially after funny jokes. Physical and neurological examination, and electroencephalography and magnetic resonance imaging were unremarkable. There was no evidence to suggest cardiogenic causes, epilepsy, or cataplexy and a diagnosis of laughing syncope was made. Laughter-induced syncope is usually a single event in the majority of cases, but may present as recurrent attacks as in our case. Some cases occur in association with underlying neurological conditions. Prognosis is good in the case of neurally mediated attacks. Laughter may not be recognized by physicians as a cause of syncope, which may lead to unnecessary investigations or misdiagnosis, and affect patients' quality of life.

Phage Therapy in the Era of Synthetic Biology.

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Barbu, E Magda; Cady, Kyle C; Hubby, Bolyn

2016-10-03

For more than a century, bacteriophage (or phage) research has enabled some of the most important discoveries in biological sciences and has equipped scientists with many of the molecular biology tools that have advanced our understanding of replication, maintenance, and expression of genetic material. Phages have also been recognized and exploited as natural antimicrobial agents and nanovectors for gene therapy, but their potential as therapeutics has not been fully exploited in Western medicine because of challenges such as narrow host range, bacterial resistance, and unique pharmacokinetics. However, increasing concern related to the emergence of bacteria resistant to multiple antibiotics has heightened interest in phage therapy and the development of strategies to overcome hurdles associated with bacteriophage therapeutics. Recent progress in sequencing technologies, DNA manipulation, and synthetic biology allowed scientists to refactor the entire bacterial genome of Mycoplasma mycoides, thereby creating the first synthetic cell. These new strategies for engineering genomes may have the potential to accelerate the construction of designer phage genomes with superior therapeutic potential. Here, we discuss the use of phage as therapeutics, as well as how synthetic biology can create bacteriophage with desirable attributes. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

Molecular Characterization of Three Lactobacillus delbrueckii subsp. bulgaricus Phages

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Casey, Eoghan; Mahony, Jennifer; O'Connell-Motherway, Mary; Bottacini, Francesca; Cornelissen, Anneleen; Neve, Horst; Heller, Knut J.; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2014-01-01

In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for ...

Phage therapy in the food industry.

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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.

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

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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.

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

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Klyczek, Karen K; Bonilla, J Alfred; Jacobs-Sera, Deborah; Adair, Tamarah L; Afram, Patricia; Allen, Katherine G; Archambault, Megan L; Aziz, Rahat M; Bagnasco, Filippa G; Ball, Sarah L; Barrett, Natalie A; Benjamin, Robert C; Blasi, Christopher J; Borst, Katherine; Braun, Mary A; Broomell, Haley; Brown, Conner B; Brynell, Zachary S; Bue, Ashley B; Burke, Sydney O; Casazza, William; Cautela, Julia A; Chen, Kevin; Chimalakonda, Nitish S; Chudoff, Dylan; Connor, Jade A; Cross, Trevor S; Curtis, Kyra N; Dahlke, Jessica A; Deaton, Bethany M; Degroote, Sarah J; DeNigris, Danielle M; DeRuff, Katherine C; Dolan, Milan; Dunbar, David; Egan, Marisa S; Evans, Daniel R; Fahnestock, Abby K; Farooq, Amal; Finn, Garrett; Fratus, Christopher R; Gaffney, Bobby L; Garlena, Rebecca A; Garrigan, Kelly E; Gibbon, Bryan C; Goedde, Michael A; Guerrero Bustamante, Carlos A; Harrison, Melinda; Hartwell, Megan C; Heckman, Emily L; Huang, Jennifer; Hughes, Lee E; Hyduchak, Kathryn M; Jacob, Aswathi E; Kaku, Machika; Karstens, Allen W; Kenna, Margaret A; Khetarpal, Susheel; King, Rodney A; Kobokovich, Amanda L; Kolev, Hannah; Konde, Sai A; Kriese, Elizabeth; Lamey, Morgan E; Lantz, Carter N; Lapin, Jonathan S; Lawson, Temiloluwa O; Lee, In Young; Lee, Scott M; Lee-Soety, Julia Y; Lehmann, Emily M; London, Shawn C; Lopez, A Javier; Lynch, Kelly C; Mageeney, Catherine M; Martynyuk, Tetyana; Mathew, Kevin J; Mavrich, Travis N; McDaniel, Christopher M; McDonald, Hannah; McManus, C Joel; Medrano, Jessica E; Mele, Francis E; Menninger, Jennifer E; Miller, Sierra N; Minick, Josephine E; Nabua, Courtney T; Napoli, Caroline K; Nkangabwa, Martha; Oates, Elizabeth A; Ott, Cassandra T; Pellerino, Sarah K; Pinamont, William J; Pirnie, Ross T; Pizzorno, Marie C; Plautz, Emilee J; Pope, Welkin H; Pruett, Katelyn M; Rickstrew, Gabbi; Rimple, Patrick A; Rinehart, Claire A; Robinson, Kayla M; Rose, Victoria A; Russell, Daniel A; Schick, Amelia M; Schlossman, Julia; Schneider, Victoria M; Sells, Chloe A; Sieker, Jeremy W; Silva, Morgan P; Silvi, Marissa M; Simon, Stephanie E; Staples, Amanda K; Steed, Isabelle L; Stowe, Emily L; Stueven, Noah A; Swartz, Porter T; Sweet, Emma A; Sweetman, Abigail T; Tender, Corrina; Terry, Katrina; Thomas, Chrystal; Thomas, Daniel S; Thompson, Allison R; Vanderveen, Lorianna; Varma, Rohan; Vaught, Hannah L; Vo, Quynh D; Vonberg, Zachary T; Ware, Vassie C; Warrad, Yasmene M; Wathen, Kaitlyn E; Weinstein, Jonathan L; Wyper, Jacqueline F; Yankauskas, Jakob R; Zhang, Christine; Hatfull, Graham F

2017-01-01

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.

Ciprofloxacin and Trimethoprim Cause Phage Induction and Virulence Modulation in Staphylococcus aureus

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Goerke, Christiane; Köller, Johanna; Wolz, Christiane

2006-01-01

In Staphylococcus aureus strains of human origin, phages which integrate into the chromosomal gene coding for β-hemolysin (hlb) are widely distributed. Most of them encode accessory virulence determinants such as staphylokinase (sak) or enterotoxins. Here, we analyzed the effects of ciprofloxacin and trimethoprim on phage induction and expression of phage-encoded virulence factors by using isolates from patients with cystic fibrosis for which the induction of hlb-converting phages was demonstrated in vivo (C. Goerke, S. Matias y Papenberg, S. Dasbach, K. Dietz, R. Ziebach, B. C. Kahl, and C. Wolz, J. Infect. Dis. 189:724-734, 2004) as well as a φ13 lysogen of phage-cured strain 8325-4. Treatment of lysogens with subinhibitory concentrations of either antibiotic resulted in (i) delysogenization of strains resembling the isolates picked up after chronic lung infection and (ii) replication of phages in the bacterial host in a dose-dependent manner. Ciprofloxacin treatment resulted in enhanced recA transcription, indicating involvement of the SOS response in phage mobilization. Induction of φ13 was linked to elevated expression of the phage-encoded virulence gene sak, chiefly due to the activation of latent phage promoters. In summary, we could show the induction of hlb-converting phages and a subsequent virulence modulation of the host bacterium by ciprofloxacin and trimethoprim. PMID:16377683

GRECOS Project (Genotyping Recurrence Risk of Stroke): The Use of Genetics to Predict the Vascular Recurrence After Stroke.

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Fernández-Cadenas, Israel; Mendióroz, Maite; Giralt, Dolors; Nafria, Cristina; Garcia, Elena; Carrera, Caty; Gallego-Fabrega, Cristina; Domingues-Montanari, Sophie; Delgado, Pilar; Ribó, Marc; Castellanos, Mar; Martínez, Sergi; Freijo, Marimar; Jiménez-Conde, Jordi; Rubiera, Marta; Alvarez-Sabín, José; Molina, Carlos A; Font, Maria Angels; Grau Olivares, Marta; Palomeras, Ernest; Perez de la Ossa, Natalia; Martinez-Zabaleta, Maite; Masjuan, Jaime; Moniche, Francisco; Canovas, David; Piñana, Carlos; Purroy, Francisco; Cocho, Dolores; Navas, Inma; Tejero, Carlos; Aymerich, Nuria; Cullell, Natalia; Muiño, Elena; Serena, Joaquín; Rubio, Francisco; Davalos, Antoni; Roquer, Jaume; Arenillas, Juan Francisco; Martí-Fábregas, Joan; Keene, Keith; Chen, Wei-Min; Worrall, Bradford; Sale, Michele; Arboix, Adrià; Krupinski, Jerzy; Montaner, Joan

2017-05-01

Vascular recurrence occurs in 11% of patients during the first year after ischemic stroke (IS) or transient ischemic attack. Clinical scores do not predict the whole vascular recurrence risk; therefore, we aimed to find genetic variants associated with recurrence that might improve the clinical predictive models in IS. We analyzed 256 polymorphisms from 115 candidate genes in 3 patient cohorts comprising 4482 IS or transient ischemic attack patients. The discovery cohort was prospectively recruited and included 1494 patients, 6.2% of them developed a new IS during the first year of follow-up. Replication analysis was performed in 2988 patients using SNPlex or HumanOmni1-Quad technology. We generated a predictive model using Cox regression (GRECOS score [Genotyping Reurrence Risk of Stroke]) and generated risk groups using a classification tree method. The analyses revealed that rs1800801 in the MGP gene (hazard ratio, 1.33; P =9×10 - 03 ), a gene related to artery calcification, was associated with new IS during the first year of follow-up. This polymorphism was replicated in a Spanish cohort (n=1.305); however, it was not significantly associated in a North American cohort (n=1.683). The GRECOS score predicted new IS ( P =3.2×10 - 09 ) and could classify patients, from low risk of stroke recurrence (1.9%) to high risk (12.6%). Moreover, the addition of genetic risk factors to the GRECOS score improves the prediction compared with previous Stroke Prognosis Instrument-II score ( P =0.03). The use of genetics could be useful to estimate vascular recurrence risk after IS. Genetic variability in the MGP gene was associated with vascular recurrence in the Spanish population. © 2017 American Heart Association, Inc.

Inhaled phage therapy: a promising and challenging approach to treat bacterial respiratory infections.

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Bodier-Montagutelli, Elsa; Morello, Eric; L'Hostis, Guillaume; Guillon, Antoine; Dalloneau, Emilie; Respaud, Renaud; Pallaoro, Nikita; Blois, Hélène; Vecellio, Laurent; Gabard, Jérôme; Heuzé-Vourc'h, Nathalie

2017-08-01

Bacterial respiratory tract infections (RTIs) are increasingly difficult to treat due to evolving antibiotic resistance. In this context, bacteriophages (or phages) are part of the foreseen alternatives or combination therapies. Delivering phages through the airways seems more relevant to accumulate these natural antibacterial viruses in proximity to their bacterial host, within the infectious site. Areas covered: This review addresses the potential of phage therapy to treat RTIs and discusses preclinical and clinical results of phages administration in this context. Recent phage formulation and aerosolization attempts are also reviewed, raising technical challenges to achieve efficient pulmonary deposition via inhalation. Expert opinion: Overall, the inhalation of phages as antibacterial treatment seems both clinically relevant and technically feasible. Several crucial points still need to be investigated, such as phage product pharmacokinetics and immunogenicity. Furthermore, given phage-specific features, appropriate regulatory and manufacturing guidelines will need to be defined. Finally, randomized controlled clinical trials should be carried out to establish phage therapy's clinical positioning in the antimicrobial arsenal against RTIs.

Differential screening of phage-ab libraries by oligonucleotide microarray technology.

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Paolo Monaci

Full Text Available A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag in the phagemid coding for each phage-displayed antibody fragment (phage-Ab present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.

Long-term risk of recurrent vascular events after young stroke: The FUTURE study

NARCIS (Netherlands)

Rutten-Jacobs, L.C.A.; Maaijwee, N.A.M.M.; Arntz, R.M.; Schoonderwaldt, H.C.; Dorresteijn, L.D.A.; Vlugt, M.J. van der; Dijk, E.J. van; Leeuw, F.E. de

2013-01-01

OBJECTIVE: Long-term data on recurrent vascular events after young stroke are limited. Our objective was to examine the long-term risk of recurrent vascular events after young stroke. METHODS: We prospectively included 724 consecutive patients with a first-ever transient ischemic attack (TIA),

Sialendoscopic Approach in Management of Juvenile Recurrent Parotitis.

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Singh, P P; Goyal, M; Goyal, A

2017-12-01

To assess the role of sialendoscopy as a diagnostic and therapeutic modality in juvenile recurrent parotitis. Juvenile recurrent parotitis (JRP) is the second most frequent salivary gland disease in childhood and is characterized by recurrent non suppurative and non obstructive parotid inflammation. These attacks influence the quality of life and can even lead to gland destruction, and there are no definitive treatment to avoid them. Sialendoscopic dilatation is emerging as the new treatment modality in this aspect.  retrospective study. Department of Otorhinolaryngology in tertiary care hospital. 17 cases of juvenile recurrent parotitis (i.e. children of age group 3-11 years presenting with complaints of recurrent parotid region swelling and pain, sometimes associated with fever) were included in the study during October 2012-September 2015. All cases underwent sialendoscopy under general anaesthesia. Diagnostic (classifying the ductal lesion) and interventional sialendoscopic procedure (dilatation with instillation of steroid) were carried out in single sitting. Follow up was done for a minimum of 6 months (range 6-36 months). 17 patients with mean age of 5.6 years and gender distribution of 47:53 (boys:girls) underwent sialendoscopy for JRP. 8 patients presented with unilateral parotitis and 9 with bilateral. The mean number of attacks in previous 1 year were 9.2. Average time for procedure was 20 min. All cases had ductal stenosis and ductal mucosa was pale in 15 cases on endoscopy. 1 patient underwent repeat endoscopy after 2 years. 50% had complete resolution of symptoms and 6 patients had one mild (swelling not associated with fever which subsided on its own) attack after treatment. Follow up period ranged from 6 months to 3 years. No complications were observed. Sialendoscopy has emerged as a viable option for assessment and treatment of JRP. Dilatation of the parotid duct and steroid instillation has significantly reduced the morbidity of this

Genomics of phages with therapeutic potential

DEFF Research Database (Denmark)

Zschach, Henrike

Bacteriophages, viruses that prey on bacteria, have been applied since the 1920’s to treat and prevent bacterial infection. After the discovery of antibiotics, this route was however largely abandoned. Now, with antimicrobial resistance in human-pathogenic bacteria on the rise and a dire need...... 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...... and mathematical modelling, can be applied to move the field towards a future of actual phage therapy in humans. It is composed of three related research projects. The first part of this thesis is an introduction to various topics and methods relevant to the research projects that jointedly make up this Ph...

Recurrent epileptic Wernicke aphasia.

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Sahaya, Kinshuk; Dhand, Upinder K; Goyal, Munish K; Soni, Chetan R; Sahota, Pradeep K

2010-04-15

We report a patient with recurrent epileptic Wernicke aphasia who prior to this presentation, had been misdiagnosed as transient ischemic attacks for several years. This case report emphasizes the consideration of epileptic nature of aphasia when a clear alternate etiology is unavailable, even when EEG fails to show a clear ictal pattern. We also present a brief discussion of previously reported ictal aphasias. Copyright 2010 Elsevier B.V. All rights reserved.

Herpes simplex virus type 2-associated recurrent aseptic (Mollaret's meningitis in genitourinary medicine clinic: a case report

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Abou-Foul AK

2014-03-01

Full Text Available Ahmad K Abou-Foul, Thajunisha M Buhary, Sedki L Gayed Department of Genitourinary Medicine, Royal Blackburn Hospital, East Lancashire Hospitals NHS Trust, Blackburn, UK Introduction: Cases of idiopathic recurrent benign aseptic meningitis were first described by Mollaret. Today, herpes simplex virus (HSV is considered the cause of most cases of Mollaret's meningitis. Case report: A 40-year-old male was referred to our genitourinary medicine clinic with recurrent genital herpetic lesions. He had HSV-2-positive genital ulcers 8 years earlier. One year after the first infection, he developed severe recurrent attacks of headache associated with meningitis symptoms. The results of all radiological and biochemical tests were normal, but the patient reported a correlation between his attacks and genital herpes flare-ups. We diagnosed the patient with Mollaret's meningitis and started him on continuous suppressive acyclovir therapy, which resulted in marked clinical improvement. Discussion: Mollaret's meningitis is a rare form of idiopathic recurrent aseptic meningitis that has a sudden onset, short duration, and spontaneous remission with unpredictable recurrence. We believe that the presence of concurrent or recurrent mucocutaneous herpetic lesions can aid its diagnosis, prior to which, affected patients usually have many unnecessary investigations and treatments. Therefore, detailed sexual history should be sought in all patients with aseptic meningitis, and clinicians should also ask about history of recurrent headaches in all patients with recurrent herpetic anogenital lesions. Continuous suppressive acyclovir therapy may reduce the frequency and severity of attacks and can dramatically improve lifestyle. Keywords: HSV-2 virus, acyclovir, Mollaret's meningitis, recurrent aseptic meningitis, HSV-2 virus, viral meningitis, acyclovir

Identification of the host determinant of two prolate-headed phages infecting lactococcus lactis

International Nuclear Information System (INIS)

Stuer-Lauridsen, Birgitte; Janzen, Thomas; Schnabl, Jannie; Johansen, Eric

2003-01-01

A gene responsible for host determination was identified in two prolate-headed bacteriophages of the c2 species infecting strains of Lactococcus lactis. The identification of the host determinant gene was based on low DNA sequence homology in a specific open reading frame (ORF) between prolate-headed phages with different host ranges. When a host carrying this ORF from one phage on a plasmid was infected with another phage, we obtained phages with an altered host range at a frequency of 10 -6 to 10 -7 . Sequencing of phage DNA originating from 10 independent single plaques confirmed that a genetic recombination had taken place at different positions between the ORF on the plasmid and the infecting phage. The adsorption of the recombinant phages to their bacterial hosts had also changed to match the phage origin of the ORF. Consequently, it is concluded that this ORF codes for the host range determinant

Sialendoscopy in juvenile recurrent parotitis: a review of the literature.

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Canzi, P; Occhini, A; Pagella, F; Marchal, F; Benazzo, M

2013-12-01

Juvenile recurrent parotitis (JRP) is the second most frequent salivary gland disease in childhood, defined as a recurrent non-suppurative and non-obstructive parotid inflammation. The recurring attacks actually represent the most dramatic and serious aspect of this pathology, since they significantly influence the quality of life, and there are no recognized therapies to avoid them. In recent years, there are reports of many international experiences related to the management of JRP by sialendoscopy. In this context, several authors have stressed the striking role of sialendoscopy in the prevention of JRP attacks. The objective of the current review is to overview the existing literature with particular regards to diagnostic and therapeutic outcomes after the application of sialendoscopy in patients suffering from JRP.

Phage Genetic Engineering Using CRISPR–Cas Systems

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Asma Hatoum-Aslan

2018-06-01

Full Text Available Since their discovery over a decade ago, the class of prokaryotic immune systems known as CRISPR–Cas have afforded a suite of genetic tools that have revolutionized research in model organisms spanning all domains of life. CRISPR-mediated tools have also emerged for the natural targets of CRISPR–Cas immunity, the viruses that specifically infect bacteria, or phages. Despite their status as the most abundant biological entities on the planet, the majority of phage genes have unassigned functions. This reality underscores the need for robust genetic tools to study them. Recent reports have demonstrated that CRISPR–Cas systems, specifically the three major types (I, II, and III, can be harnessed to genetically engineer phages that infect diverse hosts. Here, the mechanisms of each of these systems, specific strategies used, and phage editing efficacies will be reviewed. Due to the relatively wide distribution of CRISPR–Cas systems across bacteria and archaea, it is anticipated that these immune systems will provide generally applicable tools that will advance the mechanistic understanding of prokaryotic viruses and accelerate the development of novel technologies based on these ubiquitous organisms.

Recombinant lambda-phage nanobioparticles for tumor therapy in mice models.

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Ghaemi, Amir; Soleimanjahi, Hoorieh; Gill, Pooria; Hassan, Zuhair; Jahromi, Soodeh Razeghi M; Roohvand, Farzin

2010-05-12

Lambda phages have considerable potential as gene delivery vehicles due to their genetic tractability, low cost, safety and physical characteristics in comparison to other nanocarriers and gene porters. Little is known concerning lambda phage-mediated gene transfer and expression in mammalian hosts. We therefore performed experiments to evaluate lambda-ZAP bacteriophage-mediated gene transfer and expression in vitro. For this purpose, we constructed recombinant lambda-phage nanobioparticles containing a mammalian expression cassette encoding enhanced green fluorescent protein (EGFP) and E7 gene of human papillomavirus type 16 (lambda-HPV-16 E7) using Lambda ZAP- CMV XR vector. Four cell lines (COS-7, CHO, TC-1 and HEK-239) were transduced with the nanobioparticles. We also characterized the therapeutic anti-tumor effects of the recombinant lambda-HPV-16 E7 phage in C57BL/6 tumor mice model as a cancer vaccine. Obtained results showed that delivery and expression of these genes in fibroblastic cells (COS-7 and CHO) are more efficient than epithelial cells (TC-1 and HEK-239) using these nanobioparticles. Despite the same phage M.O.I entry, the internalizing titers of COS-7 and CHO cells were more than TC-1 and HEK-293 cells, respectively. Mice vaccinated with lambda-HPV-16 E7 are able to generate potent therapeutic antitumor effects against challenge with E7- expressing tumor cell line, TC-1 compared to group treated with the wild phage. The results demonstrated that the recombinant lambda-phages, due to their capabilities in transducing mammalian cells, can also be considered in design and construction of novel and safe phage-based nanomedicines.

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

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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.

Complete Genome Sequences of 44 Arthrobacter Phages.

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Klyczek, Karen K; Jacobs-Sera, Deborah; Adair, Tamarah L; Adams, Sandra D; Ball, Sarah L; Benjamin, Robert C; Bonilla, J Alfred; Breitenberger, Caroline A; Daniels, Charles J; Gaffney, Bobby L; Harrison, Melinda; Hughes, Lee E; King, Rodney A; Krukonis, Gregory P; Lopez, A Javier; Monsen-Collar, Kirsten; Pizzorno, Marie C; Rinehart, Claire A; Staples, Amanda K; Stowe, Emily L; Garlena, Rebecca A; Russell, Daniel A; Cresawn, Steven G; Pope, Welkin H; Hatfull, Graham F

2018-02-01

We report here the complete genome sequences of 44 phages infecting Arthrobacter sp. strain ATCC 21022. These phages have double-stranded DNA genomes with sizes ranging from 15,680 to 70,707 bp and G+C contents from 45.1% to 68.5%. All three tail types (belonging to the families Siphoviridae , Myoviridae , and Podoviridae ) are represented. Copyright © 2018 Klyczek et al.

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....

Genome Sequences of Gordonia Phages BaxterFox, Kita, Nymphadora, and Yeezy

OpenAIRE

Pope, Welkin H.; Bandla, Sharanya; Colbert, Alexandra K.; Eichinger, Fiona G.; Gamburg, Michelle B.; Horiates, Stavroula G.; Jamison, Jerrica M.; Julian, Dana R.; Moore, Whitney A.; Murthy, Pranav; Powell, Meghan C.; Smith, Sydney V.; Mezghani, Nadia; Milliken, Katherine A.; Thompson, Paige K.

2016-01-01

Gordonia phages BaxterFox, Kita, Nymphadora, and Yeezy are newly characterized phages of Gordonia terrae, isolated from soil samples in Pittsburgh, Pennsylvania. These phages have genome lengths between 50,346 and 53,717?bp, and encode on average 84 predicted proteins. All have G+C content of 66.6%.

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

The controlling effect of single and multiple phages on the density of Flavobacterium psychrophilum at different initial multiplicity of infection (MOI) was assessed in batch cultures to explore the potential for phage-based treatment of this important fish pathogen. A high initial phage concentr......The controlling effect of single and multiple phages on the density of Flavobacterium psychrophilum at different initial multiplicity of infection (MOI) was assessed in batch cultures to explore the potential for phage-based treatment of this important fish pathogen. A high initial phage...... 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...

Identifying Bacterial Immune Evasion Proteins Using Phage Display.

Science.gov (United States)

Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

2017-01-01

Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

Genetic characterization of ØVC8 lytic phage for Vibrio cholerae O1.

Science.gov (United States)

Solís-Sánchez, Alejandro; Hernández-Chiñas, Ulises; Navarro-Ocaña, Armando; De la Mora, Javier; Xicohtencatl-Cortes, Juan; Eslava-Campos, Carlos

2016-03-22

Epidemics and pandemics of cholera, a diarrheal disease, are attributed to Vibrio cholera serogroups O1 and O139. In recent years, specific lytic phages of V. cholera have been proposed to be important factors in the cyclic occurrence of cholera in endemic areas. However, the role and potential participation of lytic phages during long interepidemic periods of cholera in non-endemic regions have not yet been described. The purpose of this study was to isolate and characterize specific lytic phages of V. cholera O1 strains. Sixteen phages were isolated from wastewater samples collected at the Endhó Dam in Hidalgo State, Mexico, concentrated with PEG/NaCl, and purified by density gradient. The lytic activity of the purified phages was tested using different V. cholerae O1 and O139 strains. Phage morphology was visualized by transmission electron microscopy (TEM), and phage genome sequencing was performed using the Genome Analyzer IIx System. Genome assembly and bioinformatics analysis were performed using a set of high-throughput programs. Phage structural proteins were analyzed by mass spectrometry. Sixteen phages with lytic and lysogenic activity were isolated; only phage ØVC8 showed specific lytic activity against V. cholerae O1 strains. TEM images of ØVC8 revealed a phage with a short tail and an isometric head. The ØVC8 genome comprises linear double-stranded DNA of 39,422 bp with 50.8 % G + C. Of the 48 annotated ORFs, 16 exhibit homology with sequences of known function and several conserved domains. Bioinformatics analysis showed multiple conserved domains, including an Ig domain, suggesting that ØVC8 might adhere to different mucus substrates such as the human intestinal epithelium. The results suggest that ØVC8 genome utilize the "single-stranded cohesive ends" packaging strategy of the lambda-like group. The two structural proteins sequenced and analyzed are proteins of known function. ØVC8 is a lytic phage with specific activity against V. cholerae

Synthetic Phage for Tissue Regeneration

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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.

Popping the cork: mechanisms of phage genome ejection

NARCIS (Netherlands)

Molineux, I.J.; Panja, D.

2013-01-01

Sixty years after Hershey and Chase showed that nucleic acid is the major component of phage particles that is ejected into cells, we still do not fully understand how the process occurs. Advances in electron microscopy have revealed the structure of the condensed DNA confined in a phage capsid, and

Lytic phages obscure the cost of antibiotic resistance in Escherichia coli.

Science.gov (United States)

Tazzyman, Samuel J; Hall, Alex R

2015-03-17

The long-term persistence of antibiotic-resistant bacteria depends on their fitness relative to other genotypes in the absence of drugs. Outside the laboratory, viruses that parasitize bacteria (phages) are ubiquitous, but costs of antibiotic resistance are typically studied in phage-free experimental conditions. We used a mathematical model and experiments with Escherichia coli to show that lytic phages strongly affect the incidence of antibiotic resistance in drug-free conditions. Under phage parasitism, the likelihood that antibiotic-resistant genetic backgrounds spread depends on their initial frequency, mutation rate and intrinsic growth rate relative to drug-susceptible genotypes, because these parameters determine relative rates of phage-resistance evolution on different genetic backgrounds. Moreover, the average cost of antibiotic resistance in terms of intrinsic growth in the antibiotic-free experimental environment was small relative to the benefits of an increased mutation rate in the presence of phages. This is consistent with our theoretical work indicating that, under phage selection, typical costs of antibiotic resistance can be outweighed by realistic increases in mutability if drug resistance and hypermutability are genetically linked, as is frequently observed in clinical isolates. This suggests the long-term distribution of antibiotic resistance depends on the relative rates at which different lineages adapt to other types of selection, which in the case of phage parasitism is probably extremely common, as well as costs of resistance inferred by classical in vitro methods.

Isolated Cataplexy in the Differential Diagnosis of Drop Attacks: A Case of Successful Clinical Diagnosis and Treatment

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Robert T. Egel

2012-01-01

Full Text Available Drop attacks are sudden spontaneous falls that are not accompanied by alteration of consciousness and are followed by immediate recovery. Cataplexy, which is usually associated with narcolepsy, is one of the causes of drop attacks. We report a patient with the rare condition of cataplexy without associated narcolepsy (isolated cataplexy. Isolated cataplexy should be included in the differential diagnosis when a patient presents with recurrent drop attacks and normal diagnostic test results.

Identifying Risk of Future Asthma Attacks Using UK Medical Record Data: A Respiratory Effectiveness Group Initiative.

Science.gov (United States)

Blakey, John D; Price, David B; Pizzichini, Emilio; Popov, Todor A; Dimitrov, Borislav D; Postma, Dirkje S; Josephs, Lynn K; Kaplan, Alan; Papi, Alberto; Kerkhof, Marjan; Hillyer, Elizabeth V; Chisholm, Alison; Thomas, Mike

Asthma attacks are common, serious, and costly. Individual factors associated with attacks, such as poor symptom control, are not robust predictors. We investigated whether the rich data available in UK electronic medical records could identify patients at risk of recurrent attacks. We analyzed anonymized, longitudinal medical records of 118,981 patients with actively treated asthma (ages 12-80 years) and 3 or more years of data. Potential risk factors during 1 baseline year were evaluated using univariable (simple) logistic regression for outcomes of 2 or more and 4 or more attacks during the following 2-year period. Predictors with significant univariable association (P attacks included baseline-year markers of attacks (acute oral corticosteroid courses, emergency visits), more frequent reliever use and health care utilization, worse lung function, current smoking, blood eosinophilia, rhinitis, nasal polyps, eczema, gastroesophageal reflux disease, obesity, older age, and being female. The number of oral corticosteroid courses had the strongest association. The final cross-validated models incorporated 19 and 16 risk factors for 2 or more and 4 or more attacks over 2 years, respectively, with areas under the curve of 0.785 (95% CI, 0.780-0.789) and 0.867 (95% CI, 0.860-0.873), respectively. Routinely collected data could be used proactively via automated searches to identify individuals at risk of recurrent asthma attacks. Further research is needed to assess the impact of such knowledge on clinical prognosis. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

CT findings in recurrent pyogenic cholangitis

International Nuclear Information System (INIS)

Jung, Seung Hye; Lim, Jae Hoon; Ko, Young Tae; Lee, Dong Ho

1991-01-01

Recurrent pyogenic cholangitis is characterized clinically by recurrent attacks of right upper abdominal pain, fever and jaundice, and pathologically by chronic inflammation of the bile ducts with or without pigment bile duct stones. We analyzed the CT findings of 33 cases with recurrent pyogenic cholangitis. Twenty-four cases were confirmed by operation, and 9 cases were diagnosed clinically and cholangiographically. The CT findings of recurrent pyogenic cholangitis were dilatation of the intrahepatic ducts (n = 30), dilatation of the extrahepatic ducts (n = 24) intrahepatic stones (n = 16), extrahepatic stones (n = 12), stricture of the bile ducts (n = 10), wall enhancement of the bile ducts (n = 8), gallstones (n = 8), segmental atrophy of the liver (n = 7), pneumobilia (n = 4), abscess (n = 3), and segmental enhancement (n = 1) of the liver. A CT is considered helpful when sectional imaging is needed, but sonographic findings are equivocal or not confirmative; space-occupying lesions complicated with recurrent pyogenic cholangitis: hepatic resection is planned; and imaging guidance is needed for complex drainage procedures

Phage typing or CRISPR typing for epidemiological surveillance of Salmonella Typhimurium?

Science.gov (United States)

Mohammed, Manal

2017-11-07

Salmonella Typhimurium is the most dominant Salmonella serovar around the world. It is associated with foodborne gastroenteritis outbreaks but has recently been associated with invasive illness and deaths. Characterization of S. Typhimurium is therefore very crucial for epidemiological surveillance. Phage typing has been used for decades for subtyping of S. Typhimurium to determine the epidemiological relation among isolates. Recent studies however have suggested that high throughput clustered regular interspaced short palindromic repeats (CRISPR) typing has the potential to replace phage typing. This study aimed to determine the efficacy of high-throughput CRISPR typing over conventional phage typing in epidemiological surveillance and outbreak investigation of S. Typhimurium. In silico analysis of whole genome sequences (WGS) of well-documented phage types of S. Typhimurium reveals the presence of different CRISPR type among strains belong to the same phage type. Furthermore, different phage types of S. Typhimurium share identical CRISPR type. Interestingly, identical spacers were detected among outbreak and non-outbreak associated DT8 strains of S. Typhimurium. Therefore, CRISPR typing is not useful for the epidemiological surveillance and outbreak investigation of S. Typhimurium and phage typing, until it is replaced by WGS, is still the gold standard method for epidemiological surveillance of S. Typhimurium.

