Molecular weight dependence of LB morphology of poly(n-hexyl isocyanate) (PHIC).

PubMed

Morioka, Takako; Shibata, Osamu; Kawaguchi, Masami

2010-12-07

The morphologies of Langmuir-Blodgett (LB) films of two fractionated poly(n-hexyl isocyanate) (PHIC) and those of their binary mixtures were observed by AFM, together with those of an unfractionated PHIC. The low molecular weight PHIC formed random packing of bundles consisting of rigid rods, while the high molecular weight PHIC formed random packing of bundles consisting of hairy rods. Bundle interpenetration was observed only for the latter in the semidilute regime. In the bilayer region, the area occupied by the PHIC bundles in the upper layer was obviously smaller for the high molecular weight PHIC than for the low molecular weight PHIC, suggesting that the bundles of high molecular weight PHIC more easily interpenetrate than those of low molecular weight PHIC. For the blended films composed of both low and high molecular weight PHICs, the characteristic morphologies of the respective PHIC samples were no longer present. Moreover, the morphologies of the blended films appeared to resemble each other at any molar fraction owing to the ideal miscibility of the low molecular weight and high molecular weight PHICs. The morphologies of the blended films were also similar to that of the unfractionated PHIC film in the dilute regime. In the semidilute regime, the blended films became rounded owing to an increase in bundles interpenetration between PHICs as compared to that in the dilute regime, whereas the morphology of unfractionated PHIC films remained unchanged as compared to that in the dilute regime.

Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.

PubMed

Grandchamp, Nicolas; Altémir, Dorothée; Philippe, Stéphanie; Ursulet, Suzanna; Pilet, Héloïse; Serre, Marie-Claude; Lenain, Aude; Serguera, Che; Mallet, Jacques; Sarkis, Chamsy

2014-01-01

Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable.

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

PubMed Central

Auvray, F; Coddeville, M; Ritzenthaler, P; Dupont, L

1997-01-01

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction. PMID:9068626

Attachment site recognition and regulation of directionality by the serine integrases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rutherford, Karen; Yuan, Peng; Perry, Kay

Serine integrases catalyze the integration of bacteriophage DNA into a host genome by site-specific recombination between ‘attachment sites’ in the phage ( attP ) and the host ( attB ). The reaction is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded factor, nor does recombination occur between other pairings of attachment sites. A mechanistic understanding of how these enzymes achieve site-selectivity and directionality has been limited by a lack of structural models. Here, we report the structure of the C-terminal domains of a serine integrase boundmore » to an attP DNA half-site. The structure leads directly to models for understanding how the integrase-bound attP and attB sites differ, why these enzymes preferentially form attP × attB synaptic complexes to initiate recombination, and how attL × attR recombination is prevented. In these models, different domain organizations on attP vs. attB half-sites allow attachment-site specific interactions to form between integrase subunits via an unusual protruding coiled-coil motif. These interactions are used to preferentially synapse integrase-bound attP and attB and inhibit synapsis of integrase-bound attL and attR . The results provide a structural framework for understanding, testing and engineering serine integrase function.« less

Improved site-specific recombinase-based method to produce selectable marker- and vector-backbone-free transgenic cells

NASA Astrophysics Data System (ADS)

Yu, Yuan; Tong, Qi; Li, Zhongxia; Tian, Jinhai; Wang, Yizhi; Su, Feng; Wang, Yongsheng; Liu, Jun; Zhang, Yong

2014-02-01

PhiC31 integrase-mediated gene delivery has been extensively used in gene therapy and animal transgenesis. However, random integration events are observed in phiC31-mediated integration in different types of mammalian cells; as a result, the efficiencies of pseudo attP site integration and evaluation of site-specific integration are compromised. To improve this system, we used an attB-TK fusion gene as a negative selection marker, thereby eliminating random integration during phiC31-mediated transfection. We also excised the selection system and plasmid bacterial backbone by using two other site-specific recombinases, Cre and Dre. Thus, we generated clean transgenic bovine fetal fibroblast cells free of selectable marker and plasmid bacterial backbone. These clean cells were used as donor nuclei for somatic cell nuclear transfer (SCNT), indicating a similar developmental competence of SCNT embryos to that of non-transgenic cells. Therefore, the present gene delivery system facilitated the development of gene therapy and agricultural biotechnology.

Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe.

PubMed

Fogg, Paul C M; Haley, Joshua A; Stark, W Marshall; Smith, Margaret C M

2017-03-01

Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces , most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor , by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE Streptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different

Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe

PubMed Central

Haley, Joshua A.; Stark, W. Marshall

2016-01-01

ABSTRACT Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo. Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE Streptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with

Bacteriophage recombination systems and biotechnical applications.

PubMed

Nafissi, Nafiseh; Slavcev, Roderick

2014-04-01

Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed.

Simulated hatchery system to assess bacteriophage efficacy against Vibrio harveyi.

PubMed

Raghu Patil, J; Desai, Srividya Narayanamurthy; Roy, Panchali; Durgaiah, Murali; Saravanan, R Sanjeev; Vipra, Aradhana

2014-12-02

Vibriosis caused by luminous Vibrio harveyi commonly contributes to poor survival in shrimp hatcheries and aquaculture ponds. Lytic bacteriophages pathogenic for V. harveyi are currently being investigated as an alternative to antibiotics to prevent vibriosis. Here, 8 bacteriophages were isolated from oysters and clams using V. harveyi strains as baiting hosts. Among these bacteriophages, 1 strain (VHP6b) identified as broadly pathogenic for 27 V. harveyi strains examined was further characterized by electron microscopy and genome sequence analysis. Phage VHP6b possessed a tail and morphology consistent with it being a member of the family Siphoviridae, and its genome and proteome were most closely related to the Vibrio phages SSP02 and MAR10. An integrase gene essential for lysogeny was not evident. The ability of bacteriophage VHP6b to protect shrimp postlarvae against vibriosis caused by V. harveyi strain VH6 was demonstrated in a model system designed to simulate typical hatchery conditions. Bacteriophage treatment improved survival of postlarvae by 40 to 60% under these conditions, so therapies based on this or other bacteriophages may be useful in shrimp hatcheries.

Phage-host interactions analysis of newly characterized Oenococcus oeni bacteriophages: Implications for malolactic fermentation in wine.

PubMed

Costantini, Antonella; Doria, Francesca; Saiz, Juan-Carlos; Garcia-Moruno, Emilia

2017-04-04

Nowadays, only few phages infecting Oenococcus oeni, the principal lactic acid bacteria (LAB) species responsible for malolactic fermentation (MLF) in wine, have been characterized. In the present study, to better understanding the factors affecting the lytic activity of Oenococcus phages, fifteen O. oeni bacteriophages have been studied in detail, both with molecular and microbiological methods. No correlations were found between genome sizes, type of integrase genes, or morphology and the lytic activity of the 15 tested phages. Interestingly, though phage attack in a wine at the end of alcoholic fermentation seems not to be a problem, it can indeed represent a risk factor for MLF when the alcohol content is low, feature that may be a key point for choosing the appropriate time for malolactic starter inoculation. Additionally, it was observed that some phages genomes bear 2 or 3 types of integrase genes, which point to horizontal gene transfer between O. oeni bacteriophages. Copyright © 2017. Published by Elsevier B.V.

Characterization of natural polymorphic sites of the HIV-1 integrase before the introduction of HIV-1 integrase inhibitors in Germany

PubMed Central

Meixenberger, Karolin; Pouran Yousef, Kaveh; Somogyi, Sybille; Fiedler, Stefan; Bartmeyer, Barbara; von Kleist, Max; Kücherer, Claudia