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

African Journals Online (AJOL)

This plaque-forming method could be adopted to isolate E. coli phage easily, rapidly and in large quantities. Among the 18 isolated E. coli phages, 10 of them had a broad host range in E. coli and warrant further study. Key words: Escherichia coli phages, large-scale isolation, drug resistance, biological properties.

Characterizing RecA-independent induction of Shiga toxin2-encoding phages by EDTA treatment.

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Lejla Imamovic

Full Text Available BACKGROUND: The bacteriophage life cycle has an important role in Shiga toxin (Stx expression. The induction of Shiga toxin-encoding phages (Stx phages increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA. METHODOLOGY/PRINCIPAL FINDINGS: The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg(2+. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction. CONCLUSIONS/SIGNIFICANCE: Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC and in the emergence of new pathogenic strains.

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

International Nuclear Information System (INIS)

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

2015-01-01

Highlights: • Phage is an excellent seeding for bio-templates for environmentally benign vanadium oxide nanocomposite synthesis. • The synthesized bio-inorganic vanadium oxide showed photodegradation activities. • The fabricated wt phage/vanadium oxide composite exhibited bundle-like structure. • The fabricated RSTB-phage/vanadium oxide composite exhibited a ball with a fiber-like nanostructure. • The virus/vanadium oxide composite could be applied in photocatalysts, sensors and nanoelectronic applications. - Abstract: The growth of crystalline vanadium oxide using a filamentous bacteriophage template was investigated using sequential incubation in a V 2 O 5 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 V 2 O 5 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 V 2 O 5 precursors formed heterogeneous structures, which led to efficient condensation of vanadium oxide crystal formation in lines, shown by QCM-D analysis. Furthermore, re-phage/V x O x composites showed significantly enhanced photodegradation activities compared with the synthesized wt-phage-V 2 O 5 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

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.

Stochasticity in the Expression of LamB and its Affect on λ phage Infection

Science.gov (United States)

Chapman, Emily; Wu, Xiao-Lun

2006-03-01

λ phage binds to E. Coli's lamB protein and injects its DNA into the cell. The phage quickly replicates and after a latent period the bacteria bursts, emitting mature phages. We developed a mathematical model based on the known physical events that occur when a λ phage infects an E.Coli cell. The results of these models predict that the bacteria and phage populations become extinct unless the parameters of the model are very finely tuned, which is untrue in the nature. The lamB protein is part of the maltose regulon and can be repressed to minimal levels when grown in the absence of inducer. Therefore, a cell that is not expressing any lamB protein at that moment is resistant against phage infection. We studied the dynamic relationship between λ phage and E. Coli when the concentration of phage greatly outnumbers the concentration of bacteria. We study how the stochasticity of the expression of lamB affects the percentage of cells that the λ phage infects. We show that even in the case when the maltose regulon is fully induced a percentage of cells continue to persist against phage infection.

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

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......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...... 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...

Recurrent painful ophthalmoplegic neuropathy; A case report

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Semra Saygi

2014-08-01

Full Text Available Recurrent painful ophthalmoplegic neuropathy, typically seen as a serious childhood migraine attack which is followed by ptosis and diplopia due to oculomotor nerve palsy. This is regarded as a form of migraine in the previous classifications but according to the latest classification of the International Headache Society has been recognized as cranial neuralgia. Due to the poor pathological and radiological findings of oculomotor nerve during attack, it is difficult to make differential diagnosis. In this manuscript we report 11-year-old female patient with ophtalmoplegic migraine. [Cukurova Med J 2014; 39(4.000: 938-941

Genome Sequences of Gordonia Phages BaxterFox, Kita, Nymphadora, and Yeezy.

Science.gov (United States)

Pope, Welkin H; Bandla, Sharanya; Colbert, Alexandra K; Eichinger, Fiona G; Gamburg, Michelle B; Horiates, Stavroula G; Jamison, Jerrica M; Julian, Dana R; Moore, Whitney A; Murthy, Pranav; Powell, Meghan C; Smith, Sydney V; Mezghani, Nadia; Milliken, Katherine A; Thompson, Paige K; Toner, Chelsea L; Ulbrich, Megan C; Furbee, Emily C; Grubb, Sarah R; Warner, Marcie H; Montgomery, Matthew T; Garlena, Rebecca A; Russell, Daniel A; Jacobs-Sera, Deborah; Hatfull, Graham F

2016-08-11

Gordonia phages BaxterFox, Kita, Nymphadora, and Yeezy are newly characterized phages of Gordonia terrae, isolated from soil samples in Pittsburgh, Pennsylvania. These phages have genome lengths between 50,346 and 53,717 bp, and encode on average 84 predicted proteins. All have G+C content of 66.6%. Copyright © 2016 Pope et al.

Diversity of phage infection types and associated terminology: the problem with 'Lytic or lysogenic'.

Science.gov (United States)

Hobbs, Zack; Abedon, Stephen T

2016-04-01

Bacteriophages, or phages, are viruses of members of domain Bacteria. These viruses play numerous roles in shaping the diversity of microbial communities, with impact differing depending on what infection strategies specific phages employ. From an applied perspective, these especially are communities containing undesired or pathogenic bacteria that can be modified through phage-mediated bacterial biocontrol, that is, through phage therapy. Here we seek to categorize phages in terms of their infection strategies as well as review or suggest more descriptive, accurate or distinguishing terminology. Categories can be differentiated in terms of (1) whether or not virion release occurs (productive infections versus lysogeny, pseudolysogeny and/or the phage carrier state), (2) the means of virion release (lytic versus chronic release) and (3) the degree to which phages are genetically equipped to display lysogenic cycles (temperate versus non-temperate phages). We address in particular the use or overuse of what can be a somewhat equivocal phrase, 'Lytic or lysogenic', especially when employed as a means of distinguishing among phages types. We suggest that the implied dichotomy is inconsistent with both modern as well as historical understanding of phage biology. We consider, therefore, less ambiguous terminology for distinguishing between 'Lytic' versus 'Lysogenic' phage types. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Phages of lactic acid bacteria: The role of genetics in understanding phage-host interactions and their co-evolutionary processes

International Nuclear Information System (INIS)

Mahony, Jennifer; Ainsworth, Stuart; Stockdale, Stephen; Sinderen, Douwe van

2012-01-01

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.

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.

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.

Genomic analysis of WCP30 Phage of Weissella cibaria for Dairy Fermented Foods.

Science.gov (United States)

Lee, Young-Duck; Park, Jong-Hyun

2017-01-01

In this study, we report the morphogenetic analysis and genome sequence of a new WCP30 phage of Weissella cibaria , isolated from a fermented food. Based on its morphology, as observed by transmission electron microscopy, WCP30 phage belongs to the family Siphoviridae . Genomic analysis of WCP30 phage showed that it had a 33,697-bp double-stranded DNA genome with 41.2% G+C content. Bioinformatics analysis of the genome revealed 35 open reading frames. A BLASTN search showed that WCP30 phage had low sequence similarity compared to other phages infecting lactic acid bacteria. This is the first report of the morphological features and complete genome sequence of WCP30 phage, which may be useful for controlling the fermentation of dairy foods.

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

International Nuclear Information System (INIS)

Sarker, Shafiqul Alam; McCallin, Shawna; Barretto, Caroline; Berger, Bernard; Pittet, Anne-Cécile; Sultana, Shamima; Krause, Lutz; Huq, Sayeda; Bibiloni, Rodrigo; Bruttin, Anne; Reuteler, Gloria; Brüssow, Harald

2012-01-01

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 3×10 9 and 3×10 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.

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.

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.

The genome of the Erwinia amylovora phage PhiEaH1 reveals greater diversity and broadens the applicability of phages for the treatment of fire blight.

Science.gov (United States)

Meczker, Katalin; Dömötör, Dóra; Vass, János; Rákhely, Gábor; Schneider, György; Kovács, Tamás

2014-01-01

The enterobacterium Erwinia amylovora is the causal agent of fire blight. This study presents the analysis of the complete genome of phage PhiEaH1, isolated from the soil surrounding an E. amylovora-infected apple tree in Hungary. Its genome is 218 kb in size, containing 244 ORFs. PhiEaH1 is the second E. amylovora infecting phage from the Siphoviridae family whose complete genome sequence was determined. Beside PhiEaH2, PhiEaH1 is the other active component of Erwiphage, the first bacteriophage-based pesticide on the market against E. amylovora. Comparative genome analysis in this study has revealed that PhiEaH1 not only differs from the 10 formerly sequenced E. amylovora bacteriophages belonging to other phage families, but also from PhiEaH2. Sequencing of more Siphoviridae phage genomes might reveal further diversity, providing opportunities for the development of even more effective biological control agents, phage cocktails against Erwinia fire blight disease of commercial fruit crops.

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.

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

Energy Technology Data Exchange (ETDEWEB)

Mueller, Michael; Baik, Seungyun [Environmental Safety Group, Korea Institute of Science and Technology Europe (KIST-Europe) Forschungsgesellschaft mbH, Campus E 7 1, Saarbruecken (Germany); Jeon, Hojeong; Kim, Yuchan [Center for Biomaterials, Biomedical Research Institute Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Kim, Jungtae [Environmental Safety Group, Korea Institute of Science and Technology Europe (KIST-Europe) Forschungsgesellschaft mbH, Campus E 7 1, Saarbruecken (Germany); Kim, Young Jun, E-mail: youngjunkim@kist-europe.de [Environmental Safety Group, Korea Institute of Science and Technology Europe (KIST-Europe) Forschungsgesellschaft mbH, Campus E 7 1, Saarbruecken (Germany)

2015-05-15

Highlights: • Phage is an excellent seeding for bio-templates for environmentally benign vanadium oxide nanocomposite synthesis. • The synthesized bio-inorganic vanadium oxide showed photodegradation activities. • The fabricated wt phage/vanadium oxide composite exhibited bundle-like structure. • The fabricated RSTB-phage/vanadium oxide composite exhibited a ball with a fiber-like nanostructure. • The virus/vanadium oxide composite could be applied in photocatalysts, sensors and nanoelectronic applications. - Abstract: The growth of crystalline vanadium oxide using a filamentous bacteriophage template was investigated using sequential incubation in a V{sub 2}O{sub 5} 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 V{sub 2}O{sub 5} 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 V{sub 2}O{sub 5} precursors formed heterogeneous structures, which led to efficient condensation of vanadium oxide crystal formation in lines, shown by QCM-D analysis. Furthermore, re-phage/V{sub x}O{sub x} composites showed significantly enhanced photodegradation activities compared with the synthesized wt-phage-V{sub 2}O{sub 5} composite under illumination. This study demonstrates that peptide-mediated vanadium oxide mineralization is governed by a complicated interplay of peptide sequence, local structure

The True Story and Advantages of RNA Phage Capsids as Nanotools.

Science.gov (United States)

Pumpens, Paul; Renhofa, Regina; Dishlers, Andris; Kozlovska, Tatjana; Ose, Velta; Pushko, Peter; Tars, Kaspars; Grens, Elmars; Bachmann, Martin F

2016-01-01

RNA phages are often used as prototypes for modern recombinant virus-like particle (VLP) technologies. Icosahedral RNA phage VLPs can be formed from coat proteins (CPs) and are efficiently produced in bacteria and yeast. Both genetic fusion and chemical coupling have been successfully used for the production of numerous chimeras based on RNA phage VLPs. In this review, we describe advances in RNA phage VLP technology along with the history of the Leviviridae family, including its taxonomical organization, genomic structure, and important role in the development of molecular biology. Comparative 3D structures of different RNA phage VLPs are used to explain the level of VLP tolerance to foreign elements displayed on VLP surfaces. We also summarize data that demonstrate the ability of CPs to tolerate different organic (peptides, oligonucleotides, and carbohydrates) and inorganic (metal ions) compounds either chemically coupled or noncovalently added to the outer and/or inner surfaces of VLPs. Finally, we present lists of nanotechnological RNA phage VLP applications, such as experimental vaccines constructed by genetic fusion and chemical coupling methodologies, nanocontainers for targeted drug delivery, and bioimaging tools. © 2016 S. Karger AG, Basel.

Recovery of phage lambda from ultraviolet damage

International Nuclear Information System (INIS)

Devoret, R.; Blanco, M.; George, J.; Radman, M.

1975-01-01

Recovery of phage lambda from ultraviolet damage can occur, in the dark, through three types of repair processes as defined by microbiological tests: host-cell reactivation, prophage reactivation, and uv reactivation. This paper reviews the properties of the three repair processes, analyzes their dependence on the functioning of bacterial and phage genes, and discusses their relationship. Progress in the understanding of the molecular mechanisms underlying the three repair processes has been relatively slow, particularly for uv reactivation. It has been shown that host-cell reactivation is due to pyrimidine dimer excision and that prophage reactivation is due to genetic recombination (prereplicative). We provide evidence showing that neither of these mechanisms accounts for uv reactivation of phage lambda. Furthermore, uv reactivation differs from the other repair processes in that it is inducible and error-prone. Whether uv-damaged bacterial DNA is subject to a similar repair process is still an open question

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

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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.

Metagenomic recovery of phage genomes of uncultured freshwater actinobacteria.

Science.gov (United States)

Ghai, Rohit; Mehrshad, Maliheh; Mizuno, Carolina Megumi; Rodriguez-Valera, Francisco

2017-01-01

Low-GC Actinobacteria are among the most abundant and widespread microbes in freshwaters and have largely resisted all cultivation efforts. Consequently, their phages have remained totally unknown. In this work, we have used deep metagenomic sequencing to assemble eight complete genomes of the first tailed phages that infect freshwater Actinobacteria. Their genomes encode the actinobacterial-specific transcription factor whiB, frequently found in mycobacteriophages and also in phages infecting marine pelagic Actinobacteria. Its presence suggests a common and widespread strategy of modulation of host transcriptional machinery upon infection via this transcriptional switch. We present evidence that some whiB-carrying phages infect the acI lineage of Actinobacteria. At least one of them encodes the ADP-ribosylating component of the widespread bacterial AB toxins family (for example, clostridial toxin). We posit that the presence of this toxin reflects a 'trojan horse' strategy, providing protection at the population level to the abundant host microbes against eukaryotic predators.

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

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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.

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

Science.gov (United States)

O'Hara, Brendan J; Barth, Zachary K; McKitterick, Amelia C; Seed, Kimberley D

2017-06-01

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.

Analysis of whole genome sequencing for the Escherichia coli O157:H7 typing phages.

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Cowley, Lauren A; Beckett, Stephen J; Chase-Topping, Margo; Perry, Neil; Dallman, Tim J; Gally, David L; Jenkins, Claire

2015-04-08

Shiga toxin producing Escherichia coli O157 can cause severe bloody diarrhea and haemolytic uraemic syndrome. Phage typing of E. coli O157 facilitates public health surveillance and outbreak investigations, certain phage types are more likely to occupy specific niches and are associated with specific age groups and disease severity. The aim of this study was to analyse the genome sequences of 16 (fourteen T4 and two T7) E. coli O157 typing phages and to determine the genes responsible for the subtle differences in phage type profiles. The typing phages were sequenced using paired-end Illumina sequencing at The Genome Analysis Centre and the Animal Health and Veterinary Laboratories Agency and bioinformatics programs including Velvet, Brig and Easyfig were used to analyse them. A two-way Euclidian cluster analysis highlighted the associations between groups of phage types and typing phages. The analysis showed that the T7 typing phages (9 and 10) differed by only three genes and that the T4 typing phages formed three distinct groups of similar genomic sequences: Group 1 (1, 8, 11, 12 and 15, 16), Group 2 (3, 6, 7 and 13) and Group 3 (2, 4, 5 and 14). The E. coli O157 phage typing scheme exhibited a significantly modular network linked to the genetic similarity of each group showing that these groups are specialised to infect a subset of phage types. Sequencing the typing phage has enabled us to identify the variable genes within each group and to determine how this corresponds to changes in phage type.

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 ...

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

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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.

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

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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.

Survey on the phage resistance mechanisms displayed by a dairy Lactobacillus helveticus strain.

Science.gov (United States)

Zago, Miriam; Orrù, Luigi; Rossetti, Lia; Lamontanara, Antonella; Fornasari, Maria Emanuela; Bonvini, Barbara; Meucci, Aurora; Carminati, Domenico; Cattivelli, Luigi; Giraffa, Giorgio

2017-09-01

In this study the presence and functionality of phage defence mechanisms in Lactobacillus helveticus ATCC 10386, a strain of dairy origin which is sensitive to ΦLh56, were investigated. After exposure of ATCC 10386 to ΦLh56, the whole-genome sequences of ATCC 10386 and of a phage-resistant derivative (LhM3) were compared. LhM3 showed deletions in the S-layer protein and a higher expression of the genes involved in the restriction/modification (R/M) system. Genetic data were substantiated by measurements of bacteriophage adsorption rates, efficiency of plaquing, cell wall protein size and by gene expression analysis. In LhM3 two phage resistance mechanisms, the inhibition of phage adsorption and the upregulation of Type I R/M genes, take place and explain its resistance to ΦLh56. Although present in both ATCC 10386 and LhM3 genomes, the CRISPR machinery did not seem to play a role in the phage resistance of LhM3. Overall, the natural selection of phage resistant strains resulted successful in detecting variants carrying multiple phage defence mechanisms in L. helveticus. The concurrent presence of multiple phage-resistance systems should provide starter strains with increased fitness and robustness in dairy ecosystems. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed....... 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...... 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...

Predictors of migraine headache recurrence: A pooled analysis from the eletriptan database

DEFF Research Database (Denmark)

Dodick, D.W.; Lipton, R.B.; Goadsby, P.J.

2008-01-01

Objective.-To identify clinical variables associated with risk of headache recurrence within 22 hours of initial successful treatment of a migraine attack (2-hour headache response), and to analyze the effect of eletriptan in reducing the incidence of recurrence. Methods.-Data were pooled from 10...... evaluable. A multivariable logistic regression analysis identified significant predictors of headache recurrence (return to moderate/severe pain intensity within 22 hours of initial headache response). Treatment response was assessed in two high-risk subgroups, defined by the presence of significant...

Understanding the enormous diversity of bacteriophages: the tailed phages that infect the bacterial family Enterobacteriaceae

Science.gov (United States)

Grose, Julianne H.; Casjens, Sherwood R.

2014-01-01

Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae. These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships. PMID:25240328

Phage Therapy Approaches to Reducing Pathogen Persistence and Transmission in Animal Production Environments: Opportunities and Challenges.

Science.gov (United States)

Colavecchio, Anna; Goodridge, Lawrence D

2017-06-01

The era of genomics has allowed for characterization of phages for use as antimicrobials to treat animal infections with a level of precision never before realized. As more research in phage therapy has been conducted, several advantages of phage therapy have been realized, including the ubiquitous nature, specificity, prevalence in the biosphere, and low inherent toxicity of phages, which makes them a safe and sustainable technology for control of animal diseases. These unique qualities of phages have led to several opportunities with respect to emerging trends in infectious disease treatment. However, the opportunities are tempered by several challenges to the successful implementation of phage therapy, such as the fact that an individual phage can only infect one or a few bacterial strains, meaning that large numbers of different phages will likely be needed to treat infections caused by multiple species of bacteria. In addition, phages are only effective if enough of them can reach the site of bacterial colonization, but clearance by the immune system upon introduction to the animal is a reality that must be overcome. Finally, bacterial resistance to the phages may develop, resulting in treatment failure. Even a successful phage infection and lysis of its host has consequences, because large amounts of endotoxin are released upon lysis of Gram-negative bacteria, which can lead to local and systemic complications. Overcoming these challenges will require careful design and development of phage cocktails, including comprehensive characterization of phage host range and assessment of immunological risks associated with phage treatment.

Severe angina pectoris in asthma attack: a case report.

Science.gov (United States)

Nabavizadeh, Seyed Hesamedin; Farahbakhsh, Nazanin; Fazel, Ali; Mosavat, Fereshteh; Anushiravani, Amir

2016-06-01

Asthma is a chronic inflammatory disorder of the airways related to the obstruction of reversible airflow. Asthma presents as recurrent attacks of cough and dyspnea. Poor control causes recurrent admissions to the ICU, and mortality is related to poor drug compliance and follow-up. Angina pectoris is a syndrome of recurrent chest discomfort related to myocardial ischemia. The presence of these two disorders rarely has been reported. We reported a 12-year-old boy who was referred with exacerbation of asthma and developed angina pectoris during hospitalization. He had labored breathing and diffuse wheezing. During treatment of the asthma, the patient developed severe chest pain due to shunt formation and coronary hypoxia, caused by the sole administration of ventolin, since oxygen had been disconnected. After receiving appropriate therapy, both his asthma and angina recovered, and, to date, he has not experienced angina pectoris again.

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

Noninvasive positive pressure ventilation in acute asthmatic attack

Directory of Open Access Journals (Sweden)

A. Soroksky

2010-03-01

Full Text Available Asthma is characterised by reversible airway obstruction. In most patients, control of disease activity is easily achieved. However, in a small minority, asthma may be fatal. Between the two extremes lie patients with severe asthmatic attacks, refractory to standard treatment. These patients are at an increased risk of recurrent severe attacks, with respiratory failure, and mechanical ventilation. Invasive mechanical ventilation of the asthmatic patient is associated with a higher risk of complications and, therefore, is a measure of last resort. Noninvasive positive pressure ventilation (NPPV is another treatment modality that may be beneficial in patients with severe asthmatic attack who are at an increased risk of developing respiratory failure. These patients have the potential to benefit from early respiratory support in the form of NPPV. However, reports of NPPV in asthmatic patients are scarce, and its usage in asthmatic attacks is, therefore, still controversial. Only a few reports of NPPV in asthma have been published over the last decade. These studies mostly involve small numbers of patients and those who have problematic methodology. In this article we review the available evidence for NPPV in asthma and try to formulate our recommendations for NPPV application in asthma based on the available evidence and reports.

Phage Conversion for β-Lactam Antibiotic Resistance of Staphylococcus aureus from Foods.

Science.gov (United States)

Lee, Young-Duck; Park, Jong-Hyun

2016-02-01

Temperate phages have been suggested to carry virulence factors and other lysogenic conversion genes that play important roles in pathogenicity. In this study, phage TEM123 in wild-type Staphylococcus aureus from food sources was analyzed with respect to its morphology, genome sequence, and antibiotic resistance conversion ability. Phage TEM123 from a mitomycin C-induced lysate of S. aureus was isolated from foods. Morphological analysis under a transmission electron microscope revealed that it belonged to the family Siphoviridae. The genome of phage TEM123 consisted of a double-stranded DNA of 43,786 bp with a G+C content of 34.06%. A bioinformatics analysis of the phage genome identified 43 putative open reading frames (ORFs). ORF1 encoded a protein that was nearly identical to the metallo-β-lactamase enzymes that degrade β-lactam antibiotics. After transduction to S. aureus with phage TEM123, the metallo-β-lactamase gene was confirmed in the transductant by PCR and sequencing analyses. In a β-lactam antibiotic susceptibility test, the transductant was more highly resistant to β-lactam antibiotics than S. aureus S133. Phage TEM123 might play a role in the transfer of β-lactam antibiotic resistance determinants in S. aureus. Therefore, we suggest that the prophage of S. aureus with its exotoxin is a risk factor for food safety in the food chain through lateral gene transfer.

Phage-inducible chromosomal islands are ubiquitous within the bacterial universe.

Science.gov (United States)

Fillol-Salom, Alfred; Martínez-Rubio, Roser; Abdulrahman, Rezheen F; Chen, John; Davies, Robert; Penadés, José R

2018-06-06

Phage-inducible chromosomal islands (PICIs) are a recently discovered family of pathogenicity islands that contribute substantively to horizontal gene transfer, host adaptation and virulence in Gram-positive cocci. Here we report that similar elements also occur widely in Gram-negative bacteria. As with the PICIs from Gram-positive cocci, their uniqueness is defined by a constellation of features: unique and specific attachment sites, exclusive PICI genes, a phage-dependent mechanism of induction, conserved replication origin organization, convergent mechanisms of phage interference, and specific packaging of PICI DNA into phage-like infectious particles, resulting in very high transfer frequencies. We suggest that the PICIs represent two or more distinct lineages, have spread widely throughout the bacterial world, and have diverged much more slowly than their host organisms or their prophage cousins. Overall, these findings represent the discovery of a universal class of mobile genetic elements.

Novel type of specialized transduction for CTX phi or its satellite phage RS1 mediated by filamentous phage VGJ phi in Vibrio cholerae.

Science.gov (United States)

Campos, Javier; Martínez, Eriel; Marrero, Karen; Silva, Yussuan; Rodríguez, Boris L; Suzarte, Edith; Ledón, Talena; Fando, Rafael

2003-12-01

The main virulence factor of Vibrio cholerae, the cholera toxin, is encoded by the ctxAB operon, which is contained in the genome of the lysogenic filamentous phage CTX phi. This phage transmits ctxAB genes between V. cholerae bacterial populations that express toxin-coregulated pilus (TCP), the CTX phi receptor. In investigating new forms of ctxAB transmission, we found that V. cholerae filamentous phage VGJ phi, which uses the mannose-sensitive hemagglutinin (MSHA) pilus as a receptor, transmits CTX phi or its satellite phage RS1 by an efficient and highly specific TCP-independent mechanism. This is a novel type of specialized transduction consisting in the site-specific cointegration of VGJ phi and CTX phi (or RS1) replicative forms to produce a single hybrid molecule, which generates a single-stranded DNA hybrid genome that is packaged into hybrid viral particles designated HybP phi (for the VGJ phi/CTX phi hybrid) and HybRS phi (for the VGJ phi/RS1 hybrid). The hybrid phages replicate by using the VGJ phi replicating functions and use the VGJ phi capsid, retaining the ability to infect via MSHA. The hybrid phages infect most tested strains more efficiently than CTX phi, even under in vitro optimal conditions for TCP expression. Infection and lysogenization with HybP phi revert the V. cholerae live attenuated vaccine strain 1333 to virulence. Our results reinforce that TCP is not indispensable for the acquisition of CTX phi. Thus, we discuss an alternative to the current accepted evolutionary model for the emergence of new toxigenic strains of V. cholerae and the importance of our findings for the development of an environmentally safer live attenuated cholera vaccine.

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.

Lethal effects of 32P decay on transfecting activity of Bacillus subtillis phage phie DNA

International Nuclear Information System (INIS)

Loveday, K.S.

1979-01-01

Disintegration of 32 P present in the DNA of Bacillus subtilis phage phie (a phage containing double-strand DNA) results in the loss of viability of intact phage as well as transfecting activity of isolated DNA. Only 1/12 of the 32 P disintegrations per phage DNA equivalent inactivities the intact phage while nearly every disintegration inactivates the transfecting DNA. This result provides evidence for a single-strand intermediate in the transfection of B. subtilis by phie DNA

Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform.

Directory of Open Access Journals (Sweden)

Graham F Hatfull

2006-06-01

Full Text Available Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 "phamilies" of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774 of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three-encoding tape-measure proteins, lysins, and minor tail proteins-are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15% have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.

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

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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

[Dermatological features of auto-inflammatory recurrent fevers].

Science.gov (United States)

Escudier, A; Mauvais, F-X; Bastard, P; Boussard, C; Jaoui, A; Koskas, V; Lecoq, E; Michel, A; Orcel, M-C; Truelle, P-E; Wohrer, D; Piram, M

2018-02-01

Auto-inflammatory diseases are characterized by unexplained and recurrent attacks of systemic inflammation often involving the skin, joints, or serosal membranes. They are due to a dysfunction or dysregulation of the innate immunity, which is the first line of defense against pathogens. Early recognition of these diseases by the clinician, especially by pediatricians encountering such pathologies in pediatric patients, is primordial to avoid complications. Skin manifestations, common in most auto-inflammatory diseases, are helpful for prompt diagnosis. After a brief physiopathological review, we will describe auto-inflammatory recurrent fevers by their main dermatological presentations: urticarial lesions, neutrophilic dermatoses, panniculitis, other maculopapular eruptions, dyskeratosis, skin vasculitis, and oral aphthous. We finally suggest a decision tree to help clinicians better target genetic exams in patients with recurrent fevers and dermatological manifestations. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Phage FR38 Treatment on Sprague Dawley Rat Inferred from Blood Parameters and Organ Systems

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DEWI SARTIKA

2012-09-01

Full Text Available The ability of phage FR38 to lysis indigenous Salmonella P38 from feces of diarrheal patient has been studied. However, effects of phage FR38 on organ system were not revealed as yet. This study was conducted to observe the effect of phage FR38 on blood chemistry, kidney functions, and liver functions. Twelve Sprague-Dawley rats were used as a model for this study that were divided into two groups; (i control and (ii treated group with phage FR38. For treated phage group, each rat was administered by 5 ml/kg bw of 1.59•107 pfu/ml of phage intragastric. The blood parameters were analysed on day 16. The results revealed that body and organs weight, erythrocyte, hematocrit, hemoglobin, leukocyte, total protein, creatinine, SGOT, and SGPT of phage treatment rats were not significantly different with the control rats on day 16 (P > 0.05. Therefore, this study showed was no effect of phage FR38 on body weight, blood chemistry, kidney and liver functions of the rat (P > 0.05.

Phage FR38 Treatment on Sprague Dawley Rat Inferred from Blood Parameters and Organ Systems

Directory of Open Access Journals (Sweden)

DEWI SARTIKA

2012-09-01

Full Text Available The ability of phage FR38 to lysis indigenous Salmonella P38 from feces of diarrheal patient has been studied. However, effects of phage FR38 on organ system were not revealed as yet. This study was conducted to observe the effect of phage FR38 on blood chemistry, kidney functions, and liver functions. Twelve Sprague-Dawley rats were used as a model for this study that were divided into two groups; (i control and (ii treated group with phage FR38. For treated phage group, each rat was administered by 5 ml/kg bw of 1.59-107 pfu/ml of phage intragastric. The blood parameters were analysed on day 16. The results revealed that body and organs weight, erythrocyte, hematocrit, hemoglobin, leukocyte, total protein, creatinine, SGOT, and SGPT of phage treatment rats were not significantly different with the control rats on day 16 (P > 0.05. Therefore, this study showed was no effect of phage FR38 on body weight, blood chemistry, kidney and liver functions of the rat (P > 0.05.

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.

On the common modulus attack into the LUC4,6 cryptosystem

Science.gov (United States)

Wong, Tze Jin; Said, Mohd Rushdan Md; Othman, Mohamed; Koo, Lee Feng

2015-05-01

The LUC4,6 cryptosystem is a system analogy with RSA cryptosystem and extended from LUC and LUC3 cryptosystems. The process of encryption and decryption are derived from the fourth order linear recurrence sequence and based on Lucas function. This paper reports an investigation into the common modulus attack on the LUC4,6 cryptosystem. In general, the common modulus attack will be succeeded if the sender sends the plaintext to two users used same RSA-modulus and both of encryption keys of them are relatively prime to each other. However, based on the characteristics of high order Lucas sequence, the LUC4,6 cryptosystem is unattackable

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

range often displayed by phages. To identify the potential of phages as a Campylobacter reducing agent we needed to determine their infectivity on a panel of isolates representing the Campylobacter strains found in broilers as well as humans. Results: In this study, Campylobacter phages were isolated...... from the intestines of broilers and ducks and from abattoir sewage. Twelve phages were investigated to determine their ability to infect the Campylobacter Penner serotypes commonly present in Danish poultry and patients with campylobacteriosis. A total of 89% of the Campylobacter jejuni strains and 14...... range of 12 Danish Campylobacter phages. Due to their ability to infect the majority of the common serotypes in Denmark we suggest the phages can become an effective agent in the effort to reduce the incidence of campylobacteriosis in Denmark. This study provides the basis for future experiments...

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

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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.

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...... mathematical models of these processes and suggest how different types of this 'phenotypic' resistance may be elucidated. We offer preliminary in vitro studies of a previously characterized E. coli model system and Campylobacter jejuni illustrating apparent phenotypic resistance. As phenotypic resistance may...

Analysis of Lactobacillus Products for Phages and Bacteriocins That Inhibit Vaginal Lactobacilli

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Lin Tao

1997-01-01

Full Text Available Objective: Bacterial vaginosis is associated with an unexplained loss of vaginal lactobacilli. Previously, we have identified certain vaginal lactobacilli-released phages that can inhibit in vitro other vaginal lactobacilli. However, there is no apparent route for phages to be transmitted among women. The purpose of this study was to identify whether certain Lactobacillus products commonly used by women release phages or bacteriocins that can inhibit vaginal lactobacilli.

[Incidence of asthmatic attack during anesthesia in patients with a history of bronchial asthma].