2014-01-01

Introduction The aim of our study was to analyze the occurrence and evolution of HIV-1 integrase polymorphisms during the HIV-1 epidemic in Germany prior to the introduction of the first integrase inhibitor raltegravir in 2007. Materials and Methods Plasma samples from drug-naïve HIV-1 infected individuals newly diagnosed between 1986 and 2006 were used to determine PCR-based population sequences of the HIV-1 integrase (amino acids 1–278). The HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. We calculated the frequency of amino acids at each position of the HIV-1 integrase in 337 subtype B strains for the time periods 1986–1989, 1991–1994, 1995–1998, 1999–2002, and 2003–2006. Positions were defined as polymorphic if amino acid variation was >1% in any period. Logistic regression was used to identify trends in amino acid variation over time. Resistance-associated mutations were identified according to the IAS 2013 list and the HIVdb, ANRS and GRADE algorithms. Results Overall, 56.8% (158/278) amino acid positions were polymorphic and 15.8% (25/158) of these positions exhibited a significant trend in amino acid variation over time. Proportionately, most polymorphic positions (63.3%, 31/49) were detected in the N-terminal zinc finger domain of the HIV-1 integrase. Motifs and residues essential for HIV-1 integrase activity were little polymorphic, but within the minimal non-specific DNA binding region I220-D270 up to 18.1% amino acid variation was noticed, including four positions with significant amino acid variation over time (S230, D232, D256, A265). No major resistance mutations were identified, and minor resistance mutations were rarely observed without trend over time. E157Q considered by HIVdb, ANRS, and GRADE algorithms was the most frequent resistance-associated polymorphism with an overall prevalence of 2.4%. Conclusions Detailed knowledge of the evolutionary variation of HIV-1 integrase polymorphisms is important to understand

Comparison of Newly Assembled Full Length HIV-1 Integrase With Prototype Foamy Virus Integrase: Structure-Function Prospective.

PubMed

Dayer, Mohammad Reza

2016-05-01

Drug design against human immunodeficiency virus type 1 (HIV-1) integrase through its mechanistic study is of great interest in the area in biological research. The main obstacle in this area is the absence of the full-length crystal structure for HIV-1 integrase to be used as a model. A complete structure, similar to HIV-1 of a prototype foamy virus integrase in complex with DNA, including all conservative residues, is available and has been extensively used in recent investigations. The aim of this study was to determine whether the above model is precisely representative of HIV-1 integrase. This would critically determine the success of any designed drug using the model in deactivation of integrase and AIDS treatment. Primarily, a new structure for HIV-1 was constructed, using a crystal structure of prototype foamy virus as the starting structure. The constructed structure of HIV-1 integrase was simultaneously simulated with a prototype foamy virus integrase on a separate occasion. Our results indicate that the HIV-1 system behaves differently from the prototype foamy virus in terms of folding, hydration, hydrophobicity of binding site and stability. Based on our findings, we can conclude that HIV-1 integrase is vastly different from the prototype foamy virus integrase and does not resemble it, and the modeling output of the prototype foamy virus simulations could not be simply generalized to HIV-1 integrase. Therefore, our HIV-1 model seems to be more representative and more useful for future research.

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome

PubMed Central

2013-01-01

Background Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. Results We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. Conclusions We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase. PMID:24139482

A Bacteriophage-Related Chimeric Marine Virus Infecting Abalone

PubMed Central

Zhuang, Jun; Cai, Guiqin; Lin, Qiying; Wu, Zujian; Xie, Lianhui

2010-01-01

Marine viruses shape microbial communities with the most genetic diversity in the sea by multiple genetic exchanges and infect multiple marine organisms. Here we provide proof from experimental infection that abalone shriveling syndrome-associated virus (AbSV) can cause abalone shriveling syndrome. This malady produces histological necrosis and abnormally modified macromolecules (hemocyanin and ferritin). The AbSV genome is a 34.952-kilobase circular double-stranded DNA, containing putative genes with similarity to bacteriophages, eukaryotic viruses, bacteria and endosymbionts. Of the 28 predicted open reading frames (ORFs), eight ORF-encoded proteins have identifiable functional homologues. The 4 ORF products correspond to a predicted terminase large subunit and an endonuclease in bacteriophage, and both an integrase and an exonuclease from bacteria. The other four proteins are homologous to an endosymbiont-derived helicase, primase, single-stranded binding (SSB) protein, and thymidylate kinase, individually. Additionally, AbSV exhibits a common gene arrangement similar to the majority of bacteriophages. Unique to AbSV, the viral genome also contains genes associated with bacterial outer membrane proteins and may lack the structural protein-encoding ORFs. Genomic characterization of AbSV indicates that it may represent a transitional form of microbial evolution from viruses to bacteria. PMID:21079776

ФC31 Integrase-Mediated Isolation and Characterization of Novel Safe Harbors for Transgene Expression in the Pig Genome

PubMed Central

Bi, Yanzhen; Hua, Zaidong; Ren, Hongyan; Zhang, Liping; Xiao, Hongwei; Liu, Ximei; Hua, Wenjun; Mei, Shuqi; Molenaar, Adrian; Laible, Götz; Zheng, Xinmin

2018-01-01

Programmable nucleases have allowed the rapid development of gene editing and transgenics, but the technology still suffers from the lack of predefined genetic loci for reliable transgene expression and maintenance. To address this issue, we used ФC31 integrase to navigate the porcine genome and identify the pseudo attP sites suitable as safe harbors for sustained transgene expression. The combined ФC31 integrase mRNA and an enhanced green fluorescence protein (EGFP) reporter donor were microinjected into one-cell zygotes for transgene integration. Among the resulting seven EGFP-positive piglets, two had transgene integrations at pseudo attP sites, located in an intergenic region of chromosome 1 (chr1-attP) and the 6th intron of the TRABD2A gene on chromosome 3 (chr3-attP), respectively. The integration structure was determined by TAIL-PCR and Southern blotting. Primary fibroblast cells were isolated from the two piglets and examined using fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA), which demonstrated that the chr1-attP site was more potent than chr3-attP site in supporting the EGFP expression. Both piglets had green feet under the emission of UV light, and pelleted primary fibroblast cells were green-colored under natural light, corroborating that the two pseudo attP sites are beneficial to transgene expression. The discovery of these two novel safe harbors for robust and durable transgene expression will greatly facilitate the use of transgenic pigs for basic, biomedical and agricultural studies and applications. PMID:29300364

Efficacy and site-specificity of adenoviral vector integration mediated by the phage φC31 integrase.

PubMed

Robert, Marc-André; Zeng, Yue; Raymond, Benoît; Desfossé, Laurie; Mairey, Emilie; Tremblay, Jacques P; Massie, Bernard; Gilbert, Rénald

2012-12-01

Adenoviral vectors deleted of all their viral genes (helper-dependent [HD]) are efficient gene-transfer vehicles. Because transgene expression is rapidly lost in actively dividing cells, we investigated the feasibility of using phage φC31 integrase (φC31-Int) to integrate an HD carrying an attB site and the puromycin resistance gene into human cells (HeLa) and murine myoblasts (C2C12) by co-infection with a second HD-expressing φC31-Int. Because the HD genome is linear, we also investigated whether its circularization, through expression of Cre using a third HD, affects integration. Efficacy and specificity were determined by scoring the number of puromycin-resistant colonies and by sequencing integration sites. Unexpectedly, circularization of HD was unnecessary and it even reduced the integration efficacy. The maximum integration efficacy achieved was 0.5% in HeLa cells and 0.1% in C2C12 myoblasts. Up to 76% of the integration events occurred at pseudo attP sites and previously characterized hotspots were found. A small (two- to three-fold) increase in the number of γ-H2AX positive foci, accompanied by no noticeable change in γ-H2AX expression, indicated the low genotoxicity of φC31-Int. In conclusion, integration of HD mediated by φC31-Int is an attractive alternative to engineer cells, because it permits site-specific integration of large DNA fragments with low genotoxicity.

Biochemical Characterization of Novel Retroviral Integrase Proteins

PubMed Central

Ballandras-Colas, Allison; Naraharisetty, Hema; Li, Xiang; Serrao, Erik; Engelman, Alan

2013-01-01

Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their

ILG1 : a new integrase-like gene that is a marker of bacterial contamination by the laboratory Escherichia coli strain TOP10F'.