Science.gov (United States)

Nonaka, M; Sakanashi, Y; Sugahara, K; Terasaki, H

1999-07-01

We investigated retrospectively, the incidence of intraoperative asthmatic attack in patients with bronchial asthma, and compared the past 5-year period (Jan 1, 1979 to Dec 31, 1983) and the recent 5-year period (July 1, 1990 to June 30, 1994). The influence of anesthetic methods, induction agents, and intervals between the most recent attack and anesthesia were evaluated. The incidence of the attack during anesthesia in patients with asthma was similar in both period, 6.7% in the past and 7.8% in the recent period. Patients who had a history of recurrent attacks within 2 years prior to anesthesia tended to have higher incidence of intraoperative attack in both periods. Furthermore, the incidence of asthmatic attack was significantly greater in patients receiving inhalational anesthetics for induction of anesthesia than in those given intravenous anesthetics in the recent period. Although inhalational anesthetics are considered to be used safely for asthmatic patients, care should be taken when volatile anesthetics are administered to asthmatic patients during induction of anesthesia.

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

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

International Nuclear Information System (INIS)

Johansen, Annette H.; Broendsted, Lone; 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 early promoters P R and P L , O L overlaps the transcriptional start of the lytic P L promoter, and O D is located downstream of the mor gene, the first gene in the lytic gene cluster. The function of O L was verified by mutational analysis. Binding was found to be specific and cooperative. Multimeric 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 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

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

2018-03-01

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.

Association between phage types and antimicrobial resistance among bovine Staphylococcus aureus from 10 countries

DEFF Research Database (Denmark)

Vintov, J.; Aarestrup, Frank Møller; Zinn, C. E.

2003-01-01

This study was conducted to investigate the diversity of phage types and associations between penicillin resistance and phage types among 815 Staphylococcus aureus isolates from bovine mastitis in nine European countries and USA. All isolates were examined for susceptibility to antimicrobial agents...... associated with penicillin resistance in contrast to phage group I (P = 0.0023) and phage complex-80 (P = 0.0066). This study confirms that a large number of phage types of S. aureus cause bovine mastitis, but that some types predominate. In addition, these findings could indicate that the use of penicillin...... in the bovine environment has selected for specific types of S. aureus in countries with a high frequency of resistance....

“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

Phage “delay” towards enhancing bacterial escape from biofilms: a more comprehensive way of viewing resistance to bacteriophages

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Stephen T. Abedon

2017-03-01

Full Text Available In exploring bacterial resistance to bacteriophages, emphasis typically is placed on those mechanisms which completely prevent phage replication. Such resistance can be detected as extensive reductions in phage ability to form plaques, that is, reduced efficiency of plating. Mechanisms include restriction-modification systems, CRISPR/Cas systems, and abortive infection systems. Alternatively, phages may be reduced in their “vigor” when infecting certain bacterial hosts, that is, with phages displaying smaller burst sizes or extended latent periods rather than being outright inactivated. It is well known, as well, that most phages poorly infect bacteria that are less metabolically active. Extracellular polymers such as biofilm matrix material also may at least slow phage penetration to bacterial surfaces. Here I suggest that such “less-robust” mechanisms of resistance to bacteriophages could serve bacteria by slowing phage propagation within bacterial biofilms, that is, delaying phage impact on multiple bacteria rather than necessarily outright preventing such impact. Related bacteria, ones that are relatively near to infected bacteria, e.g., roughly 10+ µm away, consequently may be able to escape from biofilms with greater likelihood via standard dissemination-initiating mechanisms including erosion from biofilm surfaces or seeding dispersal/central hollowing. That is, given localized areas of phage infection, so long as phage spread can be reduced in rate from initial points of contact with susceptible bacteria, then bacterial survival may be enhanced due to bacteria metaphorically “running away” to more phage-free locations. Delay mechanisms—to the extent that they are less specific in terms of what phages are targeted—collectively could represent broader bacterial strategies of phage resistance versus outright phage killing, the latter especially as require specific, evolved molecular recognition of phage presence. The

Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays.

Science.gov (United States)

Adhikari, Meena; Strych, Ulrich; Kim, Jinsu; Goux, Heather; Dhamane, Sagar; Poongavanam, Mohan-Vivekanandan; Hagström, Anna E V; Kourentzi, Katerina; Conrad, Jacinta C; Willson, Richard C

2015-12-01

We introduce the modification of bacteriophage particles with aptamers for use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin-biotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ∼100 times lower than those of previously reported IgE assays.

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. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

The use of phage FCL-2 as an alternative to chemotherapy against columnaris disease in aquaculture

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Elina eLaanto

2015-08-01

Full Text Available Flavobacterium columnare, the causative agent of columnaris disease in fish, causes millions of dollars of losses in the US channel catfish industry alone, not to mention aquaculture industry worldwide. Novel methods are needed for the control and treatment of bacterial diseases in aquaculture to replace traditionally used chemotherapies. A potential solution could be the use of phages, i.e., bacterial viruses, host-specific and self-enriching particles that can be can easily distributed via water flow. We examined the efficacy of phages to combat columnaris disease. A previously isolated phage, FCL-2, infecting F. columnare, was characterized by sequencing. The 47 142 bp genome of the phage had G + C content of 30.2%, and the closest similarities regarding the structural proteins were found in Cellulophaga phage phiSM. Under controlled experimental conditions, two host fish species, rainbow trout (Oncorhynchus mykiss and zebrafish (Danio rerio, were used to study the success of phage therapy to prevent F. columnare infections. The survival of both fish species was significantly higher in the presence of the phage. Hundred percent of the zebrafish and 50 % of the rainbow trout survived in the phage treatment (survival without phage 0 % and 8.3 %, respectively. Most importantly, the rainbow trout population was rescued from infection by a single addition of the phage into the water in a flow-through fish tank system. Thus, F. columnare could be used as a model system to test the benefits and risks of phage therapy on a larger scale.

Phage display as a promising approach for vaccine development

OpenAIRE

Aghebati-Maleki, Leili; Bakhshinejad, Babak; Baradaran, Behzad; Motallebnezhad, Morteza; Aghebati-Maleki, Ali; Nickho, Hamid; Yousefi, Mehdi; Majidi, Jafar

2016-01-01

Bacteriophages are specific antagonists to bacterial hosts. These viral entities have attracted growing interest as optimal vaccine delivery vehicles. Phages are well-matched for vaccine design due to being highly stable under harsh environmental conditions, simple and inexpensive large scale production, and potent adjuvant capacities. Phage vaccines have efficient immunostimulatory effects and present a high safety profile because these viruses have made a constant relationship with the mamm...

A human gut phage catalog correlates the gut phageome with type 2 diabetes.

Science.gov (United States)

Ma, Yingfei; You, Xiaoyan; Mai, Guoqin; Tokuyasu, Taku; Liu, Chenli

2018-02-01

Substantial efforts have been made to link the gut bacterial community to many complex human diseases. Nevertheless, the gut phages are often neglected. In this study, we used multiple bioinformatic methods to catalog gut phages from whole-community metagenomic sequencing data of fecal samples collected from both type II diabetes (T2D) patients (n = 71) and normal Chinese adults (n = 74). The definition of phage operational taxonomic units (pOTUs) and identification of large phage scaffolds (n = 2567, ≥ 10 k) revealed a comprehensive human gut phageome with a substantial number of novel sequences encoding genes that were unrelated to those in known phages. Interestingly, we observed a significant increase in the number of gut phages in the T2D group and, in particular, identified 7 pOTUs specific to T2D. This finding was further validated in an independent dataset of 116 T2D and 109 control samples. Co-occurrence/exclusion analysis of the bacterial genera and pOTUs identified a complex core interaction between bacteria and phages in the human gut ecosystem, suggesting that the significant alterations of the gut phageome cannot be explained simply by co-variation with the altered bacterial hosts. Alterations in the gut bacterial community have been linked to the chronic disease T2D, but the role of gut phages therein is not well understood. This is the first study to identify a T2D-specific gut phageome, indicating the existence of other mechanisms that might govern the gut phageome in T2D patients. These findings suggest the importance of the phageome in T2D risk, which warrants further investigation.

Assessment of the microscreen phage-induction assay for screening hazardous wastes (1989)

Energy Technology Data Exchange (ETDEWEB)

Houk, V.S.; DeMarini, D.M.

1989-01-01

The Microscreen phage-induction assay, which quantitatively measures the induction of prophage Lambda in Escherichia coli WP2s(Lambda), was used to test 14 crude (unfractionated) hazardous industrial-waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 picograms per ml. Comparisons of the mutagenic activity of these waste samples in Salmonella and their ability to induce prophage Lambda indicate that the phage-induction assay was a more-sensitive indicator of genetic damage for this group of wastes. All but one of the wastes that were mutagenic to Salmonella were detected by the phage-induction assay, and 5 wastes not mutagenic to Salmonella were genetically active in the phage assay. The enhanced ability of the phage-induction assay to detect genotoxic activity may be related to the constituents comprising these waste samples. Partial chemical characterizations of the wastes showed high concentrations of carcinogenic metals, solvents, and chlorinated compounds, most of which are detected poorly by the Salmonella assay.

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

International Nuclear Information System (INIS)

Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

2008-01-01

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 ± 0.7 x 10 5 M -1 which indicates a strong binding close to that of antibody

Computational Modelling of Large Scale Phage Production Using a Two-Stage Batch Process

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Konrad Krysiak-Baltyn

2018-04-01

Full Text Available Cost effective and scalable methods for phage production are required to meet an increasing demand for phage, as an alternative to antibiotics. Computational models can assist the optimization of such production processes. A model is developed here that can simulate the dynamics of phage population growth and production in a two-stage, self-cycling process. The model incorporates variable infection parameters as a function of bacterial growth rate and employs ordinary differential equations, allowing application to a setup with multiple reactors. The model provides simple cost estimates as a function of key operational parameters including substrate concentration, feed volume and cycling times. For the phage and bacteria pairing examined, costs and productivity varied by three orders of magnitude, with the lowest cost found to be most sensitive to the influent substrate concentration and low level setting in the first vessel. An example case study of phage production is also presented, showing how parameter values affect the production costs and estimating production times. The approach presented is flexible and can be used to optimize phage production at laboratory or factory scale by minimizing costs or maximizing productivity.

Juvenile recurrent parotitis in children: diagnosis and treatment using sialography.

Science.gov (United States)

Narsimha Rao, Vanga V; Putta Buddi, Jai Shankar Homberhali; Kurthukoti, Ameet J

2014-01-01

Juvenile recurrent parotitis (JRP) is a nonobstructive, nonsuppurative parotid inflammation in young children. Causative factors are many such as allergy, infection, local autoimmune manifestations, and genetic inheritance have been suggested, but none have been proved. Parotid sialography is a hallmark in the diagnosis of JRP but newer modalities such as ultrasonography, computed tomography and magnetic resonance imaging-sialography are noninvasive investigative techniques. Recurrent attacks are often managed conservatively. Here we report a case of a 5-year-old child with JRP. Sailography can be used as both diagnostic and therapeutic modality.

An improved plating assay for determination of phage titer | Yang ...

African Journals Online (AJOL)

In this study, an improved plating assay was developed for detection of the number of recombinant phage Cap-T7 present in a test solution at a certain dilution point by counting the plaque forming units. The data demonstrated that the improved plating assay is fast, useful, and convenient for the determination of the phage ...

The Effect of Sleep Disordered Breathing on the Outcome of Stroke and Transient Ischemic Attack

DEFF Research Database (Denmark)

Birkbak, Johannes; Clark, Alice J; Rod, Naja Hulvej

2014-01-01

The primary objective was to systematically review the literature on how sleep disordered breathing (SDB) affects recurrence and death among stroke or transient ischemic attack (TIA) patients. A secondary objective was to evaluate how treatment of SDB with continuous positive airway pressure (CPAP...

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.

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

International Nuclear Information System (INIS)

McCallin, Shawna; Alam Sarker, Shafiqul; Barretto, Caroline; Sultana, Shamima; Berger, Bernard; Huq, Sayeda; Krause, Lutz; Bibiloni, Rodrigo; Schmitt, Bertrand; Reuteler, Gloria; Brüssow, Harald

2013-01-01

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

Temperate Streptococcus thermophilus phages expressing superinfection exclusion proteins of the Ltp type

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Yahya eAli

2014-03-01

Full Text Available Lipoprotein Ltp encoded by temperate Streptococcus thermophilus phage TP-J34 is the prototype of the wide-spread family of host cell surface-exposed lipoproteins involved in superinfection exclusion. When screening for other S. thermophilus phages expressing this type of lipoprotein, three temperate phages - TP-EW, TP-DSM20617 and TP-778 - were isolated. In this communication we present the total nucleotide sequences of TP-J34 and TP-778L. For TP-EW, a phage almost identical to TP-J34, besides the ltp gene only the two regions of deviation from TP-J34 DNA were analyzed: the gene encoding the tail protein causing an assembly defect in TP-J34 and the gene encoding the lysin, which in TP-EW contains an intron. For TP-DSM20617 only the sequence of the lysogeny module containing the ltp gene was determined. The region showed high homology to the same region of TP-778. For TP-778 we could show that absence of the attR region resulted in aberrant excision of phage DNA. The amino acid sequence of mature LtpTP-EW was shown to be identical to that of mature LtpTP-J34, whereas the amino acid sequence of mature LtpTP-778 was shown to differ from mature LtpTP-J34 in eight amino acid positions. LtpTP-DSM20617 was shown to differ from LtpTP-778 in just one amino acid position. In contrast to LtpTP-J34, LtpTP-778 did not affect infection of lactococcal phage P008 instead increased activity against phage P001 was noticed.

[Screening serum response special antibodies of U251 cell line from surface display phage antibody library].

Science.gov (United States)

Yu, Min; Tan, De-Yong; Qian, Wei; Lai, Jian-Hua; Sun, Gui-Lin

2004-05-01

U251 cell is a sensitive cell line to serum, which stops at G0 phase of cell cycle in no-serum medium, and recovers growth when the serum is added into no-serum medium. The cell can express corresponding proteins in different phase of cell cycle. Therefore it is very signification for the study of cell cycle regulation mechanism that explores these proteins. In this paper, the mouse antibody phage display library was added into the bottle in which the serum starvation U251 cells had been cultured, and the special antibody phages were absorbed. Then the absorbed antibody phages were amplified by adding E. coli TG1 and helper phage M13K07. Amplified antibody phages were added into bottle in which the serum cultured cell after serum starvation (follow named as serum recovered cells) were incubated, so that the cell absorbed the no-special antibody phages for the serum starvation cell and the special antibody phages were in supernatant. The remaining no-special antibody phages in the supernatant were discarded by repeating above program 3-4 times. The pure special antibody phages were gotten, and amplified by adding the host cell E. coli TG1 and helper phage M13K07. Then the host bacterium infected special antibody phage was spread on the plate medium with ampicillin, and the monoclonal antibody phages were gotten. Using same as above program, the monoclonal antibody phages absorbed specially for serum recovered U251 cells were obtained when the serum recovered cells instead of serum starvation cells and serum starvation cells instead of serum recovered cells. In this study, ninety-six positive monoclonal antibody phages that absorbed specially the serum starvation cells and eighty-two positive monoclonal antibody phages that absorbed specially the serum recovered cells were obtained. By using cell immunochemistry assay, two special signification antibodies were obtained. one (No.11) was the strong response in serum starvation cells, the other (No.2) was the strong

Extramedullary spinal teratoma presenting with recurrent aseptic meningitis.

Science.gov (United States)

Mpayo, Lucy L; Liu, Xiao-Hong; Xu, Man; Wang, Kai; Wang, Jiao; Yang, Li

2014-06-01

Spinal teratomas are extremely rare; they constitute meningitis. A 7-year-old boy presented with paroxysmal abdominal pain and a history of recurrent aseptic meningitis. Kernig and Brudzinski signs were present. Lumber puncture revealed pleocytosis with no evidence of bacteria growth. Imaging of the spine revealed a cystic lesion in spinal cord at thoracic level 9-11. Endoscopic excision of the cyst was successfully performed. Surgical and histopathological findings confirmed extramedullary matured teratoma. As the symptomatic attacks of spontaneous rupture of spinal teratoma resemble presentations of Mollaret meningitis, spinal teratoma should be considered in the differential diagnosis of Mollaret meningitis. We describe a rare example of spinal teratoma causing recurrent meningitis. Spine imaging should be considered in individuals with recurrent aseptic meningitis as this promotes earlier diagnosis, more appropriate treatment, and improved neurological outcome. Copyright © 2014 Elsevier Inc. All rights reserved.

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. PMID:27446059

Survival and mutagenesis in UV-irradiated phage: Multi-hit kinetics of mutation induction and lack of indirect induction by infection with UV-irradiated phage of error-prone repair

International Nuclear Information System (INIS)

Krauss, G.; Mennigmann, H.D.; Kaplan, R.W.

1980-01-01

The paper is concerned with the question of whether Weigle-reactivation (WR) and Weigle-mutagenesis (WM) can be indirectly induced by infection with UV-irradiated phage. Experiments neither with phage lambda of Escherichia coli nor with phage kappa of Serratia marcescens show such induction. In this respect phage DNA differs from F'-DNA or Hfr-DNA; possible explanations are discussed. In both systems clear plaque mutations can also be induced by UV without irradiation of the host cells; they appear, in unirradiated and irradiated host cells, with an increase in frequency which is greater than proportional to the UV dose. It is concluded that mutation induction of phage in the unirradiated host cells is due to a low level constitutive mutagenic repair; this could either be due to 'spontaneous' induction of the mutagenic SOS function or it could be a mechanism different from this one. Host irradiation would give rise to additional activity by the induced SOS function leading to WR and WM. It is further concluded that deviation of the induction kinetics from a linear dose-dependence is not due to the necessary induction of SOS functions. (author)

Genomic Characterization of a Novel Phage Found in Black Abalone (Haliotis cracherodii) Infected with Withering Syndrome

Science.gov (United States)

Closek, C. J.; Langevin, S.; Burge, C. A.; Crosson, L.; White, S.; Friedman, C. S.

2016-02-01

Withering syndrome (WS), caused by the bacterium Candidatus Xenohaliotis californiensis, a Rickettsia-like organism (RLO), infects many species of abalone. Black abalone (Haliotis cracherodii), one of two endangered species of abalone, has experienced high population losses along the California coast due to WS. Recently, we observed reduced pathogenicity and mortality events in RLO-infected abalone when a novel bacteriophage (phage) was also present. To better understand phage-bacterium dynamics and develop more informative diagnostic tools, we sequenced the genome of the novel phage associated with the RLO responsible for WS. Metagenomic sequencing libraries were prepared with extracted genomic DNA from two experimentally infected H. cracherodii and phage sequences were enriched using hydroxyapatite chromatography normalization. Normalized libraries were individually barcoded and sequenced with Illumina MiSeq. Raw sequence reads were processed using VIrominer and de novo assembly produced one single phage-like contig (35.7Kb) from the experimentally infected abalone. This highly divergent genome had closest homology with a virus associated with abalone shriveling syndrome (SS). Of the 34 predicted ORFs, overlapping homology with the SS virus ranged from 20-72%, demonstrating the phage sequenced is genetically distinct from any known phage. The phage-like sequences represented a significant portion of the total reads sequenced ( 2 million of the 12 million paired-end reads; 17%) and we obtained 94,000X coverage across the novel phage genome. Beyond characterization of this novel phage, which appears to reduce pathogenicity of the RLO, the genome enabled us to develop quantitative PCR and in situ hybridization assays as diagnostic tools. These tools allow us to detect and quantify this phage in the endangered H. cracherodii.

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

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.......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 the diversity of their phage predators. We quantify how the parameter space of coexistence exponentially decreases with diversity. For diversity to grow, an open or evolving ecosystem needs to climb a narrowing 'diversity staircase' by alternatingly adding new bacteria and phages. The unfolding coevolutionary...

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.

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

Science.gov (United States)

2016-09-01

Impedance Measurements Could Accelerate Phage-Based Identification of Bacillus anthracis And Other Bacteria Thomas Brown, Salwa Shan, Teresa...infection can be detected as early as one hour after exposing as few as 105 CFU bacteria to the stressor. We predicted that similar responses could be used... bacteria to form confluent growth and for phage-induced plaques to appear. Techniques that permit faster detection of species-specific bacteria /phage

Occurrence and predictors of persistent impaired glucose tolerance after acute ischemic stroke or transient ischemic attack

NARCIS (Netherlands)

S. Fonville (Susanne); H.M. den Hertog (Heleen); A.A.M. Zandbergen (Adrienne); P.J. Koudstaal (Peter Jan); H.F. Lingsma (Hester)

2014-01-01

textabstractBackground Impaired glucose tolerance is often present in patients with a transient ischemic attack (TIA) or ischemic stroke and doubles the risk of recurrent stroke. This impaired glucose tolerance can be transient, reflecting an acute stress response, or persistent, representing

The use of antibiotics to improve phage detection and enumeration by the double-layer agar technique

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Ferreira Eugénio C

2009-07-01

Full Text Available Abstract Background The Double-Layer Agar (DLA technique is extensively used in phage research to enumerate and identify phages and to isolate mutants and new phages. Many phages form large and well-defined plaques that are easily observed so that they can be enumerated when plated by the DLA technique. However, some give rise to small and turbid plaques that are very difficult to detect and count. To overcome these problems, some authors have suggested the use of dyes to improve the contrast between the plaques and the turbid host lawns. It has been reported that some antibiotics stimulate bacteria to produce phages, resulting in an increase in final titer. Thus, antibiotics might contribute to increasing plaque size in solid media. Results Antibiotics with different mechanisms of action were tested for their ability to enhance plaque morphology without suppressing phage development. Some antibiotics increased the phage plaque surface by up to 50-fold. Conclusion This work presents a modification of the DLA technique that can be used routinely in the laboratory, leading to a more accurate enumeration of phages that would be difficult or even impossible otherwise.

Detection of sulfur mustard adducts in human callus by phage antibodies

NARCIS (Netherlands)

Bikker, F.J.; Mars-Groenendijk, R.H.; Noort, D.; Fidder, A.; Schans, G.P. van der

2007-01-01

As part of a research program to develop novel methods for diagnosis of sulfur mustard exposure in the human skin the suitability of phage display was explored. Phage display is a relative new method that enables researchers to quickly evaluate a huge range of potentially useful antibodies, thereby

Isolation and Expression of the Lysis Genes of Actinomyces naeslundii Phage Av-1

Science.gov (United States)

Delisle, Allan L.; Barcak, Gerard J.; Guo, Ming

2006-01-01

Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these species-specific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective λ holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products. PMID:16461656

Different duration of Colchicine for preventing recurrence of Gouty arthritis

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H Karimzadeh

2006-05-01

Full Text Available Background: Gout is a Common recurrent clinical syndrome characterized by increased serum uric acid and recurrent attacks of acute arthritis. Colchicine is used for Prophylaxis against recurrence of arthritis, but the duration of its administration has mentioned variable. In this study, optimal duration of prophylactic colchicine for prevention of gouty arthritis was assessed. Methods : In a clinical trial 190 patients with gouty arthritis divided randomly to group 1,2and 3 and received colchicine for 3 to 6, 7 to 9 and 10 to 12 months then colchicine discontinued and the patients followed one year for recurrence of arthritis. Result assessed by survival analysis with Kaplan –Meier method. Results: The probability of recurrence of arthritis (in order of duration of colchicine prophylaxis was 54%, 27.5% and 23%, respectively. The difference between group one and others was statistically significant, but between group 2 and 3 was not statistically significant. Conclusion: The most suitable duration of colchicine prophylaxis that accompanied with lower recurrence rate was 7-9 months, which seems more cost -effective than 10-12 months regimen. Key words: Gout, Colchicine, Arthritis, Recurrence

Viruses in the marine environment: community dynamics, phage-host interactions and genomic structure

OpenAIRE

Lara de la Casa, Elena

2014-01-01

There are an estimated 1030 viruses in the world oceans, the majority of which are phages (viruses that infect bacteria). Extensive research has demonstrated the significant influence of marine phages on microbial abundance, community structure, genetic exchange and global biogeochemical cycles. In this thesis, we contribute to increase the knowledge about the ecological role of viruses in marine systems, but also we aimed to provide a better understanding about the interactions between phage...

Phage type and sensitivity to antibiotics of Staphylococcus aureus film-forming strains isolated from airway mucosa

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O. S. Voronkova

2014-10-01

Full Text Available Today film-forming strains of bacteria play very important role in clinical pathology. Staphylococci are ones of most dangerous of them. This bacteria can determine different pathological processes, for example, complication of airway mucosa. The ability to form a biofilm is one of the main properties of nosocomial strains. These strains should be monitored and their carriers are to be properly treated. To determine the origin of staphylococci strains we used bacteriophages from the International kit. The aim of research was to determine the phage type of staphylococci film-forming strains, that were isolated from naso-pharingial mucosa. Phage typing has been carried out for 16 film-forming strains of S. aureus. To solve this problem, we used the International phage kit by Fisher’s method. As a result, sensitivity to phages from the International kit showed 53.8% of studied strains of S. aureus. 64.3% of sensitivity strains were lysed by one of the phage, 21.4% – were by two of the phages, 14.3% – by three of the phages. Isolates were sensitive to phages: 81 – 42.9%, 75 – 35.7%, 28.6% were sensitive to phages 47 and 53. All cases of detection of sensitivity to phage 47 coincided with the ability to form biofilm. Among non-film-forming strains there was no sensitive strains for this phage. Film-forming strains resist to erythromycin (62.5%, ciprofloxacin (43.8%, gentamicin (56.3%, tetracycline (87.5%, amoxicillin (93.8%, and cefuroxime (37.5%. All cases of sensitivity to phage 47 coincided with resistance to erythromycin, amoxicillin and tetracycline. For two of these strains, we also defined resistance to gentamicin and for one of them – to ciprofloxacin. Results of research allowed to relate the bacterial cultures for determining the type. This may have implications for studying of film-forming ability, because surface structures of bacterial cell take place in this process. Belonging of an isolate to specific phage type may

Use of phages to control Vibrio splendidus infection in the juvenile sea cucumber Apostichopus japonicus.

Science.gov (United States)

Li, Zhen; Li, Xiaoyu; Zhang, Jiancheng; Wang, Xitao; Wang, Lili; Cao, Zhenhui; Xu, Yongping

2016-07-01

In the present study, we isolated 3 bacteriophages with the ability to control Vibrio splendidus, a bacterium known to cause disease in the juvenile sea cucumber. These bacteriophages were designated as vB_VspS_VS-ABTNL-1 (PVS-1), vB_VspS_VS-ABTNL-2 (PVS-2) and vB_VspS_VS-ABTNL-3 (PVS-3). The ability of the 3 phages to inhibit the growth of V. splendidus VS-ABTNL was tested in vitro using each of the 3 phages individually or in the form of a cocktail of all 3 phages in the proportion of 1:1:1. All treated cultures produced a significant (P sea cucumbers (23 ± 2 g) were randomly assigned to 1 of 6 treatments. Each treatment was housed in 3 PVC tanks (38 cm × 54 cm × 80 cm) with 20 sea cucumbers per tank. Six diets were prepared including an unsupplemented control diet, antibiotic treatment diet, 3 diets containing 1 of the 3 phages individually and a diet containing a cocktail of all 3 phages. After 60 days of feeding, all sea cucumber were challenged with V. splendidus VS-ABTNL by immersion in sea water containing a bacterial concentration of 6 × 10(6) CFU/mL for 2 days. The survival rate of sea cucumbers during the next 10 days was 18% for the unsupplemented diet, 82% for the antibiotic treatment, 82% for the phage cocktail, 65% for phage PVS-1, 58% for phage PVS-2 and 50% for phage PVS-3. There were no significant differences in weight gain, ingestion rate or feed conversion among sea cucumber fed the 4 phage treatments compared with those fed the unsupplemented diet (P > 0.05). The levels of nitric oxide synthase and acid phosphatase of sea cucumbers fed phage-containing diets were significantly (P  0.05) were detected among the 4 phage-fed treatments. An additional study was conducted in which 60 healthy sea cucumbers (23 ± 2 g) were randomly assigned to a control, an untreated group and a test group to investigate the effects of injecting phages by coelomic injection on the survival rate and enzyme activities in the coelomic fluid

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

in the two cocktails. One of these showed no similarity to publicly available phage genomes. Representatives of phages targeting E. faecalis, E. faecium, E. coli, Proteus, P. aeruginosa and S. aureus were found in both cocktails. Finally, we estimated larger overlap of the PYO2000 cocktail to PYO97 compared...

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.

Photoreactivation of cells and phages inactivated by UV of ecological wave-lengths

International Nuclear Information System (INIS)

Samojlova, K.A.; Yanovska, Eh.; Vizdalova, M.; Ceskoslovenska Akademie Ved, Brno. Biofysikalni Ustav)

1979-01-01

It has been found that the photoreactivity of infusoria Paramecium caudatum and bacteria Escherichia coli is high and practically similar if they are irradiated with short-wave (254 nm) and mean-wave (300-315 nm) UV radiation. The cells damaged with long-wave (315-400 nm) UV rays are not photoactivated. The latter is caused by the appearance of nonphotoreactivated damages since the phages jrradiated with the same UV rays are reactivated extremely weakly in the intact cells of bacteria (phage T7) or are not reactivated at all (phage lambdasub(c1 857))

Efficacy and Safety of a Bovine-Associated Staphylococcus aureus Phage Cocktail in a Murine Model of Mastitis

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Koen Breyne

2017-11-01

Full Text Available Overuse of antibiotics is a major problem in the treatment of bovine mastitis, and antibiotic treatment is frequently non-curative, thus alternative treatments are necessary. The primary aim of this study was to evaluate the efficacy of a purified phage cocktail for treatment of bovine Staphylococcus aureus mastitis in a well-defined mouse model. Candidate phages were selected based on their in vitro performance and subsequently processed into an optimally composed phage cocktail. The highest scoring phages were further tested for efficacy and resistance suppression in broth and raw milk, with and without supplemental IgG. As these in vitro results displayed significant decreases in CFU, the cocktail was purified for testing in vivo. Lactating mice were intramammarily inoculated with S. aureus N305 (ATCC 29740, a clinical bovine mastitis isolate commonly used for experimental infection of dairy cows. The phage cocktail was applied via the same route 4 h post-inoculation. Treated mammary glands were graded for gross pathological appearance and excised for bacterial and phage load quantification as well as histopathology. Observation of gross macroscopic and histopathological changes and CFU quantification demonstrated that the phage cocktail treatment significantly improved mastitis pathology and decreased bacterial counts. Phage PFU quantification indicated that the tested phage cocktail treatment was able to maintain high intramammary phage titers without spreading systemically. The in vivo results complement the in vitro data and support our concept of phage therapy as an innovative alternative or supplementation therapy to antibiotics for the treatment of bovine mastitis.

Efficacy and Safety of a Bovine-Associated Staphylococcus aureus Phage Cocktail in a Murine Model of Mastitis.

Science.gov (United States)

Breyne, Koen; Honaker, Ryan W; Hobbs, Zachary; Richter, Manuela; Żaczek, Maciej; Spangler, Taylor; Steenbrugge, Jonas; Lu, Rebecca; Kinkhabwala, Anika; Marchon, Bruno; Meyer, Evelyne; Mokres, Lucia

2017-01-01

Overuse of antibiotics is a major problem in the treatment of bovine mastitis, and antibiotic treatment is frequently non-curative, thus alternative treatments are necessary. The primary aim of this study was to evaluate the efficacy of a purified phage cocktail for treatment of bovine Staphylococcus aureus mastitis in a well-defined mouse model. Candidate phages were selected based on their in vitro performance and subsequently processed into an optimally composed phage cocktail. The highest scoring phages were further tested for efficacy and resistance suppression in broth and raw milk, with and without supplemental IgG. As these in vitro results displayed significant decreases in CFU, the cocktail was purified for testing in vivo . Lactating mice were intramammarily inoculated with S. aureus N305 (ATCC 29740), a clinical bovine mastitis isolate commonly used for experimental infection of dairy cows. The phage cocktail was applied via the same route 4 h post-inoculation. Treated mammary glands were graded for gross pathological appearance and excised for bacterial and phage load quantification as well as histopathology. Observation of gross macroscopic and histopathological changes and CFU quantification demonstrated that the phage cocktail treatment significantly improved mastitis pathology and decreased bacterial counts. Phage PFU quantification indicated that the tested phage cocktail treatment was able to maintain high intramammary phage titers without spreading systemically. The in vivo results complement the in vitro data and support our concept of phage therapy as an innovative alternative or supplementation therapy to antibiotics for the treatment of bovine mastitis.

Biology of the temperate Streptococcus thermophilus bacteriophage TP-J34 and physical characterization of the phage genome

International Nuclear Information System (INIS)

Neve, Horst; Freudenberg, Wiebke; Diestel-Feddersen, Frederike; Ehlert, Regina; Heller, Knut J.