PubMed Central

Tian, Wenzhi; Chua, Kevin; Strober, Warren; Chu, Charles C.

2002-01-01

BACKGROUND: Identification of differentially expressed genes between normal and diseased states is an area of intense current medical research that can lead to the discovery of new therapeutic targets. However, isolation of differentially expressed genes by subtraction often suffers from unreported contamination of the resulting subtraction library with clones containing DNA sequences not from the original RNA samples. MATERIALS AND METHODS: Subtraction using cDNA representational difference analysis (RDA) was performed on human B cells from normal or common variable immunodeficiency patients. The material remaining after the subtraction was cloned and individual clones were sequenced. The sequence of one clone with similarity to integrases (ILG1, integrase-like gene-1) was used to obtain the full length cDNA sequence and as a probe for the presence of this sequence in RNA or genomic DNA samples. RESULTS: After five rounds of cDNA RDA, 23.3% of the clones from the resulting subtraction library contained Escherichia coli DNA. In addition, three clones contained the sequence of a new integrase, ILG1. The full length cDNA sequence of ILG1 exhibits prokaryotic, but not eukaryotic, features. At the DNA level, ILG1 is not similar to any known gene. At the protein level, ILG1 has 58% similarity to integrases from the cryptic P4 bacteriophage family (S clade). The catalytic domain of ILG1 contains the conserved features found in site-specific recombinases. The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R-H-R-Y hallmark of these recombinases. Interestingly, ILG1 was not present in the original B cell populations. By probing genomic DNA, ILG1 could only be detected in the E. coli TOP10F' strain used in our laboratory for molecular cloning, but not in any of its precursor strains, including TOP10. Furthermore, bacteria cultured from the mouth of the laboratory worker who performed cDNA RDA were also positive for

Safe genetic modification of cardiac stem cells using a site-specific integration technique.

PubMed

Lan, Feng; Liu, Junwei; Narsinh, Kazim H; Hu, Shijun; Han, Leng; Lee, Andrew S; Karow, Marisa; Nguyen, Patricia K; Nag, Divya; Calos, Michele P; Robbins, Robert C; Wu, Joseph C

2012-09-11

Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

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

PubMed

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

2017-06-01

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

The phage integrase vector pIPI03 allows RecA-independent, site-specific labelling of Staphylococcus lugdunensis strains.

PubMed

Heilbronner, Simon; Monk, Ian R; Foster, Timothy J

2013-11-01

Staphylococcus lugdunensis is a coagulase negative staphylococcus that is a commensal of man and an opportunistic pathogen. A site-specific integrative plasmid for the use in S. lugdunensis was constructed and validated. The integrase gene ccrB of bacteriophage ϕSL01 together with its attachment site was cloned into the thermosensitive plasmid pIMAY. The resulting plasmid pIPI03 integrated RecA-independently, site-specifically and irreversibly into the S. lugdunensis chromosome. Two IPTG-inducible antibiotic resistance determinants were cloned into pIPI03 and the derivatives were used to construct strains suitable for competitive growth experiments in both in vitro and in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique

PubMed Central

Lan, Feng; Liu, Junwei; Narsinh, Kazim H.; Hu, Shijun; Han, Leng; Lee, Andrew S.; Karow, Marisa; Nguyen, Patricia K.; Nag, Divya; Calos, Michele P.; Robbins, Robert C.; Wu, Joseph C.

2012-01-01

Background Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. Methods and Results We employed the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells (hECs). Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared to unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging (BLI), and positron emission tomography (PET). Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function two weeks after cell delivery, as assessed by echocardiography (P = 0.002) and magnetic resonance imaging (P = 0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated hECs, which enhanced hindlimb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. Conclusions The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types. PMID:22965984

Prophage Integrase Typing Is a Useful Indicator of Genomic Diversity in Salmonella enterica

PubMed Central

Colavecchio, Anna; D’Souza, Yasmin; Tompkins, Elizabeth; Jeukens, Julie; Freschi, Luca; Emond-Rheault, Jean-Guillaume; Kukavica-Ibrulj, Irena; Boyle, Brian; Bekal, Sadjia; Tamber, Sandeep; Levesque, Roger C.; Goodridge, Lawrence D.