2003-01-01

The temperate Streptococcus thermophilus bacteriophage TP-J34 was identified in the lysogenic host strain J34. The majority of phage particles produced upon induction was defective and noninfectious, consisting of DNA-filled heads lacking tails. A physical map (45.6 kb) was established. Analysis of minor restriction bands of the DNA isolated from phage particles as well as the analysis of the protein pattern indicated that phage TP-J34 is a pac-type phage. This was confirmed by immunoelectron microscopy using antisera raised against virulent cos- and pac-type S. thermophilus phages. The lysogenic host J34 but not its noninducible derivate J34-12 contained phage DNA in the nonintegrated state and exhibited autolysis at elevated temperatures. Prophage-carrying strains grew homogeneously while 16 of 20 prophage-cured derivatives aggregated and sedimented rapidly. When phage TP-J34 was propagated lytically on a prophage-cured host strain, a 2.7-kb site-specific deletion occurred in the phage genome. This deletion was also identified in the prophage DNAs of relysogenized strains

Is phage therapy acceptable in the immunocompromised host?

Science.gov (United States)

Borysowski, Jan; Górski, Andrzej

2008-09-01

Over the last decade, bacteriophages (bacterial viruses) have emerged as the major alternative to antibiotics in the treatment of antibiotic-resistant infections. While a considerable body of evidence has accumulated for the efficacy and safety of phage therapy in immunocompetent patients, data remain relatively scarce regarding its use in the immunocompromised host. To our knowledge, the present article is the first to summarize all findings, of both experimental and clinical studies, that may be relevant to the employment of phage therapy in immunocompromised patients. The available data suggest that bacteriophages could also be an efficacious and safe therapeutic modality in such patients.

Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

Science.gov (United States)

Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2015-02-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system.

Science.gov (United States)

Loh, Qiuting; Leong, Siew Wen; Tye, Gee Jun; Choong, Yee Siew; Lim, Theam Soon

2015-05-15

The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display. Copyright © 2015 Elsevier Inc. All rights reserved.

The Agricultural Antibiotic Carbadox Induces Phage-mediated Gene Transfer in Salmonella

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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

Restriction of phage T4 internal protein I mutants by a strain of Escherichia coli

International Nuclear Information System (INIS)

Black, L.W.; Abremski, K.

1974-01-01

Phage T4 internal protein I(IPI), a small (ca, 10,000 MW), basic protein injected into the host with the phage DNA, is not required for infection of most hosts, but mutants defective in IPI are restricted by at least one naturally occurring strain of Escherichia coli, CT 596 (CT). Phages lacking IPI (IPI - ) appear to inject their DNA and bind it to the membrane of CT cells as well as wild-type phage T4 does, but shutoff of host protein synthesis, initiation of T4 protein synthesis, and cell killing are abnormal in the IPI - mutant infected CT host. The injection of IPI appears to be important in allowing T4 DNA to carry out early steps involved in takeover of this host. Restriction of IPI - phage growth by CT cells appears to be due, at least in part, to a defective prophage it harbors which renders the host resistant to successful infection by phage T4 which lack IPI or rII functions. Bacteria cured of this prophage can be infected by mutants defective in these functions. The resistance of CT cells to other coliphages, and the question of T-even phage internal protein diversity are discussed. (U.S.)

The factors affecting effectiveness of treatment in phages therapy, mini review

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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

Study of the phage production efficiency in the bacteria lysis processes

International Nuclear Information System (INIS)

Vidania Munoz, R. de; Garces, F.; Davila, C. A.

1979-01-01

In this work we present a search for the best production conditions of λvir andλ clear phages In E coli K12 and E coli C 6 00 infected cells respectively. By keeping fixed some parameters of the process as the bacterial and phage generation times and (he bacterial burst side, we have finder that the lysis yield is strongly dependent on the multiplicity and in a lesser degree on the infection time. It appears from the experimental results that other variables are important, as infection efficiency and approach time from phages to bacteria. We will try to describe the lysis phenomenon by a numerical model on the bases of the se experimental results. (Author) 11 refs

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

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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

An outbreak of Salmonella Enteritidis phage type 34a infection associated with a Chinese restaurant in Suffolk, United Kingdom

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Mahgoub Hamid

2004-09-01

Full Text Available Abstract Background On 30th July 2002, the Suffolk Communicable Disease Control Team received notifications of gastrointestinal illness due to Salmonella Enteritidis in subjects who had eaten food from a Chinese restaurant on 27th July. An Outbreak Control Team was formed resulting in extensive epidemiological, microbiological and environmental investigations. Methods Attempts were made to contact everybody who ate food from the restaurant on 27th July and a standard case definition was adopted. Using a pre-designed proforma information was gathered from both sick and well subjects. Food specific attack rates were calculated and two-tailed Fisher's exact test was used to test the difference between type of food consumed and the health status. Using a retrospective cohort design univariate Relative Risks and 95% Confidence Intervals were calculated for specific food items. Results Data was gathered on 52 people of whom 38 developed gastrointestinal symptoms; 16 male and 22 female. The mean age was 27 years. The mean incubation period was 30 hours with a range of 6 to 90 hours. Food attack rates were significantly higher for egg, special and chicken fried rice. Relative risk and the Confidence interval for these food items were 1.97 (1.11–3.48, 1.56 (1.23–1.97 and 1.48 (1.20–1.83 respectively. Interviews with the chef revealed that many eggs were used in the preparation of egg-fried rice, which was left at room temperature for seven hours and was used in the preparation of the other two rice dishes. Of the 31 submitted stool specimens 28 tested positive for S Enteritidis phage type 34a and one for S Enteritidis phage type 4. Conclusion In the absence of left over food available for microbiological examination, epidemiological investigation strongly suggested the eggs used in the preparation of the egg-fried rice as the vehicle for this outbreak. This investigation highlights the importance of safe practices in cooking and handling of eggs in

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.

Interaction of the phage-xanthomonas campestris (Pammel) Dowson at the eletronic microscopy level, Virazole effect and radioautographic study of the phage action on the host

International Nuclear Information System (INIS)

Sittolin, I.M.

1982-04-01

A bacteriophage from the cabbage tissue infected with Xanthomonas campestris is described. The infection process is studied through a negative staining technique (PTA) and ultrathin section. The effect of Virazole, an antivirus agent, is tested. Radioautography showed that the phage presented a reasonable domain on the bacterial host genome since the beginning of the treatment. Sorological reactions indicated the induction of specific antibodies for the phage. (M.A.C.) [pt

The genome and structural proteome of YuA, a new Pseudomonas aeruginosa phage resembling M6.

Science.gov (United States)

Ceyssens, Pieter-Jan; Mesyanzhinov, Vadim; Sykilinda, Nina; Briers, Yves; Roucourt, Bart; Lavigne, Rob; Robben, Johan; Domashin, Artem; Miroshnikov, Konstantin; Volckaert, Guido; Hertveldt, Kirsten

2008-02-01

Pseudomonas aeruginosa phage YuA (Siphoviridae) was isolated from a pond near Moscow, Russia. It has an elongated head, encapsulating a circularly permuted genome of 58,663 bp, and a flexible, noncontractile tail, which is terminally and subterminally decorated with short fibers. The YuA genome is neither Mu- nor lambda-like and encodes 78 gene products that cluster in three major regions involved in (i) DNA metabolism and replication, (ii) host interaction, and (iii) phage particle formation and host lysis. At the protein level, YuA displays significant homology with phages M6, phiJL001, 73, B3, DMS3, and D3112. Eighteen YuA proteins were identified as part of the phage particle by mass spectrometry analysis. Five different bacterial promoters were experimentally identified using a promoter trap assay, three of which have a sigma54-specific binding site and regulate transcription in the genome region involved in phage particle formation and host lysis. The dependency of these promoters on the host sigma54 factor was confirmed by analysis of an rpoN mutant strain of P. aeruginosa PAO1. At the DNA level, YuA is 91% identical to the recently (July 2007) annotated phage M6 of the Lindberg typing set. Despite this level of DNA homology throughout the genome, both phages combined have 15 unique genes that do not occur in the other phage. The genome organization of both phages differs substantially from those of the other known Pseudomonas-infecting Siphoviridae, delineating them as a distinct genus within this family.

X-ray inactivation and reactivation characteristics of the phage 'kappa'

International Nuclear Information System (INIS)

Bhattacharyya, S.C.; Samad, S.A.; Mandal, J.C.; Chatterjee, S.N.

1991-01-01

Vibrio cholerae temperate phage 'kappa' was inactivated by X-ray (60 kV) in a dose dependent manner, the inactivation dose leading to 37% survival (D 37 ) in PBS, pH 7.4 being 0.36 kGy. The phages were significantly protected against X-ray irradiation when histidine or cysteine or both were present in PBS or when phages were irradiated in nutrient broth. The maximum protection was offered when histidine (10.0 nM) and cysteine (10.0 nM) were both present in PBS (dose enhancement factor being 4.17). The X-irradiated 'kappa' phages also underwent a small but significant Weigle reactivation and also Weigle mutagenesis in the UV-irradiated V. cholerae host H218Sm r . The Weigle factor (WF) or the frequency of clear plaque mutants increased with increasing UV dose, attained a maximum at the UV dose of 2.4 Jm -2 and thereafter decreased gradually with further increase of UV dose. The X-ray dose (D)-survival (S) curves could be empirically described by the equation S=exp-(aD+bD 2 ) where 'a' and 'b' are constants depending on the irradiation conditions and good agreement between the theoretical curves and experimental data was obtained. (author). 1 5 refs., 2 fig., 1 tab

Eleven episodes of recurrent optic neuritis of the same eye for 22 years eventually diagnosed as neuromyelitis optica spectrum disorder.

Science.gov (United States)

Yew, Yih Chian; Hor, Jyh Yung; Lim, Thien Thien; Kanesalingam, Ruban; Ching, Yee Ming; Arip, Masita; Easaw, P E Samuel; Eow, Gaik Bee

2016-11-01

It is difficult to predict whether a particular attack of neuromyelitis optica spectrum disorder (NMOSD) will affect the optic nerve [optic neuritis (ON): unilateral or bilateral], spinal cord (myelitis), brain or brainstem, or a combination of the above. We report an interesting case of recurrent ON of the same eye for a total of 11 episodes in a Chinese woman. Over a period of 22 years, the attacks only involved the left eye, and never the right eye and also no myelitis. For a prolonged duration, she was diagnosed as recurrent idiopathic ON. Only until she was tested positive for aquaporin 4 antibody that her diagnosis was revised to NMOSD. Optical coherence tomography revealed thinning of the retinal nerve fibre layer (RNFL) for the affected left eye, while the RNFL thickness was within normal range for the unaffected right eye. The disability accrual in NMOSD is generally considered to be attack-related - without a clinical attack of ON, there shall be no visual impairment, and no significant subclinical thinning of RNFL. Our case is in agreement with this notion. This is in contrast to multiple sclerosis where subclinical RNFL thinning does occur. This case highlights the importance of revisiting and questioning a diagnosis of recurrent idiopathic ON particularly when new diagnostic tools are available. Copyright © 2016 Elsevier B.V. All rights reserved.

Phage-resistance linked to cell heterogeneity in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1.

Science.gov (United States)

Suárez, Viviana B; Maciel, Natalia; Guglielmotti, Daniela; Zago, Miriam; Giraffa, Giorgio; Reinheimer, Jorge

2008-12-10

The aim of this work was to study the relationship between the cell morphological heterogeneity and the phage-resistance in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1. Two morphological variants (named C and T) were isolated from this strain. Phage-resistant derivatives were isolated from them and the percentage of occurrence of confirmed phage-resistant cells was 0.001% of the total cellular population. Within these phage-resistant cell derivatives there were T (3 out of 4 total isolates) and C (1 out of 4 total isolates) variants. The study of some technological properties (e.g. proteolytic and acidifying activities) demonstrated that most of phage-resistant derivatives were not as good as the parental strain. However, for one derivative (a T variant), the technological properties were better than those of the parental strain. On the other hand, it was possible to determinate that the system of phage-resistance in the T variants was interference in adsorption step, with adsorption rates M.

Thermal-Stability and Reconstitution Ability of Listeria Phages P100 and A511

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Hanie Ahmadi

2017-12-01

Full Text Available The study evaluated the thermal-stability of Listeria phages P100 and A511 at temperatures simulating the preparation of ready-to-eat meats. The phage infectivity after heating to 71°C and holding for a minimum of 30 s, before eventually cooling to 4°C were examined. Higher temperatures of 75, 80, and 85°C were also tested to evaluate their effect on phages thermal-stability. This study found that despite minor differences in the amino acid sequences of their structural proteins, the two phages responded differently to high temperatures. P100 activity declined at least 10 log (PFU mL-1 with exposure to 71°C (30 s and falling below the limit of detection (1 log PFU mL-1 while, A511 dropped from 108 to 105 PFU mL-1. Cooling resulted in partial reconstitution of P100 phage particles to 103 PFU mL-1. Exposure to 75°C (30 s abolished A511 activity (8 log PFU mL-1 and both phages showed reconstitution during cooling phase after exposure to 75°C. P100 exhibited reconstitution after treatment at 80°C (30 s, conversely A511 showed no reconstitution activity. Heating P100 to 85°C abolished the reconstitution potential. Substantial differences were found in thermal-stability and reconstitution of the examined phages showing A511 to be more thermo-stable than P100, while P100 exhibited reconstitution during cooling after treatment at 80°C which was absent in A511. The differences in predicted melting temperatures of structural proteins of P100 and A511 were consistent with the observed differences in thermal stability and morphological changes observed with transmission electron microscopy.

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.

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.

Association between phage types and antimicrobial resistance among bovine isolates of Staphylococcus aureus in 10 countries

DEFF Research Database (Denmark)

Vintov, J.; Aarestrup, Frank Møller; Zinn, C. E.

2003-01-01

This study was conducted to investigate the diversity of phage types and associations between penicillin resistance and phage types among 815 Staphylococcus aureus isolates from bovine mastitis in nine European countries and USA. All isolates were examined for susceptibility to antimicrobial agents...... associated with penicillin resistance in contrast to phage group I (P = 0.0023) and phage complex-80 (P = 0.0066). This study confirms that a large number of phage types of S. aureus cause bovine mastitis, but that some types predominate. In addition, these findings could indicate that the use of penicillin...... in the bovine environment has selected for specific types of S. aureus in countries with a high frequency of resistance. (C) 2003 Elsevier B.V. All rights reserved....

A study of Salmonella typhi isolated in Suez Canal area. Biotyping, phage typing and colicinogenic property.

Science.gov (United States)

Shoeb, S; Khalifa, I; el Daly, O; Heiba, A; Farmer, J; Brenner, F; el Batawi, Y

1989-01-01

In this work a total of 82 strains of Salmonella typhi were isolated from Egyptian patients diagnosed as quiry enteric fever. These cases were from Ismalia, Suez and port Said Areas. The strains fell in 16 phage types. Phage types N, 40, E1, and degraded Vi were the commonest phage type in Ismailia, while phage types degraded Vi and C1 were the commonest in Port Said. Phage types Di-N, degraded Vi, A and C1 were the commonest in Suez. Chemotyping of Salmonella typhi showed that the majority of the strains belonged to chemotype I (82%), and the rest belonged to chemotype II (18%). Colicin production was negative and all the strains were susceptible to the currently used antibiotics.

Two Novel Myoviruses from the North of Iraq Reveal Insights into Clostridium difficile Phage Diversity and Biology

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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.

Comparative genomics and functional analysis of the 936 group of lactococcal Siphoviridae phages

NARCIS (Netherlands)

Murphy, James; Bottacini, Francesca; Mahony, Jennifer; Kelleher, Philip; Neve, Horst; Zomer, Aldert; Nauta, Arjen; van Sinderen, Douwe

2016-01-01

Genome sequencing and comparative analysis of bacteriophage collections has greatly enhanced our understanding regarding their prevalence, phage-host interactions as well as the overall biodiversity of their genomes. This knowledge is very relevant to phages infecting Lactococcus lactis, since they

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....

Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

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M Lisa Phipps

Full Text Available Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1 ensure efficient display; 2 maximize the ability to select high affinity ligands; and 3 minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

Science.gov (United States)

Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

TRANSDUCTION OF BACILLUS LICHENIFORMIS AND BACILLUS SUBTILIS BY EACH OF TWO PHAGES1

Science.gov (United States)

Taylor, Martha J.; Thorne, Curtis B.

1963-01-01

Taylor, Martha J. (U.S. Army Biological Laboratories, Fort Detrick, Frederick, Md.) and Curtis B. Thorne. Transduction of Bacillus licheniformis and Bacillus subtilis by each of two phages. J. Bacteriol. 86:452–461. 1963.—A second transducing bacteriophage, designated SP-15, was isolated from the same soil-sample culture filtrate that supplied the Bacillus subtilis transducing phage, SP-10, reported earlier from this laboratory. SP-10 and SP-15 differ serologically and in several other respects, but share the ability to propagate on B. subtilis W-23-Sr (streptomycin-resistant) and B. licheniformis ATCC 9945a, and to mediate general transduction in either species when propagated homologously. Attempts to transduce between the species have failed. SP-10 forms plaques readily on both W-23-Sr and 9945a; SP-15 forms minute plaques on W-23-Sr and has shown no evidence of any lytic activity on 9945a. Maximal recoveries of prototrophic colonies from mixtures of SP-10 with auxotrophs of either W-23-Sr or 9945a were obtained only when excess phage was neutralized by post-transduction treatment with specific phage antiserum. Such treatment was not necessary for maximal recovery of transductants effected by SP-15. Unlike SP-10, SP-15 propagated on W-23-Sr did not transduce B. subtilis 168 (indole−). SP-15 transduced B. licheniformis more efficiently than did SP-10. Neither phage was able to transduce B. licheniformis as efficiently as it transduced B. subtilis. The differing influences of multiplicity of infection were compared for the two phages in both species. PMID:14066421

Transient ischaemic attacks clinics provide equivalent and more efficient care than early in-hospital assessment.

Science.gov (United States)

Martínez-Martínez, M M; Martínez-Sánchez, P; Fuentes, B; Cazorla-García, R; Ruiz-Ares, G; Correas-Callero, E; Lara-Lara, M; Díez-Tejedor, E

2013-02-01

Clinics for early management of transient ischaemic attacks (TIAs) have been developed in some stroke centres, resulting in reduced recurrence rates compared to appointment-based outpatient management, thus saving on hospitalization. We analysed the care process, recurrence rates and economic impact of the first year of work in our early-management TIA clinic and compared these with our previous in-hospital study protocols for low- and moderate-risk TIA patients. This was a prospective evaluation of the management of low- to moderate-risk TIA patients, comparing a new TIA clinic model (2010) with a previous hospitalization model (2009). Demographic data, vascular risk factor profiles, diagnostic test performance, secondary prevention measures, final aetiological diagnoses and cerebrovascular recurrences at 7 and 90 days were compared between in-hospital and TIA clinic assessed patients. We also carried out an economic comparison of the costs of each model's process. Two hundred and eleven low- to moderate-risk TIA patients were included, of whom 40.8% were hospitalized. There were no differences between the TIA clinic assessed and in-hospital assessed patients in terms of risk factor diagnosis and secondary prevention measures. The stroke recurrence rate (2.4% vs. 1.2%; P = 0.65) was low and similar for both groups (CI 95%, 0.214-20.436; P = 0.52). Cost per patient was €393.28 for clinic versus €1931.18 for in-hospital management. Outpatient management resulted in a 77.8% reduction in hospitalizations. Transient ischaemic attacks clinics are efficient for the early management of low- to moderate-risk TIA patients compared to in-hospital assessment, with no higher recurrence rates and at almost one-fifth the cost. © 2012 The Author(s) European Journal of Neurology © 2012 EFNS.

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

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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

Interaction between the genomes of Lactococcus lactis and phages of the P335 species

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Kelly, William J.; Altermann, Eric; Lambie, Suzanne C.; Leahy, Sinead C.

2013-01-01

Phages of the P335 species infect Lactococcus lactis and have been particularly studied because of their association with strains of L. lactis subsp. cremoris used as dairy starter cultures. Unlike other lactococcal phages, those of the P335 species may have a temperate or lytic lifestyle, and are believed to originate from the starter cultures themselves. We have sequenced the genome of L. lactis subsp. cremoris KW2 isolated from fermented corn and found that it contains an integrated P335 species prophage. This 41 kb prophage (Φ KW2) has a mosaic structure with functional modules that are highly similar to several other phages of the P335 species associated with dairy starter cultures. Comparison of the genomes of 26 phages of the P335 species, with either a lytic or temperate lifestyle, shows that they can be divided into three groups and that the morphogenesis gene region is the most conserved. Analysis of these phage genomes in conjunction with the genomes of several L. lactis strains shows that prophage insertion is site specific and occurs at seven different chromosomal locations. Exactly how induced or lytic phages of the P335 species interact with carbohydrate cell surface receptors in the host cell envelope remains to be determined. Genes for the biosynthesis of a variable cell surface polysaccharide and for lipoteichoic acids (LTAs) are found in L. lactis and are the main candidates for phage receptors, as the genes for other cell surface carbohydrates have been lost from dairy starter strains. Overall, phages of the P335 species appear to have had only a minor role in the adaptation of L. lactis subsp. cremoris strains to the dairy environment, and instead they appear to be an integral part of the L. lactis chromosome. There remains a great deal to be discovered about their role, and their contribution to the evolution of the bacterial genome. PMID:24009606

The complete genome sequence and proteomics of Yersinia pestis phage Yep-phi.

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Zhao, Xiangna; Wu, Weili; Qi, Zhizhen; Cui, Yujun; Yan, Yanfeng; Guo, Zhaobiao; Wang, Zuyun; Wang, Hu; Deng, Haijun; Xue, Yan; Chen, Weijun; Wang, Xiaoyi; Yang, Ruifu

2011-01-01

Yep-phi, a lytic phage of Yersinia pestis, was isolated in China and is routinely used as a diagnostic phage for the identification of the plague pathogen. Yep-phi has an isometric hexagonal head containing dsDNA and a short non-contractile conical tail. In this study, we sequenced the Yep-phi genome (GenBank accession no. HQ333270) and performed proteomics analysis. The genome consists of 38 ,616 bp of DNA, including direct terminal repeats of 222 bp, and is predicted to contain 45 ORFs. Most structural proteins were identified by proteomics analysis. Compared with the three available genome sequences of lytic phages for Y. pestis, the phages could be divided into two subgroups. Yep-phi displays marked homology to the bacteriophages Berlin (GenBank accession no. AM183667) and Yepe2 (GenBank accession no. EU734170), and these comprise one subgroup. The other subgroup is represented by bacteriophage ΦA1122 (GenBank accession no. AY247822). Potential recombination was detected among the Yep-phi subgroup.

Semi-Solid and Solid Dosage Forms for the Delivery of Phage Therapy to Epithelia

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Petrovski, Steve; Chan, Hiu Tat; Angove, Michael J.; Tucci, Joseph

2018-01-01

The delivery of phages to epithelial surfaces for therapeutic outcomes is a realistic proposal, and indeed one which is being currently tested in clinical trials. This paper reviews some of the known research on formulation of phages into semi-solid dosage forms such as creams, ointments and pastes, as well as solid dosage forms such as troches (or lozenges and pastilles) and suppositories/pessaries, for delivery to the epithelia. The efficacy and stability of these phage formulations is discussed, with a focus on selection of optimal semi-solid bases for phage delivery. Issues such as the need for standardisation of techniques for formulation as well as for assessment of efficacy are highlighted. These are important when trying to compare results from a range of experiments and across different delivery bases. PMID:29495355

Radiosensitivity of the induction of early enzymes by. gamma. -irradiated T7-phages

Energy Technology Data Exchange (ETDEWEB)

Bopp, E

1975-01-01

The radiosensitivity of the ability of the bacteriophage T7 to produce polymerase and lysozyme during its reproduction cycle is investigated. B-cells of Escherichia coli were infected with /sup 60/Co-..gamma..-irradiated T7 phages. From the extracts of the cells opened by ultrasonic waves, the amount of enzymes produced is determined with the aid of special enzyme tests. The fraction of inactivated phages able to produce RNA polymerase is higher than the fraction with intact DNA double strands and higher than the fraction able to inject DNA. The lowest fraction is that of inactivated phages producing lysozyme.

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.

Emergence of new Salmonella Enteritidis phage types in Europe? Surveillance of infections in returning travellers

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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.

Phages of Lactobacillus casei/paracasei: response to environmental factors and interaction with collection and commercial strains.

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Capra, M L; Quiberoni, A; Reinheimer, J

2006-02-01

To investigate the influence of several environmental factors on the viability and cell-adsorption for two Lactobacillus casei/paracasei bacteriophages (PL-1 and J-1). Both phages showed a remarkably high specificity of species, sharing similar host spectra. Two phages and four sensitive strains were used to conform five phage/strain systems. Each showed a particular behaviour (burst size: ranging from 32 to 160 PFU/infective centre; burst time: 120-240 min and latent time: 5-90 min). For both phages, the viability was not significantly affected from pH 4 to 11 (room temperature) and from pH 5 to 10 (37 degrees C). Adsorption rates were not influenced by calcium ions, but decreased after the thermal inactivation of cells. Adsorption rates were high between 0 and 50 degrees C with maximum values at 30 degrees C and pH 6. System PL-1/Lact. paracasei A showed noticeable differences in comparison with the others, being times required to reach 90% of adsorption of 4 h and lower than 45 min, respectively. The data obtained in this work demonstrated that environmental parameters can influence the viability and cell adsorption rates of Lact. casei/paracasei phages. The extent of this influence was phage dependent. This work contributes to the enlargement of the currently scarce knowledge of phages of probiotic bacteria.

Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

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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.

Characterization and lytic activity of methicillin-resistant Staphylococcus aureus(MRSA phages isolated from NICU

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Golnar Rahimzadeh

2016-06-01

Full Text Available Background Methicillin-resistant Staphylococcus aureus (MRSA is a well-known pathogen that causes serious diseases in humans. As part of the efforts to control this pathogen, an isolated bacteriophage, Siphoviridae, which specifically targets Methicillin-resistant Staphylococcus aureus (MRSA, was characterized. Aims The objective of this study was to characterize of a virulent bacteriophage (Siphoviridae isolated from a NICU bathroom sink. Methods The MRSA strain was isolated from patient blood. The isolated strain was confirmed as MRSA using conventional methods. Phages were isolated from a NICU bathroom sink and activity was lytic as determined by spot test. Titer phage lysate was measured by the Double Layer Agar (DLA technique. The morphology was found with electron microscopy. The single-step growth curve was plotted. Results Electron microscopy showed the phage as a member of the family Siphoviridae, serogroup A and F. The isolated phage was capable of lytic activity against methicillin-resistant Staphylococcus aureus (MRSA strain as shown by spot test. By DLA, the titre of the phages was determined to be 10×108PFU/ml. The single-step growth curve showed that the latent period of the isolated bacteriophage was 30 min and the total number of viable progeny per infected host, burst size, was 2600 PFU/infected host. Conclusion In this study, two phages were isolated and characterized from a NICU bathroom sink, from the Siphoviridae family, which specifically targetsmethicillin-resistant Staphylococcus aureus (MRSA.

Development of anti-infectives using phage display: biological agents against bacteria, viruses, and parasites.

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Huang, Johnny X; Bishop-Hurley, Sharon L; Cooper, Matthew A

2012-09-01

The vast majority of anti-infective therapeutics on the market or in development are small molecules; however, there is now a nascent pipeline of biological agents in development. Until recently, phage display technologies were used mainly to produce monoclonal antibodies (MAbs) targeted against cancer or inflammatory disease targets. Patent disputes impeded broad use of these methods and contributed to the dearth of candidates in the clinic during the 1990s. Today, however, phage display 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 phage display technology as a means of discovering novel therapeutics against infectious diseases, with a focus on antimicrobial peptides and antibodies in clinical or preclinical development. We discuss the different strategies and methods used to derive, select, and develop anti-infectives from phage display libraries and then highlight case studies of drug candidates in the process of development and commercialization. Advances in screening, manufacturing, and humanization technologies now mean that phage display can make a significant contribution in the fight against clinically important pathogens.

Potential effect of some environmental factors on the phage removal during wastewater treatment. Study in vitro

International Nuclear Information System (INIS)

Benhyahya, M.; Bohatier, J.; Laveran, H.; Ettayebi, M.; Senaud, J.

2000-01-01

Great quantities of enteric viruses and bacteriophages are included in wastewaters. They represent a contamination risk of natural water systems. But this viral burden is greatly reduced in the sewage treatment plants by the combined action of numerous environmental factors. To study water quality, some groups of bacteriophages as E. coli phages and Bacteroides fragilis phages have been proposed as model viruses. On an other hand, somatic and, in particular, F-specific coliphages have several morphological, structural and chemical composition ressemblances with the enteric viruses. Two different bacteriophages (øX-174 and MS2) were used as virus models in this in vitro study to evaluate the viral adsorption on suspended clay particles. Distilled sterile water was used as reactional medium to avoid the possible interactions with the considered substrates, the kaolinite (K) and the montmorillonite (M). Phage behaviour in the water and in the recommended diluent for phages, the saline peptone, was first compared. K and M suspensions were used at 300 mg/l for a contact time of 5, 30 and 60 min. In other series K and M suspensions were prepared at 600, 300 and 100 mg/l then used to determine the phage adsorption capacity in a fixed time 30 min. Results show that the phage titers for all samples were constant in the organic diluent. They were lower in the distilled sterile water and decrease with the time. Distilled water favours most likely the grouping of virions and leads aggregates formation. The adsorption of øX-174 and MS2 onto K or M particles was instantaneous and independent of the duration contact. The clay concentration had a slight significant influence on the phage adsorption rate. Using the same phages we have studied, in a second stage, the potential effect of the dissolved matters in a filtered polluted effluent, that of sunlight radiations and that of the protozoan Tetrahymena pyriformis on the phage removal. No soon significant phage inactivation was

Phage nanofibers induce vascularized osteogenesis in 3D printed bone scaffolds.

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Wang, Jianglin; Yang, Mingying; Zhu, Ye; Wang, Lin; Tomsia, Antoni P; Mao, Chuanbin

2014-08-06

A virus-activated matrix is developed to overcome the challenge of forming vascularized bone tissue. It is generated by filling a 3D printed bioceramic scaffold with phage nanofibers displaying high-density RGD peptide. After it is seeded with mesenchymal stem cells (MSCs) and implanted into a bone defect, the phage nanofibers induce osteogenesis and angiogenesis by activating endothelialization and osteogenic differentiation of MSCs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Damages induced in lambda phage DNA by enzyme-generated triplet acetone

International Nuclear Information System (INIS)

Menck, C.F.; Cabral Neto, J.B.; Gomes, R.A.; Faljoni-Alario, A.

1985-01-01

Exposure of lambda phage to triplet acetone, generated during the aerobic oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only in alkaline conditions, so true breaks were not observed. Also, no sites sensitive to UV-endonuclease from Micrococcus luteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed. (Author) [pt

Effectiveness of the lactococcal abortive infection systems AbiA, AbiE, AbiF and AbiG against P335 type phages.

Science.gov (United States)

Tangney, Mark; Fitzgerald, Gerald F

2002-04-23

Four lactococcal abortive infection mechanisms were introduced into strains which were sensitive hosts for P335 type phages and plaque assay experiments performed to assess their effect on five lactococcal bacteriophages from this family. Results indicate that AbiA inhibits all five P335 phages tested, while AbiG affects phiP335 itself and phiQ30 but not the other P335 species phages. AbiA was shown to retard phage Q30 DNA replication as previously reported for other phages. It was also demonstrated that AbiG, previously shown to act at a point after DNA replication in the cases of c2 type and 936 type phages, acts at the level of, or prior to phage Q30 DNA replication. AbiE and AbiF had no effect on the P335 type phages examined.

Transcriptome Analysis of a Bloom-Forming Cyanobacterium Microcystis aeruginosa during Ma-LMM01 Phage Infection

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Daichi Morimoto

2018-01-01

Full Text Available Microcystis aeruginosa forms massive blooms in eutrophic freshwaters, where it is constantly exposed to lytic cyanophages. Unlike other marine cyanobacteria, M. aeruginosa possess remarkably abundant and diverse potential antiviral defense genes. Interestingly, T4-like cyanophage Ma-LMM01, which is the sole cultured lytic cyanophage infecting M. aeruginosa, lacks the host-derived genes involved in maintaining host photosynthesis and directing host metabolism that are abundant in other marine cyanophages. Based on genomic comparisons with closely related cyanobacteria and their phages, Ma-LMM01 is predicted to employ a novel infection program that differs from that of other marine cyanophages. Here, we used RNA-seq technology and in silico analysis to examine transcriptional dynamics during Ma-LMM01 infection to reveal host transcriptional responses to phage infection, and to elucidate the infection program used by Ma-LMM01 to avoid the highly abundant host defense systems. Phage-derived reads increased only slightly at 1 h post-infection, but significantly increased from 16% of total cellular reads at 3 h post-infection to 33% of all reads by 6 h post-infection. Strikingly, almost none of the host genes (0.17% showed a significant change in expression during infection. However, like other lytic dsDNA phages, including marine cyanophages, phage gene dynamics revealed three expression classes: early (host-takeover, middle (replication, and late (virion morphogenesis. The early genes were concentrated in a single ∼5.8-kb window spanning 10 open reading frames (gp054–gp063 on the phage genome. None of the early genes showed homology to the early genes of other T4-like phages, including known marine cyanophages. Bacterial RNA polymerase (σ70 recognition sequences were also found in the upstream region of middle and late genes, whereas phage-specific motifs were not found. Our findings suggest that unlike other known T4-like phages, Ma-LMM01

Takotsubo cardiomyopathy following recurrent doses of albuterol due to asthma attack: a very rare case report.

Science.gov (United States)

Parsa, Saeed Alipour; Khaheshi, Isa; Memaryan, Mehdi; Naderian, Mohammadreza

2016-11-01

To present an unusual case in order to clarify one of the most important differential diagnoses of ST-elevation myocardial infarction in a postmenopausal woman. A 72-year-old woman with asthma attack and retrosternal chest pain was presented to our emergency unit. She had used albuterol spray ten-times before admission due to accentuated dyspnea. Initial ECG revealed ST-segment elevation in leads V1-V6, I and aVL. Emergent coronary angiography indicated normal coronary arteries, and left ventriculography showed significant akinesia of apical segments. This unusual case emphasizes the fact that emergency physicians and cardiologists should be familiar with Takotsubo cardiomyopathy following overuse of β 2 -agonist due to asthma attack that can mimic ST-elevation myocardial infarction.

Stability of Staphylococcus aureus phage ISP after freeze-drying (lyophilization.

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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.

Construction and Selection of Affilin® Phage Display Libraries.

Science.gov (United States)

Settele, Florian; Zwarg, Madlen; Fiedler, Sebastian; Koscheinz, Daniel; Bosse-Doenecke, Eva

2018-01-01

Affilin ® molecules represent a new class of so-called scaffold proteins. The concept of scaffold proteins is to use stable and versatile protein structures which can be endowed with de novo binding properties and specificities by introducing mutations in surface exposed amino acid residues. Complex variations and combinations are generated by genetic methods of randomization resulting in large cDNA libraries. The selection for candidates binding to a desired target can be executed by display methods, especially the very robust and flexible phage display. Here, we describe the construction of ubiquitin based Affilin ® phage display libraries and their use in biopanning experiments for the identification of novel protein ligands.

[Reduced risk of stroke recurrence due to hypotensive medication, irrespective of the initial blood pressure

NARCIS (Netherlands)

Lenders, J.W.M.; Thien, Th.

2002-01-01

The 'Perindopril protection against recurrent stroke study' (PROGRESS) demonstrated that for patients with a history of stroke or transient ischaemic attack during the previous 5 years, a blood-pressure-lowering regimen based on the combination of a diuretic and an angiotensin-converting enzyme

Complete nucleotide sequence of Bacillus subtilis (natto) bacteriophage PM1, a phage associated with disruption of food production.

Science.gov (United States)

Umene, Kenichi; Shiraishi, Atsushi

2013-06-01

"Natto", considered a traditional food, is made by fermenting boiled soybeans with Bacillus subtilis (natto), which is a natto-producing strain related to B. subtilis. The production of natto is disrupted by phage infections of B. subtilis (natto); hence, it is necessary to control phage infections. PM1, a phage of B. subtilis (natto), was isolated during interrupted natto production in a factory. In a previous study, PM1 was classified morphologically into the family Siphoviridae, and its genome, comprising approximately 50 kbp of linear double-stranded DNA, was assumed to be circularly permuted. In the present study, the complete nucleotide sequence of the PM1 genomic DNA of 50,861 bp (41.3 %G+C) was determined, and 86 open reading frames (ORFs) were deduced. Forty-one ORFs of PM1 shared similarities with proteins deduced from the genome of phages reported so far. Twenty-three ORFs of PM1 were associated with functions related to the phage multiplication process of gene control, DNA replication/modification, DNA packaging, morphogenesis, and cell lysis. Bacillus subtilis (natto) produces a capsular polypeptide of glutamate with a γ-linkage (called poly-γ-glutamate), which appears to serve as a physical barrier to phage adsorption. One ORF of PM1 had similarity with a poly-γ-glutamate hydrolase, which is assumed to degrade the capsular barrier to allow phage progenies to infect encapsulated host cells. The genome analysis of PM1 revealed the characteristics of the phage that are consistent as Bacillus subtilis (natto)-infecting phage.

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.

Next-generation phage display: integrating and comparing available molecular tools to enable cost-effective high-throughput analysis.

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Emmanuel Dias-Neto

2009-12-01

Full Text Available Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i the counting of transducing units and (ii the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges.We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU, with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at >1,000-fold faster rate, and reduce costs approximately 250-fold for generating 10(6 ligand sequences.Our analyses demonstrates that whereas this approach correlates with the traditional colony-counting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger

Phage-based magnetoelastic sensor for the detection of Salmonella typhimurium

Science.gov (United States)

Lakshmanan, Ramji S.

In recent years, food-borne illness have garnered the attention of mainstream America with calls now coming from the media for more inspections to ensure the safety of our food supply. Food borne illness from the ingestion of S. typhimurium has been of great concern due to its common occurrence in food products of daily consumption. Annually approximately 80 million cases of food poisoning are reported in the United States alone. The ever growing need for rapid detection of pathogenic microorganisms present in food, environmental and clinical samples has invoked an increased interest in research efforts towards the development of novel diagnostic methodologies. Currently, the detection of bacteria in contaminated food relies on conventional microbiological methods that are time consuming and manpower intensive. This study presents the results of the characterization of a phage-based magnetoelastic biosensor for the detection of Salmonella typhimurium . This affinity-based biosensensor is comprised of a magnetoelastic material as the transducer and filamentous phage as the bio-recognition element. Magnetoelastic materials are ferromagnetic amorphous alloys that change dimensions in the presence of a magnetic field. This effect in combination with the reverse effect (inverse magnetostriction) is utilized in a typical sensor application. A time varying magnetic field causes these sensors to oscillate at a characteristic resonance frequency. The characteristic resonance frequency is dependent on the initial dimensions and physical properties of the material. These materials are of particular interest owing to their unique capability to perform remote (without direct wire contacts to the sensor) sensing, making in-vivo detection and detection in closed containers possible. The phage-immobilized magnetoelastic biosensor was characterized for specificity; dose response in water, spiked apple juice and in spiked milk; selectivity; and longevity. The sensor's sensitivity is

Comparative phenomenology of ataques de nervios, panic attacks, and panic disorder.

Science.gov (United States)

Lewis-Fernández, Roberto; Guarnaccia, Peter J; Martínez, Igda E; Salmán, Ester; Schmidt, Andrew; Liebowitz, Michael

2002-06-01

This article examines a clinical sample of 66 Dominican and Puerto Rican subjects who reported ataques de nervios and also psychiatric disorder, and disentangles the phenomenological experiences of ataque de nervios, panic attacks, and panic disorder. In-depth cultural interviews assessed the symptomatic phenomenology of ataque episodes from the local perspective as well as in terms of key panic features, such as recurrence, rapid peaking of symptoms, and lack of provocation. Independent diagnostic assessments of panic attacks and disorder were also used to establish the phenomenological overlap between ataque and panic. Our findings indicate that 36 percent of ataques de nervios fulfill criteria for panic attacks and between 17 percent and 33 percent for panic disorder, depending on the overlap method used. The main features distinguishing ataques that fulfill panic criteria from ataques that do not include whether the episodes were provoked by an upsetting event in the person's life and the rapidity of crescendo of the actual attack. A key finding is that ataques often share individual phenomenological features with panic episodes, but that these features usually do not "run together" during the ataque experience. This confirms previous findings that ataque is a more inclusive construct than panic disorder. The importance of these findings for the clinical diagnosis and treatment of persons with ataques is discussed.

Vaginal microbiology of women with acute recurrent vulvovaginal candidiasis.

OpenAIRE

Sobel, J D; Chaim, W

1996-01-01

Alterations in the autochthonous vaginal microflora can predispose women to recurring attacks of Candida vaginitis. Quantitative aerobic and anaerobic cultures were obtained from 24 premenopausal women with acute recurrent vulvovaginal candidiasis and from 21 healthy asymptomatic premenopausal women. Lactobacillus species constituted the predominant flora in both groups, with a mean log10 CFU/ml of 8.7, a total isolation rate of 96%, and a mean of 1.6 Lactobacillus species isolated per patien...

Probing ADAMTS13 substrate specificity using phage display.

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Karl C Desch

Full Text Available Von Willebrand factor (VWF is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2' and P11', for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13-VWF exosite interactions outside of VWF73.

Modular architecture of the T4 phage superfamily: A conserved core genome and a plastic periphery

International Nuclear Information System (INIS)

Comeau, Andre M.; Bertrand, Claire; Letarov, Andrei; Tetart, Francoise; Krisch, H.M.

2007-01-01

Among the most numerous objects in the biosphere, phages show enormous diversity in morphology and genetic content. We have sequenced 7 T4-like phages and compared their genome architecture. All seven phages share a core genome with T4 that is interrupted by several hyperplastic regions (HPRs) where most of their divergence occurs. The core primarily includes homologues of essential T4 genes, such as the virion structure and DNA replication genes. In contrast, the HPRs contain mostly novel genes of unknown function and origin. A few of the HPR genes that can be assigned putative functions, such as a series of novel Internal Proteins, are implicated in phage adaptation to the host. Thus, the T4-like genome appears to be partitioned into discrete segments that fulfil different functions and behave differently in evolution. Such partitioning may be critical for these large and complex phages to maintain their flexibility, while simultaneously allowing them to conserve their highly successful virion design and mode of replication

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.

Genetically Engineered Virulent Phage Banks in the Detection and Control of Emergent Pathogenic Bacteria

Science.gov (United States)

Blois, Hélène; Iris, François

2010-01-01

Natural outbreaks of multidrug-resistant microorganisms can cause widespread devastation, and several can be used or engineered as agents of bioterrorism. From a biosecurity standpoint, the capacity to detect and then efficiently control, within hours, the spread and the potential pathological effects of an emergent outbreak, for which there may be no effective antibiotics or vaccines, become key challenges that must be met. We turned to phage engineering as a potentially highly flexible and effective means to both detect and eradicate threats originating from emergent (uncharacterized) bacterial strains. To this end, we developed technologies allowing us to (1) concurrently modify multiple regions within the coding sequence of a gene while conserving intact the remainder of the gene, (2) reversibly interrupt the lytic cycle of an obligate virulent phage (T4) within its host, (3) carry out efficient insertion, by homologous recombination, of any number of engineered genes into the deactivated genomes of a T4 wild-type phage population, and (4) reactivate the lytic cycle, leading to the production of engineered infective virulent recombinant progeny. This allows the production of very large, genetically engineered lytic phage banks containing, in an E. coli host, a very wide spectrum of variants for any chosen phage-associated function, including phage host-range. Screening of such a bank should allow the rapid isolation of recombinant T4 particles capable of detecting (ie, diagnosing), infecting, and destroying hosts belonging to gram-negative bacterial species far removed from the original E. coli host. PMID:20569057

On the lack of host-cell reactivation of UV-irradiated phage T5

International Nuclear Information System (INIS)

Chiang, T.; Harm, W.

1976-01-01

Previously reported experiments have shown that host-cell reactivation (HCR) of UV-irradiated phage T1 in excision-repair proficient Escherichia coli cells is inhibited by superinfection with phage T5. Theoretical considerations have led to predictions concerning the dependence of repair inhibition on the multiplicity of superinfecting T5 phage and on the UV fluence to which they were exposed. These predictions have been supported by experimental results described in this paper. The fluence dependence permitted calculation of the relative UV sensitivity of the gene function responsible for repair inhibition; it was found to be about 2.3% that of the plaque-forming ability of phage T5. The T5-inhibitable step in excision repair occurs early in the infective cycle of T1. Furthermore, experiments involving the presence of 400 μg/ml chloramphenicol showed that HCR inhibition of T1 is caused by a protein produced after the FST segment of T5 (i.e. the first 8% of the T5 genome) has entered the host cell. A previously described minor T1 recovery process, occuring in both excision-repair-proficient and -deficient host-cells, is inhibited by T5 infection due to a different substance, which is most likely associated with the 'second-step-transfer' region of T5 DNA (involving the remainder of the genome). Superinfection with T4v 1 phage resulted in HCR inhibition of T1, resembling that observed after T5 superinfection. The discussion of these results suggests that inhibition of the bacterial excision repair system by T5 or T4 infection occurs at the level of UV-endonucleolytic incision, and that lack of HCR both in T-even phages and in T5 can be explained in the same manner

Construction of genetically engineered M13K07 helper phage for simultaneous phage display of gold binding peptide 1 and nuclear matrix protein 22 ScFv antibody.

Science.gov (United States)

Fatemi, Farnaz; Amini, Seyed Mohammad; Kharrazi, Sharmin; Rasaee, Mohammad Javad; Mazlomi, Mohammad Ali; Asadi-Ghalehni, Majid; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

2017-11-01

The most common techniques of antibody phage display are based on the use of M13 filamentous bacteriophages. This study introduces a new genetically engineered M13K07 helper phage displaying multiple copies of a known gold binding peptide on p8 coat proteins. The recombinant helper phages were used to rescue a phagemid vector encoding the p3 coat protein fused to the nuclear matrix protein 22 (NMP22) ScFv antibody. Transmission electron microscopy (TEM), UV-vis absorbance spectroscopy, and field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDX) analysis revealed that the expression of gold binding peptide 1 (GBP1) on major coat protein p8 significantly enhances the gold-binding affinity of M13 phages. The recombinant bacteriophages at concentrations above 5×10 4 pfu/ml red-shifted the UV-vis absorbance spectra of gold nanoparticles (AuNPs); however, the surface plasmon resonance of gold nanoparticles was not changed by the wild type bacteriophages at concentrations up to 10 12 pfu/ml. The phage ELISA assay demonstrated the high affinity binding of bifunctional bacteriophages to NMP22 antigen at concentrations of 10 5 and 10 6 pfu/ml. Thus, the p3 end of the bifunctional bacteriophages would be able to bind to specific target antigen, while the AuNPs were assembled along the coat of virus for signal generation. Our results indicated that the complex of antigen-bacteriophages lead to UV-vis spectral changes of AuNPs and NMP22 antigen in concentration range of 10-80μg/ml can be detected by bifunctional bacteriophages at concentration of 10 4 pfu/ml. The ability of bifunctional bacteriophages to bind to antigen and generate signal at the same time, makes this approach applicable for identifying different antigens in immunoassay techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

Bone and bone marrow scintigraphy in a patient with sickle cell-thalassemia and recurrent pain attacks

International Nuclear Information System (INIS)

Mikosch, P.; Gallowitsch, H.-J.; Lind, P.; Jauk, B.; Kaulfersch, W.

2003-01-01

The case of an eight years old African boy who suffers from sickle cell-thalassemia is presented. In the course of the disease frequent pain attacks occurred within the abdomen and extremities, recently also within the trunk. Local pain, at some occasions in combination with local swelling and always positive laboratory parameters for inflammation, hindered a solely clinical differentiation between bone infarcts and osteomyelitis. Bone scintigraphy, eventually in combination with bone marrow scintigraphy, can assist the clinician in the differentiation of aseptic bone infarcts versus secondary osteomyelitis. Based on the presented case scintigraphic results for bone infarcts, osteomyelitis and special scintigraphic pattern seen in sickle cell disease are presented. Furthermore, problems regarding the interpretation of the scintigraphies in relation to the delayed time after the beginning of pain attacks are discussed. (author)

Phage or foe: an insight into the impact of viral predation on microbial communities.

Science.gov (United States)

Fernández, Lucía; Rodríguez, Ana; García, Pilar

2018-05-01

Since their discovery, bacteriophages have been traditionally regarded as the natural enemies of bacteria. However, recent advances in molecular biology techniques, especially data from "omics" analyses, have revealed that the interplay between bacterial viruses and their hosts is far more intricate than initially thought. On the one hand, we have become more aware of the impact of viral predation on the composition and genetic makeup of microbial communities thanks to genomic and metagenomic approaches. Moreover, data obtained from transcriptomic, proteomic, and metabolomic studies have shown that responses to phage predation are complex and diverse, varying greatly depending on the bacterial host, phage, and multiplicity of infection. Interestingly, phage exposure may alter different phenotypes, including virulence and biofilm formation. The complexity of the interactions between microbes and their viral predators is also evidenced by the link between quorum-sensing signaling pathways and bacteriophage resistance. Overall, new data increasingly suggests that both temperate and virulent phages have a positive effect on the evolution and adaptation of microbial populations. From this perspective, further research is still necessary to fully understand the interactions between phage and host under conditions that allow co-existence of both populations, reflecting more accurately the dynamics in natural microbial communities.

Phages can constrain protist predation-driven attenuation of Pseudomonas aeruginosa virulence in multienemy communities

Science.gov (United States)

Friman, Ville-Petri; Buckling, Angus

2014-01-01

The coincidental theory of virulence predicts that bacterial pathogenicity could be a by-product of selection by natural enemies in environmental reservoirs. However, current results are ambiguous and the simultaneous impact of multiple ubiquitous enemies, protists and phages on virulence evolution has not been investigated previously. Here we tested experimentally how Tetrahymena thermophila protist predation and PNM phage parasitism (bacteria-specific virus) alone and together affect the evolution of Pseudomonas aeruginosa PAO1 virulence, measured in wax moth larvae. Protist predation selected for small colony types, both in the absence and presence of phage, which showed decreased edibility to protists, reduced growth in the absence of enemies and attenuated virulence. Although phage selection alone did not affect the bacterial phenotype, it weakened protist-driven antipredatory defence (biofilm formation), its associated pleiotropic growth cost and the correlated reduction in virulence. These results suggest that protist selection can be a strong coincidental driver of attenuated bacterial virulence, and that phages can constrain this effect owing to effects on population dynamics and conflicting selection pressures. Attempting to define causal links such as these might help us to predict the cold and hot spots of coincidental virulence evolution on the basis of microbial community composition of environmental reservoirs. PMID:24671085

Impact of reducing and oxidizing agents on the infectivity of Qβ phage and the overall structure of its capsid.

Science.gov (United States)

Loison, Pauline; Majou, Didier; Gelhaye, Eric; Boudaud, Nicolas; Gantzer, Christophe

2016-11-01

Qβ phages infect Escherichia coli in the human gut by recognizing F-pili as receptors. Infection therefore occurs under reducing conditions induced by physiological agents (e.g. glutathione) or the intestinal bacterial flora. After excretion in the environment, phage particles are exposed to oxidizing conditions and sometimes disinfection. If inactivation does not occur, the phage may infect new hosts in the human gut through the oral route. During such a life cycle, we demonstrated that, outside the human gut, cysteines of the major protein capsid of Qβ phage form disulfide bonds. Disinfection with NaClO does not allow overoxidation to occur. Such oxidation induces inactivation rather by irreversible damage to the minor proteins. In the presence of glutathione, most disulfide bonds are reduced, which slightly increases the capacity of the phage to infect E. coli in vitro Such reduction is reversible and barely alters infectivity of the phage. Reduction of all disulfide bonds by dithiothreitol leads to complete capsid destabilization. These data provide new insights into how the phages are impacted by oxidizing-reducing conditions outside their host cell and raises the possibility of the intervention of the redox during life cycle of the phage. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Large palindromes in the lambda phage genome are preserved in a rec/sup +/ host by inhibiting lambda DNA replication

Energy Technology Data Exchange (ETDEWEB)

Shurvinton, C.E.; Stahl, M.M.; Stahl, F.W.

1987-03-01

A large palindrome carried by phage lambda has been shown to prevent growth of the phage on a rec/sup +/ strain of Escherichia coli. The phage do form plaques on recBC sbcB strains, but the palindrome is not stable - deletions that either destroy the palindrome or diminish its size overgrow the original engineered palindrome-containing phage. The authors have prepared stocks of lambda carrying a palindrome that is 2 x 4200 base pairs long. lambda phage were density labeled by UV induction of lysogens grown in minimal medium containing (/sup 13/C) glucose and /sup 15/NH/sub 4/Cl. These phage stocks are produced by induction of a lysogen in which the two halves of the palindrome are stored at opposite ends of the prophage and are of sufficient titer (10/sup 9/ phage per ml) to enable one-step growth experiments with replication-blocked phage. They find that the large palindrome as well as a lesser palindrome of 2 x 265 base pairs are recovered intact among particles carrying unreplicated chromosomes following such an infection of a rec/sup +/ host. they propose that DNA replication drives the extrusion of palindromic sequences in vivo, forming secondary structures that are substrates for the recBC and sbcB gene products.

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

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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.

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.

Directory of Open Access Journals (Sweden)

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.

W-reactivation of phage lambda in X-irradiated mutants of Escherichia coli K-12

Energy Technology Data Exchange (ETDEWEB)

Martignoni, K D; Haselbacher, I [Muenchen Univ. (Germany, F.R.). Strahlenbiologisches Inst.

1980-07-01

The survival of UV irradiated phage lambda was increased on X-irradiated E.coli K-12 host cells over that on unirradiated cells. The frequency of c mutants among the surviving phages was increased to a similar extent by the X-ray exposure of the host cells as by UV light. This W-reactivation of phage lambda occurred in uvrA, polA, and recB mutants besides the wild type at about equal X-ray doses, but at a reduced reactivation efficiency compared with the wild type. W-reactivation was undetectable in recA mutants. While maximal UV induced W-reactivation occured 30 min after irradiation, the maximal X-ray induced reactivation was found immediately after irradiation. Chloramphenicol (100 ..mu..g/ml) and nitrofurantoin (50 ..mu..g/ml) inhibited W-reactivation of phage lambda if added before irradiation of the host cells, indicating the necessity of protein synthesis for W-reactivation.

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

Recurrent cerebral thrombosis

International Nuclear Information System (INIS)

Iwamoto, Toshihiko; Abe, Shin-e; Kubo, Hideki; Hanyu, Haruo; Takasaki, Masaru

1992-01-01

Neuroradiological techniques were used to elucidate pathophysiology of recurrent cerebral thrombosis. Twenty-two patients with cerebral thrombosis who suffered a second attack under stable conditions more than 22 days after the initial stroke were studied. Hypertension, diabetes mellitus, and hypercholesterolemia were also seen in 20, 8, and 12 patients, respectively. The patients were divided into three groups according to their symptoms: (I) symptoms differed between the first and second strokes (n=12); (II) initial symptoms were suddenly deteriorated (n=6); and (III) symptoms occurring in groups I and II were seen (n=4). In group I, contralateral hemiparesis or suprabulbar palsy was often associated with the initial hemiparesis. The time of recurrent stroke varied from 4 months to 9 years. CT and MRI showed not only lacunae in both hemispheres, but also deep white-matter ischemia of the centrum semi-ovale. In group II, hemiparesis or visual field defect was deteriorated early after the initial stroke. In addition, neuroimaging revealed that infarction in the posterior cerebral artery was progressed on the contralateral side, or that white matter lesion in the middle artery was enlarged in spite of small lesion in the left cerebral hemisphere. All patients in group III had deterioration of right hemiparesis associated with aphasia. CT, MRI, SPECT, and angiography indicated deep white-matter ischemia caused by main trunk lesions in the left hemisphere. Group III seemed to be equivalent to group II, except for laterality of the lesion. Neuroradiological assessment of the initial stroke may help to predict the mode of recurrence, although pathophysiology of cerebral thrombosis is complicated and varies from patient to patient. (N.K.)

Assessment of the Microscreen phage-induction assay for screening hazardous wastes

Energy Technology Data Exchange (ETDEWEB)

Houk, V.S.; DeMarini, D.M.

1987-09-01

The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s(lambda), was used to test 14 crude (unfractionated) hazardous industrial waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 picograms per ml. Comparisons between the mutagenicity of these waste samples in Salmonella and their ability to induce prophage lambda indicate that the Microscreen phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, the Microscreen assay detected as genotoxic 5 additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed along with some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.

Commensal E. coli Stx2 lysogens produce high levels of phages after spontaneous prophage induction

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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.

Isolation and characterization of φkm18p, a novel lytic phage with therapeutic potential against extensively drug resistant Acinetobacter baumannii.

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Gwan-Han Shen

Full Text Available AIMS: To isolate phages against extensively drug resistant Acinetobacter baumannii (XDRAB and characterize the highest lytic capability phage as a model to evaluate the potential on phage therapy. METHODS AND RESULTS: Eight phages were isolated from hospital sewage and showed narrow host spectrum. Phage φkm18p was able to effectively lyse the most XDRAB. It has a dsDNA genome of 45 kb in size and hexagonal head of about 59 nm in diameter and no tail. Bacterial population decreased quickly from 10(8 CFU ml(-1 to 10(3 CFU ml(-1 in 30 min by φkm18p. The 185 kDa lysis protein encoded by φkm18p genome was detected when the extracted protein did not boil before SDS-PAGE; it showed that the lysis protein is a complex rather than a monomer. Phage φkm18p improved human lung epithelial cells survival rates when they were incubated with A. baumannii. Combination of phages (φkm18p, φTZ1 and φ314 as a cocktail could lyse all genotype-varying XDRAB isolates. CONCLUSION: Infections with XDRAB are extremely difficult to treat and development of a phage cocktails therapy could be a therapeutic alternative in the future. Phage φkm18p is a good candidate for inclusion in phage cocktails.

Phage induction by UV and mitomycin C in Pseudomonas mori, the pathogen of bacterial blight of mulberry

International Nuclear Information System (INIS)

Sato, Mamoru

1979-01-01

Phage induction by ultraviolet radiation (UV) and mitomycin C (MMC) in some lysogenic strains of Pseudomonas mori, the pathogen of bacterial blight of mulberry, was examined. Among 5 strains tested, in the strains S 6804 and S 6805, phage was induced by both UV and MMC, and in the strain M 5, only by MMC. In the strains S 6807 and S 6808, it was not induced by both these inducers. The rate of phage production in the strain 6805 was highest when it was exposed to UV (15 W UV lamp, 40 cm) for 5 seconds, by which about 90% of the bacteria were killed, and decreased rapidly by further extending the exposure time. The bacteria suspended in 0.02 M magnesium solution were more sensitive in responding to UV than those suspended in nutrient broth, but after the UV treatment, nutrient broth was more favorable than magnesium solution for phage production. The MMC added to nutrient broth induced phage production at the concentration from 0.5 to 5 μg/ml. The strains induced by either UV or MMC their temperate phages after about 3 hours of latent period. The phage induction by UV was almost completely suppressed by 40 minute exposure to fluorescent light (a 15 W fluorescent lamp, 10 cm) or by 5 minute exposure to sunlight, given within 45 minutes after the UV treatment, i.e. within 1/4 of the latent period. Thus, the photoreversion of the UV effect on phage induction was observed in Ps. mori as well as in Ps. pyocyanea and E. coli. (Kaihara, S.)

Calculating Adversarial Risk from Attack Trees: Control Strength and Probabilistic Attackers

NARCIS (Netherlands)

Pieters, Wolter; Davarynejad, Mohsen

2015-01-01

Attack trees are a well-known formalism for quantitative analysis of cyber attacks consisting of multiple steps and alternative paths. It is possible to derive properties of the overall attacks from properties of individual steps, such as cost for the attacker and probability of success. However, in

Chronic Recurrent Non-specific Parotitis: A Case Report and Review.

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Mahalakshmi, Saibaba; Kandula, Srinivas; Shilpa, Patil; Kokila, Ganganna

2017-01-01

Chronic recurrent non-specific parotitis is characterized by recurrent episodes of swelling and pain of unknown etiology in the parotid gland. Sialography is a hallmark in the diagnosis of salivary gland disorders; newer imaging modalities like CT-Sialography, sialoendoscopy and MRI can be used. Various treatment modalities have been tried, from conservative approach to surgical excision depending on the recurrence rate and severity of the condition. Although symptomatic treatment with antibiotics and analgesic, injection of intraductal medicament, aggressive treatment like duct ligation or excision of gland are some of the treatment modalities, there is no established algorithm as to which treatment method should be opted in such clinical situation. A 20 years old male patient reported with pain and salty taste in the mouth that had began before a week. Examination revealed an elevated right parotid papilla; ropy, cloudy appearing saliva was oozing out on milking the gland. Unstimulated and stimulated whole salivary flow rate was assessed using drooling method. Sialography was used as a diagnostic and a therapeutic aid. In our case, sialography as a treatment showed a good response with no recurrence after two years of follow-up. We highlighted the role of sialography as a therapeutic aid. Recurrent attacks significantly affect the quality of life and also lead to progressive gland destruction. Preventing or reducing the frequency of recurrence remains the goal of therapeutic procedure. Hence, conventional sialography is useful in the diagnosis and also effective as a therapeutic aid in recurrent parotitis.

Use of phages against antibiotic-resistant Staphylococcus aureus isolated from bovine mastitis.

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Dias, R S; Eller, M R; Duarte, V S; Pereira, Â L; Silva, C C; Mantovani, H C; Oliveira, L L; Silva, E de A M; De Paula, S O

2013-08-01

Bovine mastitis is the primary disease of dairy cattle worldwide and it causes large economic losses. Among several microorganisms that are the causative agents of this disease, Staphylococcus aureus is the most prevalent. Although antibiotic therapy is still the most widely used procedure for the treatment of bovine mastitis, alternative means of treatment are necessary due to the presence of antibiotic residues in milk, which is a growing concern because of its interference with the production of milk derivatives and the selection of resistant bacterial strains. The use of bacteriophages as a tool for the control of pathogens is an alternative treatment to antibiotic therapy. In this work, to obtain phages with the potential for use in phage therapy as a treatment for mastitis, we isolated and identified the bacteria from the milk of mastitis-positive cows. A total of 19% of the animals from small and medium farms of the Zona da Mata Mineira, Brazil, was positive for bovine mastitis, and bacteria of the genus Staphylococcus were the most prevalent pathogens. The majority of the S. aureus isolates tested was resistant to penicillin and ampicillin. In parallel, we isolated 10 bacteriophages able to infect some of these S. aureus isolates. We determined that these phages contained DNA genomes of approximately 175 kb in length, and the protein profiles indicated the presence of 4 major proteins. Electron microscopy revealed that the phages are caudate and belong to the Myoviridae family. The isolates exhibited interesting features for their use in phage therapy such as a high lytic potential, a wide range of hosts, and thermostability, all of which favor their use in the field.

Phage display peptide libraries: deviations from randomness and correctives

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Ryvkin, Arie; Ashkenazy, Haim; Weiss-Ottolenghi, Yael; Piller, Chen; Pupko, Tal; Gershoni, Jonathan M

2018-01-01

Abstract Peptide-expressing phage display libraries are widely used for the interrogation of antibodies. Affinity selected peptides are then analyzed to discover epitope mimetics, or are subjected to computational algorithms for epitope prediction. A critical assumption for these applications is the random representation of amino acids in the initial naïve peptide library. In a previous study, we implemented next generation sequencing to evaluate a naïve library and discovered severe deviations from randomness in UAG codon over-representation as well as in high G phosphoramidite abundance causing amino acid distribution biases. In this study, we demonstrate that the UAG over-representation can be attributed to the burden imposed on the phage upon the assembly of the recombinant Protein 8 subunits. This was corrected by constructing the libraries using supE44-containing bacteria which suppress the UAG driven abortive termination. We also demonstrate that the overabundance of G stems from variant synthesis-efficiency and can be corrected using compensating oligonucleotide-mixtures calibrated by mass spectroscopy. Construction of libraries implementing these correctives results in markedly improved libraries that display random distribution of amino acids, thus ensuring that enriched peptides obtained in biopanning represent a genuine selection event, a fundamental assumption for phage display applications. PMID:29420788

Classification of etiologic subtypes for transient ischemic attacks. Clinical significance of lacunar transient ischemic attack

International Nuclear Information System (INIS)

Ohara, Tomoyuki; Yamamoto, Yasumasa; Nagakane, Yoshinari; Tanaka, Eijiro; Morii, Fukiko; Koizumi, Takashi

2011-01-01

Lacunar transient ischemic attack (lacunar TIA) may have been underestimated because of diagnostic difficulties. The aim of our study was to classify TIAs by etiologic subtypes, especially using defined criteria for diagnosis of lacunar TIA and clarify clinical characteristics of lacunar TIA.105 TIA patients out of consecutive 1,244 patients with acute ischemic stroke admitted to our hospital between January 2007 and June 2010 were enrolled in the present study. TIA was defined as an acute focal neurological deficit lasting less than 24 hours, suspected to be of cerebrovascular origin regardless of ischemic lesions on MRI. TIAs were classified to 5 etiologic subtypes; cardioembolic TIA, atherothrombotic TIA, lacunar TIA, other etiologies, and undetermined etiology and clinical characteristics in each subtype and the incidence of recurrent stroke after TIA were investigated. Lacunar TIA was diagnosed if the following criteria were fulfilled; presence of lacunar infarct on MRI and/or the presence of unilateral dysfunction of at least two of three body parts (face, arm, leg) in the absence of cortical dysfunction presumed due to subcortical ischemia. Absence of cardiac sources of embolism and large artery atherosclerosis. In 105 patients with TIA, lacunar TIA was the most frequent etiology (31%) followed by cardioembolic TIA (27%), atherothrombotic TIA (19%), undetermined etiology (18%), and other etiologies (6%). In patients with lacunar TIA, history of repeated TIA was more frequent and systolic blood pressure on admission was higher significantly than in cardioembolic TIA. Six of 105 patients had experienced recurrent stroke after TIA during admission. Among these 6 patients, 3 patients were diagnosed as lacunar infarctions. Lacunar TIA was most common TIA subtype in the present study. It is critical to identify lacunar TIA on admission because some patients with lacunar TIAs experience early recurrent stroke. (author)

Cardiac myxoma: An uncommon cause of recurrent stroke in uncommon age

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Harish Kumar

2015-01-01

Full Text Available Atrial fibrillation is the most common cause of cardiogenic emboli in stroke, responsible for over 50% cases of total stroke patients. Myxoma is responsible only in few cases. A stroke caused by left atrial myxoma commonly occur in young females. This patient presented with the repeated attack of stroke in the age of 80 years. However, it is the most common benign cardiac tumor found more frequently. In young adults with stroke or transient ischemic attack than in older patients. Age of the patient and unusual cause of recurrent stroke make this case report interesting. An 80-year-old male with no other conventional vascular risk factors such as hypertension, diabetes, or hyperlipidemia presented with left hemiparesis. Infarction over the right middle cerebral artery was disclosed on a magnetic resonance imaging study. The patient was a known case of right hemiparesis 3 years back, which was improved. The cause of repeated attack of stroke was left atrial myxoma, diagnosed by two-dimensional echocardiography.

Identification of keratinocyte specific markers using phage display and mass spectrometry

DEFF Research Database (Denmark)

Jensen, K.B.; Jensen, O.N.; Ravn, P.

2003-01-01

and mass spectrometry that allows identification of cell type-specific protein markers. The most important features of the method are (i) reduction of experimental noise originating from background binding of phage particles and (ii) isolation of affinity binders after a single round of selection, which...... 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....

Removal of phages and viral pathogens in a full-scale MBR: Implications for wastewater reuse and potable water.

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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.

Bad Phages in Good Bacteria: Role of the Mysterious orf63 of λ and Shiga Toxin-Converting Φ24B Bacteriophages

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Aleksandra Dydecka

2017-08-01

Full Text Available Lambdoid bacteriophages form a group of viruses that shares a common schema of genome organization and lifecycle. Some of them can play crucial roles in creating the pathogenic profiles of Escherichia coli strains. For example, Shiga toxin-producing E. coli (STEC acquired stx genes, encoding Shiga toxins, via lambdoid prophages (Stx phages. The results obtained so far present the evidence for the relation between the exo-xis region of the phage genome and lambdoid phage development, however molecular mechanisms of activities of the exo-xis genes' products are still unknown. In view of this, we decided to determine the influence of the uncharacterized open reading frame orf63 of the exo-xis region on lambdoid phages development using recombinant prophages, λ and Stx phage Φ24B. We have demonstrated that orf63 codes for a folded protein, thus, it is a functional gene. NMR spectroscopy and analytical gel filtration were used to extend this observation further. From backbone chemical shifts, Orf63 is oligomeric in solution, likely a trimer and consistent with its small size (63 aa., is comprised of two helices, likely intertwined to form the oligomer. We observed that the deletion of phage orf63 does not impair the intracellular lambdoid phage lytic development, however delays the time and decreases the efficiency of prophage induction and in consequence results in increased survival of E. coli during phage lytic development. Additionally, the deletion of phage orf63 negatively influences expression of the major phage genes and open reading frames from the exo-xis region during prophage induction with hydrogen peroxide. We conclude, that lambdoid phage orf63 may have specific functions in the regulation of lambdoid phages development, especially at the stage of the lysis vs. lysogenization decision. Besides, orf63 probably participates in the regulation of the level of expression of essential phage genes and open reading frames from the exo

Biological control of Aeromonas salmonicida subsp. salmonicida infection in rainbow trout (Oncorhynchus mykiss) using Aeromonas phage PAS-1.

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Kim, J H; Choresca, C H; Shin, S P; Han, J E; Jun, J W; Park, S C

2015-02-01

The potential control efficacy of Aeromonas phage PAS-1 was evaluated against Aeromonas salmonicida subsp. salmonicida infection in rainbow trout (Oncorhynchus mykiss) model in this study. The phage was co-cultured with the virulent A. salmonicida subsp. salmonicida strain AS05 that possesses the type III secretion system (TTSS) ascV gene, and efficient bacteriolytic activity was observed against the bacteria. The administration of PAS-1 in rainbow trout demonstrated that the phage was cleared from the fish within 200 h post-administration, and a temporal neutralizing activity against the phage was detected in the sera of phage-administrated fish. The administration of PAS-1 (multiplicity of infection: 10 000) in A. salmonicida subsp. salmonicida infected rainbow trout model showed notable protective effects, with increased survival rates and mean times to death. These results demonstrated that Aeromonas phage PAS-1 could be considered as an alternative biological control agent against A. salmonicida subsp. salmonicida infections in rainbow trout culture. © 2013 Blackwell Verlag GmbH.

Probing ADAMTS13 Substrate Specificity using Phage Display

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Desch, Karl C.; Kretz, Colin; Yee, Andrew; Gildersleeve, Robert; Metzger, Kristin; Agrawal, Nidhi; Cheng, Jane; Ginsburg, David

2015-01-01

Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73. PMID:25849793

Familial Hemiplegic Migraine and Recurrent Episodes of Psychosis

DEFF Research Database (Denmark)

LaBianca, Sonja; Jensen, Rigmor; van den Maagdenberg, Arn M J M

2015-01-01

Familial hemiplegic migraine (FHM) is a rare autosomal dominant form of migraine with motor aura. We present a case report of a father and son with very similar attacks of hemiplegic migraine and recurrent episodes of accompanying psychoses. Previously, such episodes led to hospitalization...... and extended clinical examinations, which further worsened the psychoses. Since the episodes were recognized as related to the hemiplegic migraine, a treatment strategy combining sleep and sedation was initiated and progression onto psychosis was almost completely avoided in both father and son. Genetic...

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...

Genome of a SAR116 bacteriophage shows the prevalence of this phage type in the oceans.

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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.

Recurrent Vestibular Migraine Vertigo Attacks Associated With the Development of Profound Bilateral Vestibulopathy: A Case Series.

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Wester, Jacob L; Ishiyama, Akira; Ishiyama, Gail

2017-09-01

Bilateral vestibulopathy (BVP) is a debilitating condition characterized by gait ataxia, oscillopsia, and imbalance. Case series of patients with migraine-linked vertigo spells and profound BVP. PATIENT 1:: A 69-year-old man presented with a history of recurrent severe vertigo spells lasting up to 3 days in duration associated with prostrating migraine headaches starting at age 60. His symptoms were misdiagnosed as an anxiety syndrome. At age 68, electronystagmography (ENG) revealed bilaterally absent caloric responses and complete BVP. His hearing was normal. PATIENT 2:: A 51-year-old man presented with a history of "earthquake-like" vertigo, sharp head pain, and phonophobia. These episodes occurred a handful of times over a 7-year period. Previous ENG testing at age 43 was normal. However, his ENG at age 48 revealed complete BVP. He was started on acetazolamide and noted improved balance, although subsequent ENG was unchanged. PATIENT 3:: A 49-year-old woman presented with a history of recurrent migraines with visual aura associated with vertigo lasting 1 hour. ENG at age 50 revealed complete BVP. Subjectively, she noted improved balance with acetazolamide and subsequent ENG demonstrated mild improvement. PATIENT 4:: A 43-year-old man presented with a 5-year history of optical migraines and recurrent vertigo spells, lasting 30 seconds, which was misdiagnosed as positional vertigo. He additionally had a 10-year history of oscillopsia. ENG at age 61 revealed complete BVP. In these cases, vestibular migraine was linked to recurrent vertigo spells that eventually led to complete bilateral vestibulopathy.

Isolation and characterization of ZZ1, a novel lytic phage that infects Acinetobacter baumannii clinical isolates

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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

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.

Characterization of Bacillus phage-K2 isolated from chungkookjang, a fermented soybean foodstuff.

Science.gov (United States)

Kim, Eun Ju; Hong, Jeong Won; Yun, Na-Rae; Lee, Young Nam

2011-01-01

An investigation of a virulent Bacillus phage-K2 (named Bp-K2) isolated from chungkookjang (a fermented soybean foodstuff) was made. Bp-K2 differed in infectivity against a number of Bacillus subtilis strains including starter strains of chungkookjang and natto, being more infectious to Bacillus strains isolated from the chungkookjang, but much less active against a natto strain. Bp-K2 is a small DNA phage whose genome size is about 21 kb. Bp-K2 is a tailed bacteriophage with an isometric icosahedral head (50 nm long on the lateral side, 80 nm wide), a long contractile sheath (85-90 nm × 28 nm), a thin tail fiber (80-85 nm long, 10 nm wide), and a basal plate (29 nm long, 47 nm wide) with a number of spikes, but no collar. The details of the structures of Bp-K2 differ from natto phage ϕBN100 as well as other known Bacillus phages such as SPO1-like or ϕ 29-like viruses. These data suggest that Bp-K2 would be a new member of the Myoviridae family of Bacillus bacteriophages.

Characterization and Testing the Efficiency of Acinetobacter baumannii Phage vB-GEC_Ab-M-G7 as an Antibacterial Agent

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Ia Kusradze

2016-10-01

Full Text Available Acinetobacter baumannii is a gram-negative, non-motile bacterium that, due to its multidrug resistance, has become a major nosocomial pathogen .The increasing number of multidrug resistant (MDR strains has renewed interest in phage therapy. The aim of our study was to assess the effectiveness of phage administration in Acinetobacter baumannii wound infections in an animal model to demonstrate phage therapy as non-toxic, safe and alternative antibacterial remedy. Using classical methods for the study of bacteriophage properties, we characterized phage vB-GEC_Ab-M-G7 as a dsDNA myovirus with a 90kb genome size. Important characteristics of vB-GEC_Ab-M-G7include a short latent period and large burst size, wide host range, resistance to chloroform and thermal and pH stability. In a rat wound model, phage application effectively decreased the number of bacteria isolated from the wounds of successfully treated animals. This study highlights the effectiveness of the phage therapy and provides further insight into treating infections caused by MDR strains using phage administration.

Bluetooth security attacks comparative analysis, attacks, and countermeasures

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Haataja, Keijo; Pasanen, Sanna; Toivanen, Pekka

2013-01-01

This overview of Bluetooth security examines network vulnerabilities and offers a comparative analysis of recent security attacks. It also examines related countermeasures and proposes a novel attack that works against all existing Bluetooth versions.

Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes.

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Edgar, Rotem; Friedman, Nir; Molshanski-Mor, Shahar; Qimron, Udi

2012-02-01

Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that resensitizes pathogens to approved antibiotics therefore holds key advantages. We present a proof of principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, the genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages carrying the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene's sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage's ability to kill pathogens in the infected host, but instead, on its ability to deliver genetic constructs into the bacteria and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phage will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.

Complete Genome Sequence of EtG, the First Phage Sequenced from Erwinia tracheiphila.

Science.gov (United States)

Andrade-Domínguez, Andrés; Kolter, Roberto; Shapiro, Lori R

2018-02-22

Erwinia tracheiphila is the causal agent of bacterial wilt of cucurbits. Here, we report the genome sequence of the temperate phage EtG, which was isolated from an E. tracheiphila -infected cucumber plant. Phage EtG has a linear 30,413-bp double-stranded DNA genome with cohesive ends and 45 predicted open reading frames. Copyright © 2018 Andrade-Domínguez et al.

'Twisted tape sign': Its significance in recurrent sigmoid volvulus

International Nuclear Information System (INIS)

Gopal, K.; Lim, Y.; Banerjee, B.

2005-01-01

Aim: Sigmoid volvulus is a common cause of intestinal obstruction in the elderly. Mild attacks of sigmoid volvulus may be more difficult to diagnose due to the lack of severity of symptoms which may resolve spontaneously only to recur after an interval. This study was a review of patients to assess the incidence of the 'twisted tape sign' and to evaluate the significance of its presence in cases of recurrent sigmoid volvulus. Methods and materials: A retrospective study over eight years revealed six cases of surgically confirmed recurrent sigmoid volvulus. Case records and barium enemas of all patients were reviewed. Results: Six patients were identified, including four men and two women, with a median age of 56 years. Diagnostic difficulties were encountered in four (67%) patients with a delay ranging between 10 and 37 months with a mean 17.3 months. Twisted tape sign was confirmed on all barium examinations retrospectively. Conclusion: Recognition of twisted tape sign on barium enema examination along with an appropriate clinical history would suggest a diagnosis of recurrent sigmoid volvulus

Identification of tumor associated single-chain Fv by panning and screening antibody phage library using tumor cells

Science.gov (United States)

Nie, Yong-Zhan; He, Feng-Tian; Li, Zhi-Kui; Wu, Kai-Chun; Cao, Yun-Xin; Chen, Bao-Jun; Fan, Dai-Ming

2002-01-01

AIM: To study the feasibility of panning and screening phage-displaying recombinant single-chain variable fragment (ScFv) of anti-tumor monoclonal antibodies for fixed whole cells as the carriers of mAb-binding antigens. METHODS: The recombinant phage displaying libraries for anti-colorectal tumor mAb MC3Ab, MC5Ab and anti-gastric tumor mAb MGD1 was constructed. Panning and screening were carried out by means of modified fixation of colorectal and gastric tumor cells expressed the mAb-binding antigens. Concordance of binding specificity to tumor cells between phage clones and parent antibodies was analyzed. The phage of positive clones was identified with competitive ELISA, and infected by E. coli HB2151 to express soluble ScFv. RESULTS: The ratio of positive clones to MC3-ScF-MC5-ScFv and MGD1-ScFv were 60%, 24% and 30%. MC3-ScFv had Mr 32000 confirmed by Western blot. The specificity to antigen had no difference between 4 positive recombinant phage antibodies and MC3Ab. CONCLUSION: The modified process of fixing whole tumor cells is efficient, convenient and feasible to pan and screen the phage-displaying ScFv of anti-tumor monoclonal antibodies. PMID:12174367

The genome and proteome of a virulent Escherichia coli O157:H7 bacteriophage closely resembling Salmonella phage Felix O1

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Waddell Thomas E

2009-04-01

Full Text Available Abstract Based upon whole genome and proteome analysis, Escherichia coli O157:H7-specific bacteriophage (phage wV8 belongs to the new myoviral genus, "the Felix O1-like viruses" along with Salmonella phage Felix O1 and Erwinia amylovora phage φEa21-4. The genome characteristics of phage wV8 (size 88.49 kb, mol%G+C 38.9, 138 ORFs, 23 tRNAs are very similar to those of phage Felix O1 (86.16 kb, 39.0 mol%G+C, 131 ORFs and 22 tRNAs and, indeed most of the proteins have their closest homologs within Felix O1. Approximately one-half of the Escherichia coli O157:H7 mutants resistant to phage wV8 still serotype as O157:H7 indicating that this phage may recognize, like coliphage T4, two different surface receptors: lipopolysaccharide and, perhaps, an outer membrane protein.

One-step production of phage-silicon nanoparticles by PLAL as fluorescent nanoprobes for cell identification

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De Plano, Laura M.; Scibilia, Santi; Rizzo, Maria Giovanna; Crea, Sara; Franco, Domenico; Mezzasalma, Angela M.; Guglielmino, Salvatore P. P.

2018-03-01

Silicon nanoparticles (SiNPs) are widely used as promising nanoplatform owing to their high specific surface area, optical properties and biocompatibility. Silicon nanoparticles find possible application in biomedical environment for their potential quantum effects and the functionalization with biomaterials, too. In this work, we propose a new approach for bio-functionalization of SiNPs and M13-engineered bacteriophage, displaying specific peptides that selectively recognize peripheral blood mononuclear cells (PBMC). The "one-step" functionalization is conducted during the laser ablation of silicon plate in buffer solution with engineered bacteriophages, to obtain SiNPs binding bacteriophages (phage-SiNPs). The interaction between SiNPs and bacteriophage is investigated. Particularly, the optical and morphological characterizations of phage-SiNPs are performed by UV-Vis spectroscopy, scanning electron microscopy operating in transmission mode (STEM) and X-ray spectroscopy (EDX). The functionality of phage-SiNPs is investigated through the photoemissive properties in recognition test on PBMC. Our results showed that phage-SiNPs maintain the capability and the activity to bind PBMC within 30 min. The fluorescence of phage-SiNPs allowed to obtain an optical signal on cell type targets. Finally, the proposed strategy demonstrated its potential use in in vitro applications and could be exploited to realize an optical biosensor to detect a specific target.

Analysis of risk factors and risk assessment for ischemic stroke recurrence

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Xiu-ying LONG

2016-08-01

Full Text Available Objective To screen the risk factors for recurrence of ischemic stroke and to assess the risk of recurrence. Methods Essen Stroke Risk Score (ESRS was used to evaluate the risk of recurrence in 176 patients with ischemic stroke (96 cases of first onset and 80 cases of recurrence. Univariate and multivariate stepwise Logistic regression analysis was used to screen risk factors for recurrence of ischemic stroke.  Results There were significant differences between first onset group and recurrence group on age, the proportion of > 75 years old, hypertension, diabetes, coronary heart disease, peripheral angiopathy, transient ischemic attack (TIA or ischemic stroke, drinking and ESRS score (P < 0.05, for all. First onset group included one case of ESRS 0 (1.04%, 8 cases of 1 (8.33%, 39 cases of 2 (40.63%, 44 cases of 3 (45.83%, 4 cases of 4 (4.17%. Recurrence group included 2 cases of ESRS 3 (2.50%, 20 cases of 4 (25% , 37 cases of 5 (46.25% , 18 cases of 6 (22.50% , 3 cases of 7 (3.75% . There was significant difference between 2 groups (Z = -11.376, P = 0.000. Logistic regression analysis showed ESRS > 3 score was independent risk factor for recurrence of ischemic stroke (OR = 31.324, 95%CI: 3.934-249.430; P = 0.001.  Conclusions ESRS > 3 score is the independent risk factor for recurrence of ischemic stroke. It is important to strengthen risk assessment of recurrence of ischemic stroke. To screen and control risk factors is the key to secondary prevention of ischemic stroke. DOI: 10.3969/j.issn.1672-6731.2016.07.011

Bacteriophage interactions with Vibrio anguillarum and the potential for phage therapy in marine aquaculture

DEFF Research Database (Denmark)

Rørbo, Nanna Iben

is widespread in the Vibrio community which underscore the lysogenic phages influence on bacterial evolution and functional properties. Highly genetically similar Vibrio phages, termed H20-like prophages, were isolated across large geographical scales being present both as freeliving phages and as prophages...... in V. anguillarum genomes. The H20-like phages’ widespread presence suggests a mutualistic interaction which selects for co-existence with V. anguillarum. In aquaculture, especially the larvae and fry are vulnerable to pathogens, and they are not susceptible to alternatives to antibiotics, e...

Molecular insights on the recognition of a Lactococcus lactis cell wall pellicle by the phage 1358 receptor binding protein.

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Farenc, Carine; Spinelli, Silvia; Vinogradov, Evgeny; Tremblay, Denise; Blangy, Stéphanie; Sadovskaya, Irina; Moineau, Sylvain; Cambillau, Christian

2014-06-01

The Gram-positive bacterium Lactococcus lactis is used for the production of cheeses and other fermented dairy products. Accidental infection of L. lactis cells by virulent lactococcal tailed phages is one of the major risks of fermentation failures in industrial dairy factories. Lactococcal phage 1358 possesses a host range limited to a few L. lactis strains and strong genomic similarities to Listeria phages. We report here the X-ray structures of phage 1358 receptor binding protein (RBP) in complex with monosaccharides. Each monomer of its trimeric RBP is formed of two domains: a "shoulder" domain linking the RBP to the rest of the phage and a jelly roll fold "head/host recognition" domain. This domain harbors a saccharide binding crevice located in the middle of a monomer. Crystal structures identified two sites at the RBP surface, ∼8 Å from each other, one accommodating a GlcNAc monosaccharide and the other accommodating a GlcNAc or a glucose 1-phosphate (Glc1P) monosaccharide. GlcNAc and GlcNAc1P are components of the polysaccharide pellicle that we identified at the cell surface of L. lactis SMQ-388, the host of phage 1358. We therefore modeled a galactofuranose (Galf) sugar bridging the two GlcNAc saccharides, suggesting that the trisaccharidic motif GlcNAc-Galf-GlcNAc (or Glc1P) might be common to receptors of genetically distinct lactococcal phages p2, TP091-1, and 1358. Strain specificity might therefore be elicited by steric clashes induced by the remaining components of the pellicle hexasaccharide. Taken together, these results provide a first insight into the molecular mechanism of host receptor recognition by lactococcal phages. Siphophages infecting the Gram-positive bacterium Lactococcus lactis are sources of milk fermentation failures in the dairy industry. We report here the structure of the pellicle polysaccharide from L. lactis SMQ-388, the specific host strain of phage 1358. We determined the X-ray structures of the lytic lactococcal phage

Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

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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.

SNP-PHAGE – High throughput SNP discovery pipeline

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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

Inhibition of bacterial conjugation by phage M13 and its protein g3p: quantitative analysis and model.

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Abraham Lin

Full Text Available Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage, these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

Scaling Up: Adapting a Phage-Hunting Course to Increase Participation of First-Year Students in Research.

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Staub, Nancy L; Poxleitner, Marianne; Braley, Amanda; Smith-Flores, Helen; Pribbenow, Christine M; Jaworski, Leslie; Lopatto, David; Anders, Kirk R

2016-01-01

Authentic research experiences are valuable components of effective undergraduate education. Research experiences during the first years of college are especially critical to increase persistence in science, technology, engineering, and mathematics fields. The Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) model provides a high-impact research experience to first-year students but is usually available to a limited number of students, and its implementation is costly in faculty time and laboratory space. To offer a research experience to all students taking introductory biology at Gonzaga University (n = 350/yr), we modified the traditional two-semester SEA-PHAGES course by streamlining the first-semester Phage Discovery lab and integrating the second SEA-PHAGES semester into other courses in the biology curriculum. Because most students in the introductory course are not biology majors, the Phage Discovery semester may be their only encounter with research. To discover whether students benefit from the first semester alone, we assessed the effects of the one-semester Phage Discovery course on students' understanding of course content. Specifically, students showed improvement in knowledge of bacteriophages, lab math skills, and understanding experimental design and interpretation. They also reported learning gains and benefits comparable with other course-based research experiences. Responses to open-ended questions suggest that students experienced this course as a true undergraduate research experience. © 2016 N. L. Staub et al. CBE—Life Sciences Education © 2016 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

[Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

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Han, Dong-gang; Duan, Xiao-yi; Guo, You-min; Zhou, Qi; Wang, Quan-ying; Yang, Guang-xiao

2010-01-01

To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

Transcription Profiling of Bacillus subtilis Cells Infected with AR9, a Giant Phage Encoding Two Multisubunit RNA Polymerases.

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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.

Characterization and complete genome sequence analysis of a novel virulent Siphoviridae phage against Staphylococcus aureus isolated from bovine mastitis in Xinjiang, China.

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Zhang, Qian; Xing, Shaozhen; Sun, Qiang; Pei, Guangqian; Cheng, Shi; Liu, Yannan; An, Xiaoping; Zhang, Xianglilan; Qu, Yonggang; Tong, Yigang

2017-06-01

Bovine mastitis is one of the most costly diseases in dairy cows worldwide. It can be caused by over 150 different microorganisms, where Staphylococcus aureus is the most frequently isolated and a major pathogen responsible for heavy economic losses in dairy industry. Although antibiotic therapy is most widely used, alternative treatments are necessary due to the increasing antibiotic resistance. Using phage for pathogen control is a promising tool in the fight against antibiotic resistance. Mainly using high-throughput sequencing, bioinformatics and our proposed phage termini identification method, we have isolated and characterized a novel virulent phage, designated as vB_SauS_IMEP5, from manure collected from dairy farms in Shihezi, Xinjiang, China, for use as a biocontrol agent against Staphylococcus aureus infections. Its latent period was about 30 min and its burst size was approximately 272PFU/cell. Phage vB_SauS_IMEP5 survives in a wide pH range between 3 and 12. A treatment at 70 °C for 20 min can inactive the phage. Morphological analysis of vB_SauS_IMEP5 revealed that phage vB_SauS_IMEP5 morphologically resembles phages in the family Siphoviridae. Among our tested multiplicity of infections (MOIs), the optimal multiplicity of infection (MOI) of this phage was determined to be 0.001, suggesting that phage vB_SauS_IMEP5 has high bacteriolytic potential and good efficiency for reducing bacterial growth. The complete genome of IME-P5 is a 44,677-bp, linear, double-stranded DNA, with a G+C content of 34.26%, containing 69 putative ORFs. The termini of genome were determined with next-generation sequencing data using our previously proposed termini identification method, which suggests that this phage has non-redundant termini with 9nt 3' protruding cohesive ends. The genomic and proteomic characteristics of IMEP5 demonstrate that this phage does not belong to any of the previously recognized Siphoviridae Staphylococcus phage groups, suggesting the

Properties and genomic analysis of Lactococcus garvieae lysogenic bacteriophage PLgT-1, a new member of Siphoviridae, with homology to Lactococcus lactis phages.

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Hoai, Truong Dinh; Nishiki, Issei; Yoshida, Terutoyo

2016-08-15

The lysogenic phage PLgT-1 is highly prevalent in Lactococcus garvieae, which is a serious bacterial pathogen in marine fish. Therefore, information regarding this phage is one of the key factors to predict the evolution of this bacterium. However, many properties of this phage, its complete genome sequence, and its relationship with other viral communities has not been investigated to date. Here, we demonstrated that the phage PLgT-1 was not only induced by an induction agent (Mitomycin C), but could be released frequently during cell division in a nutrient-rich environment or in natural seawater. Integration of PLgT-1 into non-lysogenic bacteria via transduction changed the genotype, resulting in the diversification of L. garvieae. The complete DNA sequence of PLgT-1 was also determined. This phage has a dsDNA genome of 40,273bp with 66 open reading frames (ORFs). Of these, the biological functions of 24 ORFs could be predicted but those of 42 ORFs are unknown. Thus, PLgT-1 is a novel phage with several novel proteins encoded in its genome. The strict MegaBLAST search program for the PLgT-1 genome revealed that this phage had no similarities with other previously investigated phages specific to L. garvieae (WP-2 and GE1). Notably, PLgT-1 was relatively homologous with several phages of Lactococcus lactis and 17 of the 24 predicted proteins encoded in PLgT-1 were homologous with the deduced proteins of various phages from these dairy bacteria. Comparative genome analysis revealed that the L. garvieae phage PLgT-1 was most closely related to the L. lactis phage TP712. However, they differed from each other in genome size and gene arrangement. The results obtained in this study suggest that the lysogenic phage PLgT-1 is a new member of the family Siphoviridae and has been involved in horizontal gene exchange with microbial communities, especially with L. lactis and its phages. Copyright © 2016 Elsevier B.V. All rights reserved.

Risk factors of short-term stroke recurrence in patients with minor ischemic cerebrovascular events

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Kavian Ghandehari

2013-03-01

Full Text Available BACKGROUND: Assessing the risk of recurrent ischemic events in patients with transient ischemic attack (TIA and minor ischemic stroke (MIS is of a great importance in clinical practice. METHODS: Consecutive patients with TIA or MIS who were visited in Ghaem Hospital, (Mashhad, Iran were enrolled in a prospective cohort study during 2010 to 2011. Diagnosis of TIA or MIS was accomplished by a stroke neurologist. Only those who presented within 24 hours from the onset of symptoms were recruited. MIS was considered as an ischemic stroke with National Institutes of Health Stroke Scale (NIHSS < 4. The endpoint of the study was a new ischemic cerebrovascular event or vascular death in 90 days and additionally in 3 days. The decision to admit and type of treatment in each case was left to the discretion of the stroke neurologist. The association between 20 potential factors with recurrent ischemic events in 3 and 90 days was investigated using univariate and multivariate analysis (MVA. RESULTS: 393 TIA patients (238 males and 155 females and 118 MIS patients (77 males and 41 females were enrolled in the study. Stroke occurred in 117 (23.2% patients, TIA in 99 (19.6%, and there was 11 (2.2% vascular deaths within 3 months in the total 511 patients with minor ischemic events. Crescendo TIAs and multiple TIAs were associated with greater risk of stroke in 3 days in a univariate analysis (OR = 5.12, P < 0.001 and (OR = 3.98, P = 0.003, respectively. Patients with index stroke had 11.5% lower risk of recurrent stroke in 3 days than patients with index TIA in multivariate analysis (OR = 0.115, P = 0.039. Diabetes was independently associated with 3 months stroke recurrence in the patients with minor ischemic events (OR = 2.65, P = 0.039. CONCLUSION: Multiple and crescendo TIAs are the main predictors of stroke recurrence, derived from the univariate analysis of the patients with minor ischemic events.   Keywords: Transient Ischemic Attacks, Infarction, Brain

Acinetobacter phage genome is similar to Sphinx 2.36, the circular DNA copurified with TSE infected particles.

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Longkumer, Toshisangba; Kamireddy, Swetha; Muthyala, Venkateswar Reddy; Akbarpasha, Shaikh; Pitchika, Gopi Krishna; Kodetham, Gopinath; Ayaluru, Murali; Siddavattam, Dayananda

2013-01-01

While analyzing plasmids of Acinetobacter sp. DS002 we have detected a circular DNA molecule pTS236, which upon further investigation is identified as the genome of a phage. The phage genome has shown sequence similarity to the recently discovered Sphinx 2.36 DNA sequence co-purified with the Transmissible Spongiform Encephalopathy (TSE) particles isolated from infected brain samples collected from diverse geographical regions. As in Sphinx 2.36, the phage genome also codes for three proteins. One of them codes for RepA and is shown to be involved in replication of pTS236 through rolling circle (RC) mode. The other two translationally coupled ORFs, orf106 and orf96, code for coat proteins of the phage. Although an orf96 homologue was not previously reported in Sphinx 2.36, a closer examination of DNA sequence of Sphinx 2.36 revealed its presence downstream of orf106 homologue. TEM images and infection assays revealed existence of phage AbDs1 in Acinetobacter sp. DS002.

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.

Incidence of Transient Ischemic Attack in Auckland, New Zealand, in 2011 to 2012.

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Barber, P Alan; Krishnamurthi, Rita; Parag, Varsha; Anderson, Neil E; Ranta, Annemarei; Kilfoyle, Dean; Wong, Edward; Green, Geoff; Arroll, Bruce; Bennett, Derrick A; Witt, Emma; Rush, Elaine; Minsun Suh, Flora; Theadom, Alice; Rathnasabapathy, Yogini; Te Ao, Braden; Parmar, Priyakumari; Feigin, Valery L

2016-09-01

There have been few recent population-based studies reporting the incidence (first ever) and attack rates (incident and recurrent) of transient ischemic attack (TIA). The fourth Auckland Regional Community Stroke study (ARCOS IV) used multiple overlapping case ascertainment methods to identify all hospitalized and nonhospitalized cases of TIA that occurred in people ≥16 years of age usually resident in Auckland (population ≥16 years of age is 1.12 million), during the 12 months from March 1, 2011. All first-ever and recurrent new TIAs (any new TIA 28 days after the index event) during the study period were recorded. There were 785 people with TIA (402 [51.2%] women, mean [SD] age 71.5 [13.8] years); 614 (78%) of European origin, 84 (11%) Māori/Pacific, and 75 (10%) Asian/Other. The annual incidence of TIA was 40 (95% confidence interval, 36-43), and attack rate was 63 (95% confidence interval, 59-68), per 100 000 people, age standardized to the World Health Organization world population. Approximately two thirds of people were known to be hypertensive or were being treated with blood pressure-lowering agents, half were taking antiplatelet agents and just under half were taking lipid-lowering therapy before the index TIA. Two hundred ten (27%) people were known to have atrial fibrillation at the time of the TIA, of whom only 61 (29%) were taking anticoagulant therapy, suggesting a failure to identify or treat atrial fibrillation. This study describes the burden of TIA in an era of aggressive primary and secondary vascular risk factor management. Education programs for medical practitioners and patients around the identification and management of atrial fibrillation are required. © 2016 American Heart Association, Inc.

Phage-based surface plasmon resonance strategies for the detection of pathogens

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Tawil, Nancy

We start by reviewing the basic principles and recent advances in biosensing technologies using optical, electrochemical and acoustic platforms for phage-based diagnostics. Although much notable work has been done, a low cost, specific, sensitive optical method for detecting low concentrations of pathogens, in a few minutes, has not been established. We conclude from the limited body of work on the subject that improving immobilization strategies and finding more suitable phage recognition elements would allow for a more sensitive approach. Our aim was to better describe the attachment process of MRSA specific phages on gold surfaces, and the subsequent biodetection of their bacterial hosts by surface plasmon resonance (SPR). With the knowledge that the adsorption characteristics of thiol-containing molecules are necessary for applications involving the attachment of recognition elements to a functionalized surface, we start by providing comparative details on the kinetics of self-assembly of L-cysteine and 11-mercaptoundecanoic acid (MUA) monolayers on gold using SPR[1]. Our purpose, in carrying out these measurements was to establish each molecule's validity and applicability as a linker element for use in biosensing. We find that monolayer formation, for both L-cysteine and MUA, is described by the Langmuir isotherm at low concentrations only. For L-cysteine, both the amine and thiol groups contribute to the initial attachment of the molecule, followed by the replacement of the amine-gold complexes initially formed with more stable thiol-gold complexes. The reorganization of L-cysteine creates more space on the gold surface, and the zwitterionic form of the molecule permits the physisorption of a second layer through electrostatic interactions. On the other hand, MUA deposits randomly onto the surface of gold as a SAM and slowly reorganizes into a denser, vertical state. Surface plasmon resonance was then used for the real-time monitoring of the attachment of

Injected phage-displayed-VP28 vaccine reduces shrimp Litopenaeus vannamei mortality by white spot syndrome virus infection.

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Solís-Lucero, G; Manoutcharian, K; Hernández-López, J; Ascencio, F

2016-08-01

White spot syndrome virus (WSSV) is the most important viral pathogen for the global shrimp industry causing mass mortalities with huge economic losses. Recombinant phages are capable of expressing foreign peptides on viral coat surface and act as antigenic peptide carriers bearing a phage-displayed vaccine. In this study, the full-length VP28 protein of WSSV, widely known as potential vaccine against infection in shrimp, was successfully cloned and expressed on M13 filamentous phage. The functionality and efficacy of this vaccine immunogen was demonstrated through immunoassay and in vivo challenge studies. In ELISA assay phage-displayed VP28 was bind to Litopenaeus vannamei immobilized hemocyte in contrast to wild-type M13 phage. Shrimps were injected with 2 × 10(10) cfu animal(-1) single dose of VP28-M13 and M13 once and 48 h later intramuscularly challenged with WSSV to test the efficacy of the vaccine against the infection. All dead challenged shrimps were PCR WSSV-positive. The accumulative mortality of the vaccinated and challenged shrimp groups was significantly lower (36.67%) than the unvaccinated group (66.67%). Individual phenoloxidase and superoxide dismutase activity was assayed on 8 and 48 h post-vaccination. No significant difference was found in those immunological parameters among groups at any sampled time evaluated. For the first time, phage display technology was used to express a recombinant vaccine for shrimp. The highest percentage of relative survival in vaccinated shrimp (RPS = 44.99%) suggest that the recombinant phage can be used successfully to display and deliver VP28 for farmed marine crustaceans. Copyright © 2016 Elsevier Ltd. All rights reserved.

Typing of Panton-Valentine leukocidin-encoding phages carried by methicillin-susceptible and methicillin-resistant Staphylococcus aureus from Italy.

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Sanchini, A; Del Grosso, M; Villa, L; Ammendolia, M G; Superti, F; Monaco, M; Pantosti, A

2014-11-01

Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) but can also be found in methicillin-susceptible S. aureus (MSSA) sharing pathogenic and epidemiological characteristics of CA-MRSA. PVL is encoded by two co-transcribed genes that are carried by different staphylococcal bacteriophages. We applied an extended PCR-based typing scheme for the identification of two morphological groups (elongated-head group and icosahedral-head group I phages) and specific PVL phage types in S. aureus isolates recovered in Italy. We examined 48 PVL-positive isolates (25 MSSA and 23 MRSA) collected from different hospital laboratories from April 2005 to May 2011. spa typing, multilocus sequence typing and staphylococcal cassette chromosome mec typing were applied to categorize the isolates. Phage typeability was 48.0% in MSSA and 91.3% in MRSA, highlighting the limitation of the PCR typing scheme when applied to PVL-positive MSSA. Five different PVL phages and two variants of a known phage were detected, the most prevalent being ΦSa2usa, recovered in 15 out of 48 (31.2%) isolates, and carried by both MSSA and MRSA belonging to CC8 and CC5. The recently described ΦTCH60 was recovered in four isolates. A PVL phage (ΦSa119) from an ST772 MRSA, that was not detected using the previous typing scheme, was sequenced, and new primers were designed for the identification of the icosahedral-head group II PVL phages present in ST772 and ST59 MRSA. A comprehensive PVL-phage typing can contribute to the understanding of the epidemiology and evolution of PVL-positive MSSA and MRSA. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

Recurrent Candida albicans Ventriculitis Treated with Intraventricular Liposomal Amphotericin B

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Demet Toprak

2015-01-01

Full Text Available Central nervous system (CNS infection with Candida is rare but significant because of its high morbidity and mortality. When present, it is commonly seen among immunocompromised and hospitalized patients. Herein, we describe a case of a four-year-old boy with acute lymphoblastic leukemia (ALL who experienced recurrent Candida albicans meningitis. The patient was treated successfully with intravenous liposomal amphotericin B at first attack, but 25 days after discharge he was readmitted to hospital with symptoms of meningitis. Candida albicans was grown in CFS culture again and cranial magnetic resonance imaging (MRI showed ventriculitis. We administered liposomal amphotericin B both intravenously and intraventricularly and favorable result was achieved without any adverse effects. Intraventricular amphotericin B may be considered for the treatment of recurrent CNS Candida infections in addition to intravenous administration.

Genome Sequence of Gordonia Phage BetterKatz

Science.gov (United States)

Berryman, Emily N.; Forrest, Kaitlyn M.; McHale, Lilliana; Wertz, Anthony T.; Zhuang, Zenas; Kasturiarachi, Naomi S.; Pressimone, Catherine A.; Schiebel, Johnathon G.; Furbee, Emily C.; Grubb, Sarah R.; Warner, Marcie H.; Montgomery, Matthew T.; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah; Hatfull, Graham F.

2016-01-01

BetterKatz is a bacteriophage isolated from a soil sample collected in Pittsburgh, Pennsylvania using the host Gordonia terrae 3612. BetterKatz’s genome is 50,636 bp long and contains 75 predicted protein-coding genes, 35 of which have been assigned putative functions. BetterKatz is not closely related to other sequenced Gordonia phages. PMID:27516497

HostPhinder: A Phage Host Prediction Tool

DEFF Research Database (Denmark)

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

2016-01-01

The current dramatic increase of antibiotic resistant bacteria has revitalised the interest in bacteriophages as alternative antibacterial treatment. Meanwhile, the development of bioinformatics methods for analysing genomic data places high-throughput approaches for phage characterization within...... bacterial hosts. HostPhinder is available as an interactive web service [1] and as a stand alone download from the Docker registry [2]....

[Inventory building of phages against extensively drug-resistant Acinetobacter baumannii isolated from wounds of patients with severe burn and related characteristic analysis].

Science.gov (United States)

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

2016-09-20

To build inventory of phages against extensively drug-resistant Acinetobacter Baumannii isolated from wounds of inpatients of burn ICU and analyze related characteristics. In 2014 and 2015, 131 strains of extensively drug-resistant Acinetobacter Baumannii were isolated from wounds of inpatients of burn ICU from one hospital in Chongqing. In 2015, 98 strains of extensively drug-resistant Acinetobacter Baumannii were isolated from wounds of inpatients of burn ICU from 6 hospitals in Guangdong province. Above-mentioned 229 strains were collected for conducting experiments as follows: (1) Multilocus sequence typing (MLST) of strains isolated from Chongqing and Guangdong province was analyzed. (2) Sewage co-culture method was applied for isolation of phages with above-mentioned strains and sewage from Chongqing and Guangdong province. Numbers of isolated phages and times of successful isolation and unsuccessful isolation were recorded. (3) The most prevalent subtypes of strains from Chongqing and Guangdong province in 2015 were collected, and their phages respectively underwent cross infection with all strains from Chongqing and those from Guangdong province. The lysis ability of phage was observed when phage underwent cross infection with the same subtype of strain or not the same, and the lytic ratio was calculated. (4) Fluid of phage in one type was randomly selected and equally divided into 3 parts, and its titer was determined by double dilution method. Then each part of phage fluid was subdivided into 3 small parts, which were cultured with LB fluid medium and respectively stored under the condition of -20 ℃, 4 ℃, and room temperature. After being stored for 1 month and 2 months, the titer of phage was determined for evaluating stability of phage. Data were processed with Fisher's exact test, chi-square test, and one-way analysis of variance. (1) The major type of strains from Chongqing in 2014 was ST368 (45%, 31/69), and major types of strains from Chongqing

Distinguishing attack and second-preimage attack on encrypted message authentication codes (EMAC)

Science.gov (United States)

Ariwibowo, Sigit; Windarta, Susila

2016-02-01

In this paper we show that distinguisher on CBC-MAC can be applied to Encrypted Message Authentication Code (EMAC) scheme. EMAC scheme in general is vulnerable to distinguishing attack and second preimage attack. Distinguishing attack simulation on AES-EMAC using 225 message modifications, no collision have been found. According to second preimage attack simulation on AES-EMAC no collision found between EMAC value of S1 and S2, i.e. no second preimage found for messages that have been tested. Based on distinguishing attack simulation on truncated AES-EMAC we found collision in every message therefore we cannot distinguish truncated AES-EMAC with random function. Second-preimage attack is successfully performed on truncated AES-EMAC.

Evolution of DWI signal abnormalities after transient ischemic attack and minor ischaemic stroke

LENUS (Irish Health Repository)

Merwick, A

2011-05-01

Background: Diffusion weighted imaging (DWI) signal abnormality after transient ischaemic attack (TIA) predicts early stroke, independently of other risk markers included in the ABCD3-I score. Early stroke recurrence detected on follow-up DWI after the acute-phase DWI may identify patients at high risk for subsequent clinicalstrokesstroke, cognitive impairment, and seizures. We aimed to determine the evolution of acute DWI lesions and rate of new ischaemic lesion (NIL) occurrence on follow-up DWI after TIA and minor stroke.\\r\

Comparative genomics defines the core genome of the growing N4-like phage genus and identifies N4-like Roseophage specific genes

Directory of Open Access Journals (Sweden)

Jacqueline Zoe-Munn Chan

2014-10-01

Full Text Available Two bacteriophages, RPP1 and RLP1, infecting members of the marine Roseobacter clade were isolated from seawater. Their linear genomes are 74.7 and 74.6 kb and encode 91 and 92 coding DNA sequences, respectively. Around 30% of these are homologous to genes found in Enterobacter phage N4. Comparative genomics of these two new Roseobacter phages and twenty-three other sequenced N4-like phages (three infecting members of the Roseobacter lineage and twenty infecting other Gammaproteobacteria revealed that N4-like phages share a core genome of 14 genes responsible for control of gene expression, replication and virion proteins. Phylogenetic analysis of these genes placed the five N4-like roseophages (RN4 into a distinct subclade. Analysis of the RN4 phage genomes revealed they share a further 19 genes of which nine are found exclusively in RN4 phages and four appear to have been acquired from their bacterial hosts. Proteomic analysis of the RPP1 and RLP1 virions identified a second structural module present in the RN4 phages similar to that found in the Pseudomonas N4-like phage LIT1. Searches of various metagenomic databases, included the GOS database, using CDS sequences from RPP1 suggests these phages are widely distributed in marine environments in particular in the open ocean environment.

Genomic, proteomic, morphological, and phylogenetic analyses of vB_EcoP_SU10, a podoviridae phage with C3 morphology.

Directory of Open Access Journals (Sweden)

Mohammadali Khan Mirzaei

Full Text Available A recently isolated phage, vB_EcoP_SU10 (SU10, with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM, scanning electron microscopy (SEM, and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome. The TEM images reveal that SU10 have a quite long tail for being a Podoviridae phage, and that the tail also changes conformation upon infection. The ultra-thin section electron microscopy images of phages at the stage of replication within the host cell show that the phages form a honeycomb-like structure under packaging of genomes and assembly of mature capsids. This implies a tight link between the replication and cutting of the concatemeric genome, genome packaging, and capsid assembly. We have also performed a phylogenetic analysis of the structural genes common between Podoviridae phages of the C1 and C3 morphotypes. The result shows that the structural genes have coevolved, and that they form two distinct groups linked to their morphotypes. The structural genes of C1 and C3 phages appear to have diverged around 280 million years ago applying a molecular clock calibrated according to the presumed split between the Escherichia - Salmonella genera.

Genetic exchanges caused by ultraviolet photoproducts in phage lamda DNA molecules: the role of DNA replication

International Nuclear Information System (INIS)

Lin, P.F.; Howard-Flanders, P.; Yale Univ., New Haven, Conn.

1976-01-01

Genetic recombination induced by structural damage in DNA molecules was investigated in E. coli K12(lamda) lysogens infected with genetically marked phage lamda. Photoproducts were induced in the phage DNA before infection by exposing them either to 313 nm light in the presence of acetophenone or to 254 nm light. To test the role of the replication of the damage phage DNA on the frequency of the induced recombination , both heteroimmune and homoimmune crosses were performed, and scored for P + recombinants. In heteroimmune crosses, recombination was less frequent in infected cells exposed to visible light and in wild type cells able to perform excision repair than in excision-defective lysogens. Therefore, much of the induced recombination can be attributed to the pyrimidine dimers in the phage DNA. In homoimmune crosses, replication of the phage DNA containing ultraviolet photoproducts was represented by lamda immunity, and was further blocked by the lack of the P gene product needed for replication. The 254 nm photoproducts increased the frequency of recombination in these homoimmune crosses, even though phage DNA replication was blocked. Irradiation with 313 nm light and acetophenone M, which produces dimers and unknown photoproducts, was not as effective per dimer as the 254 nm light. It is concluded from these results that certain unidentified 254 nm photoproducts can cause recombination even in the absence of DNA replication. They are not pyrimidine dimers, as they are not susceptible to excision repair or photoreactivation. In contrast, pyrimidine dimers appear to cause recombination only when the DNA containing them undergoes replication. (orig./MG) [de

The XXIIIrd Phage/Virus Assembly Meeting.

Science.gov (United States)

Serwer, Philip

2014-01-01

The XXIIIrd Phage/Virus Assembly (PVA) meeting returned to its birthplace in Lake Arrowhead, CA on September 8-13, 2013 (Fig. 1). The original meeting occurred in 1968, organized by Bob Edgar (Caltech, Pasadena, CA USA), Fred Eiserling (University of California, Los Angeles, Los Angeles, CA USA) and Bill Wood (Caltech, Pasadena, CA USA). The organizers of the 2013 meeting were Bill Gelbart (University of California, Los Angeles, Los Angeles, CA USA) and Jack Johnson (Scripps Research Institute, La Jolla, CA USA). This meeting specializes in an egalitarian format. Students are distinguished from senior faculty primarily by the signs of age. With the exception of historically based introductory talks, all talks were allotted the same time and freedom. This tradition began when the meeting was phage-only and has been continued now that all viruses are included. Many were the animated conversations about basic questions. New and international participants were present, a sign that the field has significant attraction, as it should, based on details below. The meeting was also characterized by a sense of humor and generally good times, a chance to both enjoy the science and forget the funding malaise to which many participants are exposed. I will present some of the meeting content, without attempting to be comprehensive.

PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.

Science.gov (United States)

Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred

2018-01-01

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

Molecular Interaction between Lipoteichoic Acids and Lactobacillus delbrueckii Phages Depends on d-Alanyl and α-Glucose Substitution of Poly(Glycerophosphate) Backbones▿

Science.gov (United States)

Räisänen, Liisa; Draing, Christian; Pfitzenmaier, Markus; Schubert, Karin; Jaakonsaari, Tiina; von Aulock, Sonja; Hartung, Thomas; Alatossava, Tapani

2007-01-01

Lipoteichoic acids (LTAs) have been shown to act as bacterial counterparts to the receptor binding proteins of LL-H, LL-H host range mutant LL-H-a21, and JCL1032. Here we have used LTAs purified by hydrophobic interaction chromatography from different phage-resistant and -sensitive strains of Lactobacillus delbrueckii subsp. lactis. Nuclear magnetic resonance analyses revealed variation in the degree of α-glucosyl and d-alanyl substitution of the 1,3-linked poly(glycerophosphate) LTAs between the phage-sensitive and phage-resistant strains. Inactivation of phages was less effective if there was a high level of d-alanine residues in the LTA backbones. Prior incubation of the LTAs with α-glucose-specific lectin inhibited the LL-H phage inactivation. The overall level of decoration or the specific spatial combination of α-glucosyl-substituted, d-alanyl-substituted, and nonsubstituted glycerol residues may also affect phage adsorption. PMID:17416656

Relative roles of uvrA and recA genes in the recovery of Escherichia coli and phage lambda after ultraviolet irradiation

International Nuclear Information System (INIS)

Salaj-Smic, E.; Petranovic, D.; Petranovic, M.; Trgovcevic, Z.

1980-01-01

The action of the host-cell repair system on recovery from uv damage to bacterial and phage DNA was studied. lambda cI857 ind red lysogens were used. These lysogens, although noninducible by uv light, can be induced by raising the temperature from 30 to 42 0 C. Sensitivity of the phage in relation to its host was analyzed in various bacterial backgrounds. Relative sensitivity of the phage and its host is the same if the uv survival curve for colonies is 80 times steeper than for plaques. This same relative sensitivity is observed if the host cell does not possess any mechanism for DNA repair (uvrA recA background). In the uvrA recA + background, the plaque survival is not significantly increased above the level observed in the uvrA recA double mutant. recA-dependent recombinational postreplication repair does not operate on the phage DNA in the cytoplasm; relative sensitivity of the phage is therefore much higher than that of the host. If the lysogenic induction is delayed, a marked increase in the plaque count is seen so the phage shows the same relative sensitivity as the bacterial cell. Short-patch excision repair operates on both phage and bacterial DNA but less efficiently on phage DNA. In the wild-type (uvrA + recA + ) host, the highest survival of plaques and colonies is obtained. Relative sensitivity of the phage is nevertheless 50 times higher then that of the bacterial cell. This may mean the recA gene product is involved in copy-choice excision and/or long-patch excision and/or incision-promoted recombination repair of the phage DNA but it remains unable to mediate its recombinational postreplication repair

Investigation of the protective effect of whey proteins on lactococcal phages during heat treatment at various pH.

Science.gov (United States)

Geagea, Hany; Gomaa, Ahmed I; Remondetto, Gabriel; Moineau, Sylvain; Subirade, Muriel

2015-10-01

The incorporation of whey protein concentrates (WPC) into cheese is a risky process due to the potential contamination with thermo-resistant phages of lactic acid bacteria (LAB). Furthermore, whey proteins can protect phages during heat treatment, thereby increasing the above risk. The main objective of this work was to understand this protective effect in order to better control LAB phages and maximize whey recycling in the cheese industry. First, the inactivation of a previously characterized thermo-resistant lactococcal virulent phage (P1532) was investigated at 95 °C in WPC, in individual whey components β-lactoglobulin, α-lactalbumin, and bovine serum albumin as well as under different heat and pH conditions. The structural changes of the tested proteins were also monitored by transmission FTIR spectroscopy. Phage inactivation results indicated that the protective effect of whey proteins was pH and time dependent at 95 °C and was not restricted to one component. FTIR spectra suggest that the protection is related to protein molecular structures and to the level of protein aggregates, which was more pronounced in acidic conditions. Moreover, the molecular structure of the three proteins tested was differently influenced by pH and the duration of the heat treatment. This work confirms the protective effect of WPC on phages during heat treatment and offers the first hint to explain such phenomenon. Finding the appropriate treatment of WPC to reduce the phage risk is one of the keys to improving the cheese manufacturing process. Copyright © 2015 Elsevier B.V. All rights reserved.

Early risk of recurrent stroke in patients with symptomatic carotid near-occlusion: Results from CAOS, a multicenter registry study.

Science.gov (United States)

García-Pastor, Andrés; Gil-Núñez, Antonio; Ramírez-Moreno, José María; González-Nafría, Noelia; Tejada, Javier; Moniche, Francisco; Portilla-Cuenca, Juan Carlos; Martínez-Sánchez, Patricia; Fuentes, Blanca; Gamero-García, Miguel Ángel; Alonso de Leciñana, María; Cánovas-Verge, David; Aladro, Yolanda; Parkhutik, Vera; Lago-Martín, Aida; de Arce-Borda, Ana María; Usero-Ruíz, María; Delgado-Mederos, Raquel; Pampliega, Ana; Ximenez-Carrillo, Álvaro; Bártulos-Iglesias, Mónica; Castro-Reyes, Enrique

2017-10-01

Background The risk of recurrent stroke among patients with symptomatic carotid near-occlusion is not well established, and management of the condition remains controversial. Symptomatic carotid near-occlusion with full collapse has been identified as a strong predictor of early recurrence. We aimed to analyze the 90-day risk of recurrent ipsilateral ischemic stroke in medically treated patients with symptomatic carotid near-occlusion. Methods We performed a multicenter, nationwide, prospective study from January 2010 to May 2016. Patients with angiography-confirmed symptomatic carotid near-occlusion were included. The primary endpoint was ipsilateral ischemic stroke or transient ischemic attack (TIA) within 90 days after the presenting event. For this analysis, patients who underwent revascularization within 90 days after stroke were excluded. Results The study population comprised 141 patients from 17 Spanish centers; 83 patients were treated medically. Primary endpoint occurred in eight patients, resulting in a cumulative rate of 10.6% (95% CI, 3.7-17.5). Previous history of stroke or transient ischemic attack was identified as an independent predictor for recurrence in the multivariate Cox regression analysis (HR, 4.37 [95% CI, 1.05-18.18]; p = 0.043), while the presence of full collapse was not associated with an increased risk (HR, 0.81 [95% CI, 0.17-3.92]; p = 0.793). The risk of recurrence was also not affected by the presence of significant stenosis or occlusion of the contralateral carotid artery, or by the collateral circulation. Conclusions Patients with symptomatic carotid near-occlusion seem to have an increased risk of early ipsilateral recurrent stroke. Our results contrast with the low risk of symptomatic carotid near-occlusion reported to date. Full collapse did not increase the risk of recurrent stroke in our study.

The revival of Phage Therapy to fight Antimicrobial Resistance – Part II: What about patent protection and alternative incentives?

DEFF Research Database (Denmark)

Minssen, Timo

2014-01-01

Three days ago I commented on a couple of legal issues raised in the recent Nature report “Phage therapy gets revitalized” by Sara Reardon. One challenge concerns the reluctance of pharma companies to broadly invest in the development of phage therapies. As pointed out in the report, this does....... Like in Europe, the first door to patentability that phage-related technology would need to pass concerns patent eligibility. In the last years the US Supreme Court has rendered an astonishing number of fundamental patent-decisions, including not less than four (!) landmark judgments on patent...... occurring viruses, such as phages, and the processes in which these are used. Myriad and Prometheus could thus have a fundamental impact on many patent portfolios relating to phage therapy and thus business involvement. In that context, it is important to realize that an important component of proper use...

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.

Chloroform-Treated Filamentous Phage as a Bioreceptor for Piezoelectric Sensors

National Research Council Canada - National Science Library

Olsen, Eric V; Sykora, Jennifer C; Sorokulova, Iryna B; Petrenko, Valery A; Chen, I-Hsuan; Barbaree, James M; Vodyanoy, Vitaly J

2005-01-01

... and S. typhimurium. ELISA confirmed affinity-selected phage specificity for streptavidin. Spheroid induction was optimized to achieve greatest conversion yields as a function of solvent exposure time and concentration...

Cooperating attackers in neural cryptography.

Science.gov (United States)

Shacham, Lanir N; Klein, Einat; Mislovaty, Rachel; Kanter, Ido; Kinzel, Wolfgang

2004-06-01

A successful attack strategy in neural cryptography is presented. The neural cryptosystem, based on synchronization of neural networks by mutual learning, has been recently shown to be secure under different attack strategies. The success of the advanced attacker presented here, called the "majority-flipping attacker," does not decay with the parameters of the model. This attacker's outstanding success is due to its using a group of attackers which cooperate throughout the synchronization process, unlike any other attack strategy known. An analytical description of this attack is also presented, and fits the results of simulations.

Crony Attack: Strategic Attack’s Silver Bullet

Science.gov (United States)

2006-11-01

physical assets or financial assets. The form of crony attack that most closely resembles classic strategic attack is to deny, degrade, or destroy a money...February 1951. Reprinted in Airpower Studies Coursebook , Air Command and Staff College, Maxwell AFB, AL, 2002, 152–58. Hirsch, Michael. “NATO’s Game of

Isolation and characterization of Bacteroides host strain HB-73 used to detect sewage specific phages in Hawaii.

Science.gov (United States)

Vijayavel, Kannappan; Fujioka, Roger; Ebdon, James; Taylor, Huw

2010-06-01

Previous studies have shown that Escherichia coli and enterococci are unreliable indicators of fecal contamination in Hawaii because of their ability to multiply in environmental soils. In this study, the method of detecting Bacteroides phages as specific markers of sewage contamination in Hawaii's recreational waters was evaluated because these sewage specific phages cannot multiply under environmental conditions. Bacteroides hosts (GB-124, GA-17), were recovered from sewage samples in Europe and were reported to be effective in detecting phages from sewage samples obtained in certain geographical areas. However, GB-124 and GA-17 hosts were ineffective in detecting phages from sewage samples obtained in Hawaii. Bacteroides host HB-73 was isolated from a sewage sample in Hawaii, confirmed as a Bacteroides sp. and shown to recover phages from multiple sources of sewage produced in Hawaii at high concentrations (5.2-7.3 x 10(5) PFU/100 mL). These Bacteroides phages were considered as potential markers of sewage because they also survived for three days in fresh stream water and two days in marine water. Water samples from Hawaii's coastal swimming beaches and harbors, which were known to be contaminated with discharges from streams, were shown to contain moderate (20-187 CFU/100 mL) to elevated (173-816 CFU/100 mL) concentrations of enterococci. These same samples contained undetectable levels (Hawaii and the most likely source of these enterococci is from environmental soil rather than from sewage. 2010 Elsevier Ltd. All rights reserved.

A leucine repeat motif in AbiA is required for resistance of Lactococcus lactis to phages representing three species.

Science.gov (United States)

Dinsmore, P K; O'Sullivan, D J; Klaenhammer, T R

1998-05-28

The abiA gene encodes an abortive bacteriophage infection mechanism that can protect Lactococcus species from infection by a variety of bacteriophages including three unrelated phage species. Five heptad leucine repeats suggestive of a leucine zipper motif were identified between residues 232 and 266 in the predicted amino acid sequence of the AbiA protein. The biological role of residues in the repeats was investigated by incorporating amino acid substitutions via site-directed mutagenesis. Each mutant was tested for phage resistance against three phages, phi 31, sk1, and c2, belonging to species P335, 936, and c2, respectively. The five residues that comprise the heptad repeats were designated L234, L242, A249, L256, and L263. Three single conservative mutations of leucine to valine in positions L235, L242, and L263 and a double mutation of two leucines (L235 and L242) to valines did not affect AbiA activity on any phages tested. Non-conservative single substitutions of charged amino acids for three of the leucines (L235, L242, and L256) virtually eliminated AbiA activity on all phages tested. Substitution of the alanine residue in the third repeat (A249) with a charged residue did not affect AbiA activity. Replacement of L242 with an alanine elimination phage resistance against phi 31, but partial resistance to sk1 and c2 remained. Two single proline substitutions for leucines L242 and L263 virtually eliminated AbiA activity against all phages, indicating that the predicted alpha-helical structure of this region is important. Mutations in an adjacent region of basic amino acids had various effects on phage resistance, suggesting that these basic residues are also important for AbiA activity. This directed mutagenesis analysis of AbiA indicated that the leucine repeat structure is essential for conferring phage resistance against three species of lactococcal bacteriophages.

Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light

International Nuclear Information System (INIS)

Wood, R.D.

1985-01-01

Experiments were performed to examine the role of cyclobutyl pyrimidine dimers in the process of mutagenesis by ultraviolet light. Lambda phage DNA was irradiated with u.v. and then incubated with an Escherichia coli photoreactivating enzyme, which monomerizes cyclobutyl pyrimidine dimers upon exposure to visible light. The photoreactivated DNA was packaged into lambda phage particles, which were used to infect E. coli uvr - host cells that had been induced for SOS functions by ultraviolet irradiation. Photoreactivation removed most toxic lesions from irradiated phage, but did not change the frequency of induction of mutations to the clear-plaque phenotype. This implies that cyclobutyl pyrimidine dimers can be lethal, but usually do not serve as sites of mutations in the phage. The DNA sequences of mutants, derived from photoreactivated DNA showed that almost two-thirds (16/28) were transitions, the same fraction found for u.v. mutagenesis without photoreactivation. Thus the lesion inducing transitions is not the cyclobutyl pyrimidine dimer. Photoreactivation of SOS-induced host cells before infection with u.v.-irradiated phage reduced mutagenesis substantially. In this case, photoreversal of cyclobutyl dimers serves to reduce expression of the SOS functions that are required in targeted u.v. mutagenesis. (author)

Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

Science.gov (United States)

Bidlingmaier, Scott; Su, Yang; Liu, Bin

2015-01-01

Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

A Raman-spectroscopy-based approach for detection and discrimination of Streptococcus thermophilus and Lactobacillus bulgaricus phages at low titer in raw milk.

Science.gov (United States)

Tayyarcan, Emine Kübra; Acar Soykut, Esra; Boyaci, Ismail Hakki

2018-04-11

In this study, a method combining Raman spectroscopy with chemometric analysis was developed for detection of phage presence in raw milk and discrimination of Streptococcus thermophilus and Lactobacillus bulgaricus phages which are among the main phages causing problems in dairy industry. For this purpose, S. thermophilus and L. bulgaricus phages were added into raw milk separately, and then some pretreatments such as fat separation, removal of casein, and filtration were applied to the raw milk samples. Raman spectra of the samples were collected and then analyzed using principal component analysis in order to discriminate these phages in raw milk. In the next step, dilutions of S. thermophilus phages in pretreated raw milk were prepared, and Raman spectra were collected. These spectra were analyzed by using partial least squares method to quantify phages in low titer. Consequently, it has been demonstrated that S. thermophilus and L. bulgaricus phages, which have titers sufficient to fail the fermentation (~ 10 7  pfu/mL) and have lower titers (10 2 -10 3  pfu/mL), could be discriminated from antibiotic and each other. Additionally, low concentrations of S. thermophilus phages (10 2  pfu/mL) could be detected through Raman spectroscopy with a short analysis time (60 min) and high coefficient of determination (R 2 ) values for both calibration (0.985) and validation (0.906) with a root mean square error of calibration of 70.54 and root mean square error of prediction of 165.47. However, a lower success was achieved with L. bulgaricus phages and the obtained coefficient of determination values were not sufficiently high (0.649).

TfR Binding Peptide Screened by Phage Display Technology ...

African Journals Online (AJOL)

Purpose: To screen an hTfR affinity peptide and investigate its activity in vitro. Methods: hTfR ... Keywords: Peptide, hTfR, Transferrin receptor, Phage display technology, Enhanced green ..... mediated uptake of peptides that bind the human.

Phage-mediated counting by the naked eye of miRNA molecules at attomolar concentrations in a Petri dish

Science.gov (United States)

Zhou, Xin; Cao, Peng; Zhu, Ye; Lu, Wuguang; Gu, Ning; Mao, Chuanbin

2015-10-01

The ability to count biomolecules such as cancer-biomarker miRNAs with the naked eye is seemingly impossible in molecular diagnostics. Here, we show an ultrasensitive naked-eye-counting strategy for quantifying miRNAs by employing T7 phage--a bacteria-specific virus nanoparticle--as a surrogate. The phage is genetically engineered to become fluorescent and capable of binding a miRNA-capturing gold nanoparticle (GNP) in a one-to-one manner. Target miRNAs crosslink the resultant phage-GNP couple and miRNA-capturing magnetic microparticles, forming a sandwich complex containing equimolar phage and miRNA. The phage is then released from the complex and developed into one macroscopic fluorescent plaque in a Petri dish by plating it in a host bacterial medium. Counting the plaques by the naked eye enables the quantification of miRNAs with detection limits of ~3 and ~5 aM for single-target and two-target miRNAs, respectively. This approach offers ultrasensitive and convenient quantification of disease biomarkers by the naked eye.

Occurrence and predictors of persistent impaired glucose tolerance after acute ischemic stroke or transient ischemic attack

OpenAIRE

Fonville, Susanne; Hertog, Heleen; Zandbergen, Adrienne; Koudstaal, Peter Jan; Lingsma, Hester

2014-01-01

textabstractBackground Impaired glucose tolerance is often present in patients with a transient ischemic attack (TIA) or ischemic stroke and doubles the risk of recurrent stroke. This impaired glucose tolerance can be transient, reflecting an acute stress response, or persistent, representing undiagnosed impaired glucose metabolism possibly requiring treatment. We aimed to assess the occurrence of persistent impaired glucose tolerance after a stroke or TIA and to develop a prediction model to...

Recurrent episcleritis in relation to menstruation: a case report.

Science.gov (United States)

Rajoo, Sangeetha Govinda; Gandhewar, Jaishree

2011-09-01

To describe a case of recurrent episcleritis associated with a patient's menstrual cycle. A retrospective case review of a 39-year-old woman who presented with a 12-year history of recurrent episcleritis in relation to her menstruation. She was seen during an acute attack and started on a reducing regime of topical steroids for 5 weeks. She was then advised to use it a week before and after menstruation. Examination and investigations revealed episcleritis with a negative systems review. After starting the treatment, she was symptom free when reviewed at 4, 8, 14, and 33 weeks. Now, the patient uses topical steroids only 1 week before menstruation. Literature review revealed no recent case reports and provided insufficient evidence to understand this relationship. We recommend increased awareness and reporting because there is a need for more studies to understand this relationship and to provide evidence for management.

Effect of urgent treatment of transient ischaemic attack and minor stroke on early recurrent stroke (EXPRESS study): a prospective population-based sequential comparison.

Science.gov (United States)

Rothwell, Peter M; Giles, Matthew F; Chandratheva, Arvind; Marquardt, Lars; Geraghty, Olivia; Redgrave, Jessica N E; Lovelock, Caroline E; Binney, Lucy E; Bull, Linda M; Cuthbertson, Fiona C; Welch, Sarah J V; Bosch, Shelley; Alexander, Faye C; Carasco-Alexander, Faye; Silver, Louise E; Gutnikov, Sergei A; Mehta, Ziyah

2007-10-20

The risk of recurrent stroke is up to 10% in the week after a transient ischaemic attack (TIA) or minor stroke. Modelling studies suggest that urgent use of existing preventive treatments could reduce the risk by 80-90%, but in the absence of evidence many health-care systems make little provision. Our aim was to determine the effect of more rapid treatment after TIA and minor stroke in patients who are not admitted direct to hospital. We did a prospective before (phase 1: April 1, 2002, to Sept 30, 2004) versus after (phase 2: Oct 1, 2004, to March 31, 2007) study of the effect on process of care and outcome of more urgent assessment and immediate treatment in clinic, rather than subsequent initiation in primary care, in all patients with TIA or minor stroke not admitted direct to hospital. The study was nested within a rigorous population-based incidence study of all TIA and stroke (Oxford Vascular Study; OXVASC), such that case ascertainment, investigation, and follow-up were complete and identical in both periods. The primary outcome was the risk of stroke within 90 days of first seeking medical attention, with independent blinded (to study period) audit of all events. Of the 1278 patients in OXVASC who presented with TIA or stroke (634 in phase 1 and 644 in phase 2), 607 were referred or presented direct to hospital, 620 were referred for outpatient assessment, and 51 were not referred to secondary care. 95% (n=591) of all outpatient referrals were to the study clinic. Baseline characteristics and delays in seeking medical attention were similar in both periods, but median delay to assessment in the study clinic fell from 3 (IQR 2-5) days in phase 1 to less than 1 (0-3) day in phase 2 (prisk of recurrent stroke in the patients referred to the study clinic was 10.3% (32/310 patients) in phase 1 and 2.1% (6/281 patients) in phase 2 (adjusted hazard ratio 0.20, 95% CI 0.08-0.49; p=0.0001); there was no significant change in risk in patients treated elsewhere. The

Recurrent epistaxis caused by an intranasal supernumerary tooth in a young adult.

Science.gov (United States)

Al Dhafeeri, Hamed O; Kavarodi, Abdulmajid; Al Shaikh, Khalil; Bukhari, Ahmed; Al Hussain, Omair; El Baramawy, Ahmed

2014-01-01

Male, 27. Recurrent epistaxis. Nasal bleeding. -. -. Pediatrics and Neonatology. Congenital defects/diseases. Recurrent epistaxis is a common disorder among children and young adults. We report an unusual cause, intranasal supernumerary tooth causing friction with Little's area of the nasal septum. A 22-year-old male presented with recurrent, mild, unilateral left-sided epistaxis once to twice per month for 3 years. This usually occurred after minor nasal trauma or rubbing his nose. The patient also suffered from recurrent tonsillitis. There was neither history of blood transfusion or nasal packing, nor a history suggestive of bleeding diathesis. Anterior rhinoscopy revealed ivory white nasal mass antero-inferiorly in the left nasal cavity touching Little's area. There was no bleeding. Nasal endoscopy showed a white cylindrical bony mass 1 cm long arising from the floor of the nose, with no attachment to the nasal septum or the lateral wall of the nose. Examination of the right nasal cavity was unremarkable. Nasal teeth result from the ectopic eruption of supernumerary teeth and may cause a variety of symptoms including recurrent epistaxis. Their clinical and radiologic presentation is so characteristic that their diagnosis is not difficult. CT scan is helpful in planning management. Early extraction prevents further complications and prevents further attacks of epistaxis.

Composite Dos Attack Model

Directory of Open Access Journals (Sweden)

Simona Ramanauskaitė

2012-04-01

Full Text Available Preparation for potential threats is one of the most important phases ensuring system security. It allows evaluating possible losses, changes in the attack process, the effectiveness of used countermeasures, optimal system settings, etc. In cyber-attack cases, executing real experiments can be difficult for many reasons. However, mathematical or programming models can be used instead of conducting experiments in a real environment. This work proposes a composite denial of service attack model that combines bandwidth exhaustion, filtering and memory depletion models for a more real representation of similar cyber-attacks. On the basis of the introduced model, different experiments were done. They showed the main dependencies of the influence of attacker and victim’s properties on the success probability of denial of service attack. In the future, this model can be used for the denial of service attack or countermeasure optimization.

Isolation and Characterization of phiLLS, a Novel Phage with Potential Biocontrol Agent against Multidrug-Resistant Escherichia coli

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Luis Amarillas

2017-07-01

Full Text Available Foodborne diseases are a serious and growing problem, and the incidence and prevalence of antimicrobial resistance among foodborne pathogens is reported to have increased. The emergence of antibiotic-resistant bacterial strains demands novel strategies to counteract this epidemic. In this regard, lytic bacteriophages have reemerged as an alternative for the control of pathogenic bacteria. However, the effective use of phages relies on appropriate biological and genomic characterization. In this study, we present the isolation and characterization of a novel bacteriophage named phiLLS, which has shown strong lytic activity against generic and multidrug-resistant Escherichia coli strains. Transmission electron microscopy of phiLLS morphology revealed that it belongs to the Siphoviridae family. Furthermore, this phage exhibited a relatively large burst size of 176 plaque-forming units per infected cell. Phage phiLLS significantly reduced the growth of E. coli under laboratory conditions. Analyses of restriction profiles showed the presence of submolar fragments, confirming that phiLLS is a pac-type phage. Phylogenetic analysis based on the amino acid sequence of large terminase subunits confirmed that this phage uses a headful packaging strategy to package their genome. Genomic sequencing and bioinformatic analysis showed that phiLLS is a novel bacteriophage that is most closely related to T5-like phages. In silico analysis indicated that the phiLLS genome consists of 107,263 bp (39.0 % GC content encoding 160 putative ORFs, 16 tRNAs, several potential promoters and transcriptional terminators. Genome analysis suggests that the phage phiLLS is strictly lytic without carrying genes associated with virulence factors and/or potential immunoreactive allergen proteins. The bacteriophage isolated in this study has shown promising results in the biocontrol of bacterial growth under in vitro conditions, suggesting that it may prove useful as an alternative

Phage-mediated dispersal of biofilm and distribution of bacterial virulence genes is induced by quorum sensing.

Directory of Open Access Journals (Sweden)

Friederike S Rossmann

2015-02-01

Full Text Available The microbiome and the phage meta-genome within the human gut are influenced by antibiotic treatments. Identifying a novel mechanism, here we demonstrate that bacteria use the universal communication molecule AI-2 to induce virulence genes and transfer them via phage release. High concentrations (i.e. 100 μM of AI-2 promote dispersal of bacteria from already established biofilms, and is associated with release of phages capable of infecting other bacteria. Enterococcus faecalis V583ΔABC harbours 7 prophages in its genome, and a mutant deficient in one of these prophages (i.e. prophage 5 showed a greatly reduced dispersal of biofilm. Infection of a probiotic E. faecalis strain without lytic prophages with prophage 5 resulted in increased biofilm formation and also in biofilm dispersal upon induction with AI-2. Infection of the probiotic E. faecalis strain with phage-containing supernatants released through AI-2 from E. faecalis V583ΔABC resulted in a strong increase in pathogenicity of this strain. The polylysogenic probiotic strain was also more virulent in a mouse sepsis model and a rat endocarditis model. Both AI-2 and ciprofloxacin lead to phage release, indicating that conditions in the gastrointestinal tract of hospitalized patients treated with antibiotics might lead to distribution of virulence genes to apathogenic enterococci and possibly also to other commensals or even to beneficial probiotic strains.

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

more than doubled at the national level from 12,000-13,000 kg of active compound in 1996-1998 to 29,000 kg of active compound in 2004. Instead, tetracycline-resistant S. Typhimurium phage types became more prevalent. This suggests that the spread of already established or new resistant clones, rather......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 type...

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.

Science.gov (United States)

Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E

2008-04-01

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.

Uses of Phage Display in Agriculture: A Review of Food-Related Protein-Protein Interactions Discovered by Biopanning over Diverse Baits

Directory of Open Access Journals (Sweden)

Rekha Kushwaha

2013-01-01

Full Text Available This review highlights discoveries made using phage display that impact the use of agricultural products. The contribution phage display made to our fundamental understanding of how various protective molecules serve to safeguard plants and seeds from herbivores and microbes is discussed. The utility of phage display for directed evolution of enzymes with enhanced capacities to degrade the complex polymers of the cell wall into molecules useful for biofuel production is surveyed. Food allergies are often directed against components of seeds; this review emphasizes how phage display has been employed to determine the seed component(s contributing most to the allergenic reaction and how it has played a central role in novel approaches to mitigate patient response. Finally, an overview of the use of phage display in identifying the mature seed proteome protection and repair mechanisms is provided. The identification of specific classes of proteins preferentially bound by such protection and repair proteins leads to hypotheses concerning the importance of safeguarding the translational apparatus from damage during seed quiescence and environmental perturbations during germination. These examples, it is hoped, will spur the use of phage display in future plant science examining protein-ligand interactions.

Uses of phage display in agriculture: a review of food-related protein-protein interactions discovered by biopanning over diverse baits.

Science.gov (United States)

Kushwaha, Rekha; Payne, Christina M; Downie, A Bruce

2013-01-01

This review highlights discoveries made using phage display that impact the use of agricultural products. The contribution phage display made to our fundamental understanding of how various protective molecules serve to safeguard plants and seeds from herbivores and microbes is discussed. The utility of phage display for directed evolution of enzymes with enhanced capacities to degrade the complex polymers of the cell wall into molecules useful for biofuel production is surveyed. Food allergies are often directed against components of seeds; this review emphasizes how phage display has been employed to determine the seed component(s) contributing most to the allergenic reaction and how it has played a central role in novel approaches to mitigate patient response. Finally, an overview of the use of phage display in identifying the mature seed proteome protection and repair mechanisms is provided. The identification of specific classes of proteins preferentially bound by such protection and repair proteins leads to hypotheses concerning the importance of safeguarding the translational apparatus from damage during seed quiescence and environmental perturbations during germination. These examples, it is hoped, will spur the use of phage display in future plant science examining protein-ligand interactions.

Bacteriophage prevalence in the genus Azospirillum and analysis of the first genome sequence of an Azospirillum brasilense integrative phage.

Science.gov (United States)

Boyer, Mickaël; Haurat, Jacqueline; Samain, Sylvie; Segurens, Béatrice; Gavory, Frédérick; González, Víctor; Mavingui, Patrick; Rohr, René; Bally, René; Wisniewski-Dyé, Florence

2008-02-01

The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus Azospirillum. Upon induction by mitomycin C, the release of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (phiAb-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the phiAb-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of phiAb-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd.

Phage adsorption and lytic propagation in Lactobacillus plantarum: Could host cell starvation affect them?

OpenAIRE

Briggiler Marc?, Mari?ngeles; Reinheimer, Jorge; Quiberoni, Andrea

2015-01-01

Background Bacteriophages constitute a great threat to the activity of lactic acid bacteria used in industrial processes. Several factors can influence the infection cycle of bacteriophages. That is the case of the physiological state of host cells, which could produce inhibition or delay of the phage infection process. In the present work, the influence of Lactobacillus plantarum host cell starvation on phage B1 adsorption and propagation was investigated. Result First, cell growth kinetics ...

Dependence of the degree of antibacterial and antiphage action of ozone on cell and phage particle concentrations in nutrient media

Energy Technology Data Exchange (ETDEWEB)

Grits, N.V.; Fomichev, A.Iu.

1985-05-01

The work was aimed at studying the inactivating effect of ozone on Escherichia coli K-12 AB1157, Pseudomonas aeruginosa PA01, Erwinia herbicola EH103 and their phages T4, SM and I4. The degree of bacterial and phage inactivation was found to increase with a decrease in their initial concentration during the treatment. The effect depends on differences in the quantity of ozone per cell or per phage particle in the reaction medium. This conclusion is based on the fact that, irrespective of the suspension density, the amount of surviving bacteria and phages plotted versus O3 concentration and recalculated per one bacterial cell or phage particle is described graphically by one and the same curve typical of a strain under study. This technique for assessing the sensitivity of microbiological objects to ozone can be used in order to compare experimental data obtained in different laboratories.

Pyocin-sensitivity testing as a method of typing Pseudomonas aeruginosa: use of "phage-free" preparations of pyocin.

Science.gov (United States)

Rampling, A; Whitby, J L; Wildy, P

1975-11-01

A method for pyocin-sensitivity typing by means of "phage-free" preparations of pyocin is described. The method was tested on 227 isolates of P. aeruginosa, collected from 34 different foci of infection in hospitals in the British Isles and the results were compared with those for combined serological and phage typing of all strains and pyocin production of 105 of the isolates. It is concluded that pyocin-sensitivity typing is a simple and reliable method giving a high degree of discrimination, comparable to that of combined serological and phage typing, and it is suitable for use in routine hospital laboratories.

The usefulness of percutaneous transluminal angioplasty of the middle cerebral artery stenosis in patients with transient ischemic attack

Energy Technology Data Exchange (ETDEWEB)

Lee, Young Chul; Lim, Hyo Soon; Kim, Jae Kyu; Seo, Jeong Jin; Jeong, Gwang Woo; Kang, Heoung Keun [Chonnam National Univ. Medical School, Kwangju (Korea, Republic of)

2001-06-01

To determine the effectiveness of percutaneous transluminal angioplasty(PTA) of atherosclerotic middle cerebral artery(MCA) stenosis in patients with transient ischemic attack (TIA). Ten patients with TIA who had undergone PTA were retrospectively investigated. In all ten, angiography revealed stenosis of the MCA. Mechanical dilatation was performed at the stenotic portion, and the angiographic findings after PTA, as well as peri/post-angioplastic complications, were evaluated. Four to 64 (mean, 23.5) months later, neurologic symptoms and the nature and timing of recurrent attacks were also assessed. The degree of stenosis before PTA was 50-75% in six patients and greater than 75% in four. Complete or partial angiographic recanalization of the stenotic segment occurred in nine patients (90%). During follow-up, seven patients recovered without recurrent TIA or cerebral stroke; one reported a tingling sensation and one experienced vertebrobasilar insufficiency. Motor aphasia developed in one patient after PTA, but after systemic heparinization, improved within 24 hours. One patient who suffered intracranial hemorrhage due to vascular rupture during PTA did three days later. PTA for atherosclerotic MCA stenosis in patients with TIA is an effective therapeutic method.

The usefulness of percutaneous transluminal angioplasty of the middle cerebral artery stenosis in patients with transient ischemic attack

International Nuclear Information System (INIS)

Lee, Young Chul; Lim, Hyo Soon; Kim, Jae Kyu; Seo, Jeong Jin; Jeong, Gwang Woo; Kang, Heoung Keun

2001-01-01

To determine the effectiveness of percutaneous transluminal angioplasty(PTA) of atherosclerotic middle cerebral artery(MCA) stenosis in patients with transient ischemic attack (TIA). Ten patients with TIA who had undergone PTA were retrospectively investigated. In all ten, angiography revealed stenosis of the MCA. Mechanical dilatation was performed at the stenotic portion, and the angiographic findings after PTA, as well as peri/post-angioplastic complications, were evaluated. Four to 64 (mean, 23.5) months later, neurologic symptoms and the nature and timing of recurrent attacks were also assessed. The degree of stenosis before PTA was 50-75% in six patients and greater than 75% in four. Complete or partial angiographic recanalization of the stenotic segment occurred in nine patients (90%). During follow-up, seven patients recovered without recurrent TIA or cerebral stroke; one reported a tingling sensation and one experienced vertebrobasilar insufficiency. Motor aphasia developed in one patient after PTA, but after systemic heparinization, improved within 24 hours. One patient who suffered intracranial hemorrhage due to vascular rupture during PTA did three days later. PTA for atherosclerotic MCA stenosis in patients with TIA is an effective therapeutic method

Phage-Mediated Immuno-PCR for Ultrasensitive Detection of Cry1Ac Protein Based on Nanobody.

Science.gov (United States)

Liu, Yuanyuan; Jiang, Dongjian; Lu, Xin; Wang, Wei; Xu, Yang; He, Qinghua

2016-10-11

The widespread use of Cry proteins in transgenic plants for insect control has raised concerns about the environment and food safety in the public. An effective detection method for introduced Cry proteins is of significance for environmental risk assessment and product quality control. This paper describes a novel phage mediated immuno-PCR (iPCR) for the ultrasensitive determination of Cry proteins based on nanobodies. Three nanobodies against Cry1Ac protein were obtained from a naı̈ve phage displayed nanobody library without animal immunization process and were applied to the iPCR assay for Cry1Ac. The phage-mediated iPCR for Cry1Ac based on nanobodies showed a dynamic range of 0.001-100 ng/mL and a limit detection of 0.1 pg/mL. Specific measurement of this established method was performed by testing cross-reativity of other Cry1Ac analogues, and the result showed negligible cross-reactivity with other test Cry proteins (Cry1Ab, Cry1F, Cry3B). Furthermore, the phage-mediated iPCR based on nanobody should be easily applicable to the detection of many other Cry proteins.

Changes in DNA base sequence induced by gamma-ray mutagenesis of lambda phage and prophage

Energy Technology Data Exchange (ETDEWEB)

Tindall, K.R.; Stein, J.; Hutchinson, F.

1988-04-01

Mutations in the cI (repressor) gene were induced by gamma-ray irradiation of lambda phage and of prophage, and 121 mutations were sequenced. Two-thirds of the mutations in irradiated phage assayed in recA host cells (no induction of the SOS response) were G:C to A:T transitions; it is hypothesized that these may arise during DNA replication from adenine mispairing with a cytosine product deaminated by irradiation. For irradiated phage assayed in host cells in which the SOS response had been induced, 85% of the mutations were base substitutions, and in 40 of the 41 base changes, a preexisting base pair had been replaced by an A:T pair; these might come from damaged bases acting as AP (apurinic or apyrimidinic) sites. The remaining mutations were 1 and 2 base deletions. In irradiated prophage, base change mutations involved the substitution of both A:T and of G:C pairs for the preexisting pairs; the substitution of G:C pairs shows that some base substitution mechanism acts on the cell genome but not on the phage. In the irradiated prophage, frameshifts and a significant number of gross rearrangements were also found.

Heart Attack Recovery FAQs

Science.gov (United States)

... recommendations to make a full recovery. View an animation of a heart attack . Heart Attack Recovery Questions ... Support Network Popular Articles 1 Understanding Blood Pressure Readings 2 Sodium and Salt 3 Heart Attack Symptoms ...

A tale of tails: Sialidase is key to success in a model of phage therapy against K1-capsulated Escherichia coli

International Nuclear Information System (INIS)

Bull, J.J.; Vimr, E.R.; Molineux, I.J.

2010-01-01

Prior studies treating mice infected with Escherichia coli O18:K1:H7 observed that phages requiring the K1 capsule for infection (K1-dep) were superior to capsule-independent (K1-ind) phages. We show that three K1-ind phages all have low fitness when grown on cells in serum whereas fitnesses of four K1-dep phages were high. The difference is serum-specific, as fitnesses in broth overlapped. Sialidase activity was associated with all K1-dep virions tested but no K1-ind virions, a phenotype supported by sequence analyses. Adding endosialidase to cells infected with K1-ind phage increased fitness in serum by enhancing productive infection after adsorption. We propose that virion sialidase activity is the primary determinant of high fitness on cells grown in serum, and thus in a mammalian host. Although the benefit of sialidase is specific to K1-capsulated bacteria, this study may provide a scientific rationale for selecting phages for therapeutic use in many systemic infections.

'Twisted tape sign': Its significance in recurrent sigmoid volvulus

Energy Technology Data Exchange (ETDEWEB)

Gopal, K. [Tameside and Glossop Acute Services NHS Trust, Fountain Street, Ashton-under-Lyne OL6 9RW (United Kingdom)]. E-mail: karthikgopal73@yahoo.co.uk; Lim, Y. [Tameside and Glossop Acute Services NHS Trust, Fountain Street, Ashton-under-Lyne OL6 9RW (United Kingdom); Banerjee, B. [Tameside and Glossop Acute Services NHS Trust, Fountain Street, Ashton-under-Lyne OL6 9RW (United Kingdom)

2005-11-01

Aim: Sigmoid volvulus is a common cause of intestinal obstruction in the elderly. Mild attacks of sigmoid volvulus may be more difficult to diagnose due to the lack of severity of symptoms which may resolve spontaneously only to recur after an interval. This study was a review of patients to assess the incidence of the 'twisted tape sign' and to evaluate the significance of its presence in cases of recurrent sigmoid volvulus. Methods and materials: A retrospective study over eight years revealed six cases of surgically confirmed recurrent sigmoid volvulus. Case records and barium enemas of all patients were reviewed. Results: Six patients were identified, including four men and two women, with a median age of 56 years. Diagnostic difficulties were encountered in four (67%) patients with a delay ranging between 10 and 37 months with a mean 17.3 months. Twisted tape sign was confirmed on all barium examinations retrospectively. Conclusion: Recognition of twisted tape sign on barium enema examination along with an appropriate clinical history would suggest a diagnosis of recurrent sigmoid volvulus.

Entanglement generation secure against general attacks

Science.gov (United States)

Pirker, Alexander; Dunjko, Vedran; Dür, Wolfgang; Briegel, Hans J.

2017-11-01

We present a security proof for establishing private entanglement by means of recurrence-type entanglement distillation protocols over noisy quantum channels. We consider protocols where the local devices are imperfect, and show that nonetheless a confidential quantum channel can be established, and used to e.g. perform distributed quantum computation in a secure manner. While our results are not fully device independent (which we argue to be unachievable in settings with quantum outputs), our proof holds for arbitrary channel noise and noisy local operations, and even in the case where the eavesdropper learns the noise. Our approach relies on non-trivial properties of distillation protocols which are used in conjunction with de-Finetti and post-selection-type techniques to reduce a general quantum attack in a non-asymptotic scenario to an i.i.d. setting. As a side result, we also provide entanglement distillation protocols for non-i.i.d. input states.

Unstable lysogeny and pseudolysogeny in Vibrio harveyi siphovirus-like phage 1.

Science.gov (United States)

Khemayan, Krit; Pasharawipas, Tirasak; Puiprom, Orapim; Sriurairatana, Siriporn; Suthienkul, Orasa; Flegel, Timothy W

2006-02-01

Exposure of Vibrio harveyi (strain VH1114) to V. harveyi siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage.

A Taxonomic Review of Clostridium difficile Phages and Proposal of a Novel Genus, “Phimmp04likevirus”

Directory of Open Access Journals (Sweden)

Katherine R. Hargreaves

2015-05-01

Full Text Available Currently, only three phages that infect the medically important bacterium Clostridium difficile have been discussed by the International Committee of Viral Taxonomy (ICTV. They are all myoviruses, and have been assigned to the genus “phicd119likevirus”. An additional nine phages have since been described in the literature with their genome data available. The Phicd119likevirus is named after the type species: the myovirus ΦCD119 which was the first C. difficile phage to be sequenced. The two additional myoviruses, ϕCD27 and φC2, also fall into this genus based on the similarity of their genome and morphological characteristics. The other nine phages have not been assigned to this genus, and four of them do not fit the criteria for the current taxonomic grouping. We have applied protein clustering analysis to determine their phylogenetic relationships. From these results we propose an additional myoviridae genus, that we term “phiMMP04likevirus”.

Seven Deadliest Wireless Technologies Attacks

CERN Document Server

Haines, Brad

2010-01-01

How can an information security professional keep up with all of the hacks, attacks, and exploits? One way to find out what the worst of the worst are is to read the seven books in our Seven Deadliest Attacks Series. Not only do we let you in on the anatomy of these attacks but we also tell you how to get rid of them and how to defend against them in the future. Countermeasures are detailed so that you can fight against similar attacks as they evolve. Attacks featured in this book include:Bluetooth AttacksCredit Card, Access Card, and Passport AttacksBad Encryption

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...

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

International Nuclear Information System (INIS)

Zhao Haiyan; Sequeira, Reuben D.; Galeva, Nadezhda A.; Tang Liang

2011-01-01

Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus may be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.

Microcystin-LR nanobody screening from an alpaca phage display nanobody library and its expression and application.

Science.gov (United States)

Xu, Chongxin; Yang, Ying; Liu, Liwen; Li, Jianhong; Liu, Xiaoqin; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Liu, Xianjin

2018-04-30

Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC 50 ) were 0.87, 1.17 and 1.47μg/L, their detection limits (IC 10 ) were 0.06, 0.08 and 0.12μg/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7-116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0-106.5%, coefficient of variations (CVs) were 3.4-10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple

Blocking of Brute Force Attack

OpenAIRE

M.Venkata Krishna Reddy

2012-01-01

A common threat Web developers face is a password-guessing attack known as a brute-force attack. A brute-force attack is an attempt to discover a password by systematically trying every possible combination of letters, numbers, and symbols until you discover the one correct combination that works. If your Web site requires user authentication, you are a good target for a brute-force attack. An attacker can always discover a password through a brute-force attack, but the downside is that it co...

Ménage à trois in the human gut: interactions between host, bacteria and phages.

Science.gov (United States)

Mirzaei, Mohammadali Khan; Maurice, Corinne F

2017-07-01

The human gut is host to one of the densest microbial communities known, the gut microbiota, which contains bacteria, archaea, viruses, fungi and other microbial eukaryotes. Bacteriophages in the gut are largely unexplored, despite their potential to regulate bacterial communities and thus human health. In addition to helping us understand gut homeostasis, applying an ecological perspective to the study of bacterial and phage communities in the gut will help us to understand how this microbial system functions. For example, temporal studies of bacteria, phages and host immune cells in the gut during health and disease could provide key information about disease development and inform therapeutic treatments, whereas understanding the regulation of the replication cycles of phages could help harness the gut microbiota to improve disease outcomes. As the most abundant biological entities in our gut, we must consider bacteriophages in our pursuit of personalized medicine.

Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus

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da Silva Ayla

2010-05-01

Full Text Available Abstract Background The effects of acetic acid, a common food preservative, on the bacteriophage-encoded enterotoxin A (SEA expression and production in Staphylococcus aureus was investigated in pH-controlled batch cultures carried out at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Also, genomic analysis of S. aureus strains carrying sea was performed to map differences within the gene and in the temperate phage carrying sea. Results The sea expression profile was similar from pH 7.0 to 5.5, with the relative expression peaking in the transition between exponential and stationary growth phase and falling during stationary phase. The levels of sea mRNA were below the detection limit at pH 5.0 and 4.5, confirmed by very low SEA levels at these pH values. The level of relative sea expression at pH 6.0 and 5.5 were nine and four times higher, respectively, in the transitional phase than in the exponential growth phase, compared to pH 7.0 and pH 6.5, where only a slight increase in relative expression in the transitional phase was observed. Furthermore, the increase in sea expression levels at pH 6.0 and 5.5 were observed to be linked to increased intracellular sea gene copy numbers and extracellular sea-containing phage copy numbers. The extracellular SEA levels increased over time, with highest levels produced at pH 6.0 in the four growth phases investigated. Using mitomycin C, it was verified that SEA was at least partially produced as a consequence of prophage induction of the sea-phage in the three S. aureus strains tested. Finally, genetic analysis of six S. aureus strains carrying the sea gene showed specific sea phage-groups and two versions of the sea gene that may explain the different sea expression and production levels observed in this study. Conclusions Our findings suggest that the increased sea expression in S. aureus caused by acetic acid induced the sea-encoding prophage, linking SEA production to the lifecycle of the phage.

Bacteriophages of Leuconostoc, Oenococcus and Weissella

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Witold P. Kot

2014-04-01

Full Text Available Leuconostoc (Ln., Weissella and Oenococcus form a group of related genera of lactic acid bacteria, which once all shared the name Leuconostoc. They are associated with plants, fermented vegetable products, raw milk, dairy products, meat and fish. Most of industrially relevant Leuconostoc strains can be classified as either Ln. mesenteroides or Ln. pseudomesenteroides. They are important flavor producers in dairy fermentations and they initiate nearly all vegetable fermentations. Therefore bacteriophages attacking Leuconostoc strains may negatively influence the production process. Bacteriophages attacking Leuconostoc strains were first reported in 1946. Since then, the majority of described Leuconostoc phages was isolated from either dairy products or fermented vegetable products. Both lytic and temperate phages of Leuconostoc were reported. Most of Leuconostoc phages examined using electron microscopy belong to the Siphoviridae family and differ in morphological details. Hybridization and comparative genomic studies of Leuconostoc phages suggest that they can be divided into several groups, however overall diversity of Leuconostoc phages is much lower as compared to e.g. lactococcal phages. Several fully sequenced genomes of Leuconostoc phages have been deposited in public databases. Lytic phages of Leuconostoc can be divided into two host species-specific groups with similarly organized genomes that shared very low nucleotide similarity. Phages of dairy Leuconostoc have rather limited host-ranges. The receptor binding proteins of two lytic Ln. pseudomesenteroides phages have been identified. Molecular tools for detection of dairy Leuconostoc phages have been developed. The rather limited data on phages of Oenococcus and Weissella show that i lysogeny seems to be abundant in Oenococcus strains, and ii several phages infecting Weissella cibaria are also able to productively infect strains of other Weissella species and even strains of the genus

Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

Directory of Open Access Journals (Sweden)

Wadim L. Matochko

2013-01-01

Full Text Available Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries. Our paper focuses on error analysis of 7-mer peptide libraries sequenced by Illumina method. Low theoretical complexity of this phage library, as compared to complexity of long genetic reads and genomes, allowed us to describe this library using convenient linear vector and operator framework. We describe a phage library as N×1 frequency vector n=ni, where ni is the copy number of the ith sequence and N is the theoretical diversity, that is, the total number of all possible sequences. Any manipulation to the library is an operator acting on n. Selection, amplification, or sequencing could be described as a product of a N×N matrix and a stochastic sampling operator (Sa. The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of Sa and use them to define the sequencing operator (Seq. Sequencing without any bias and errors is Seq=Sa IN, where IN is a N×N unity matrix. Any bias in sequencing changes IN to a nonunity matrix. We identified a diagonal censorship matrix (CEN, which describes elimination or statistically significant downsampling, of specific reads during the sequencing process.

An orthologue of the cor gene is involved in the exclusion of temperate lambdoid phages. Evidence that Cor inactivates FhuA receptor functions

International Nuclear Information System (INIS)

Uc-Mass, Augusto; Loeza, Eva Jacinto; Garza, Mireya de la; Guarneros, Gabriel; Hernandez-Sanchez, Javier; Kameyama, Luis

2004-01-01

A new set of lambdoid phages (mEp) classified into different immunity groups was previously described. Phages mEp213, mEp237, and mEp410 were unable to grow in mEp167 lysogenic cells, presumably due to an exclusion mechanism expressed constitutively by the mEp167 repressed prophage. In this work, to analyze the exclusion phenomenon, we constructed a genomic library from mEp167 phage in a pPROEX derivative plasmid. A DNA fragment containing an open reading frame for a 77 amino acid polypeptide was selected by its ability to confer resistance to heteroimmune phage infection. This ORF shows high amino acid sequence identity with putative Cor proteins of phages HK022, φ80 and N15. Cells expressing the mEp167 cor gene from a plasmid (Cor + phenotype) excluded 13 of 20 phages from different infection immunity groups. This exclusion was observed in both tonB - and tonB + cells. Lambdoid mEp phages that were excluded in these cells were unable to infect cells defective in the outer membrane FhuA receptor (fhuA - ). Thus, Cor-mediated exclusion was only observed in fhuA + cells. Phage production after DNA transfection or the spontaneous induction of mEp prophage in Cor + cells was not blocked. In addition, ferrichrome uptake, which is mediated by FhuA, was inhibited in Cor + cells. Our results show that not only phage infection via FhuA but also a FhuA transport activity (ferrichrome uptake) are inhibited by Cor, presumably by inactivation of FhuA