2017-01-01

Salmonella enterica is a bacterial species that is a major cause of illness in humans and food-producing animals. S. enterica exhibits considerable inter-serovar diversity, as evidenced by the large number of host adapted serovars that have been identified. The development of methods to assess genome diversity in S. enterica will help to further define the limits of diversity in this foodborne pathogen. Thus, we evaluated a PCR assay, which targets prophage integrase genes, as a rapid method to investigate S. enterica genome diversity. To evaluate the PCR prophage integrase assay, 49 isolates of S. enterica were selected, including 19 clinical isolates from clonal serovars (Enteritidis and Heidelberg) that commonly cause human illness, and 30 isolates from food-associated Salmonella serovars that rarely cause human illness. The number of integrase genes identified by the PCR assay was compared to the number of integrase genes within intact prophages identified by whole genome sequencing and phage finding program PHASTER. The PCR assay identified a total of 147 prophage integrase genes within the 49 S. enterica genomes (79 integrase genes in the food-associated Salmonella isolates, 50 integrase genes in S. Enteritidis, and 18 integrase genes in S. Heidelberg). In comparison, whole genome sequencing and PHASTER identified a total of 75 prophage integrase genes within 102 intact prophages in the 49 S. enterica genomes (44 integrase genes in the food-associated Salmonella isolates, 21 integrase genes in S. Enteritidis, and 9 integrase genes in S. Heidelberg). Collectively, both the PCR assay and PHASTER identified the presence of a large diversity of prophage integrase genes in the food-associated isolates compared to the clinical isolates, thus indicating a high degree of diversity in the food-associated isolates, and confirming the clonal nature of S. Enteritidis and S. Heidelberg. Moreover, PHASTER revealed a diversity of 29 different types of prophages and 23

Prophage Integrase Typing Is a Useful Indicator of Genomic Diversity in Salmonella enterica.

PubMed

Colavecchio, Anna; D'Souza, Yasmin; Tompkins, Elizabeth; Jeukens, Julie; Freschi, Luca; Emond-Rheault, Jean-Guillaume; Kukavica-Ibrulj, Irena; Boyle, Brian; Bekal, Sadjia; Tamber, Sandeep; Levesque, Roger C; Goodridge, Lawrence D

2017-01-01

Salmonella enterica is a bacterial species that is a major cause of illness in humans and food-producing animals. S. enterica exhibits considerable inter-serovar diversity, as evidenced by the large number of host adapted serovars that have been identified. The development of methods to assess genome diversity in S. enterica will help to further define the limits of diversity in this foodborne pathogen. Thus, we evaluated a PCR assay, which targets prophage integrase genes, as a rapid method to investigate S. enterica genome diversity. To evaluate the PCR prophage integrase assay, 49 isolates of S. enterica were selected, including 19 clinical isolates from clonal serovars (Enteritidis and Heidelberg) that commonly cause human illness, and 30 isolates from food-associated Salmonella serovars that rarely cause human illness. The number of integrase genes identified by the PCR assay was compared to the number of integrase genes within intact prophages identified by whole genome sequencing and phage finding program PHASTER. The PCR assay identified a total of 147 prophage integrase genes within the 49 S. enterica genomes (79 integrase genes in the food-associated Salmonella isolates, 50 integrase genes in S . Enteritidis, and 18 integrase genes in S . Heidelberg). In comparison, whole genome sequencing and PHASTER identified a total of 75 prophage integrase genes within 102 intact prophages in the 49 S. enterica genomes (44 integrase genes in the food-associated Salmonella isolates, 21 integrase genes in S . Enteritidis, and 9 integrase genes in S . Heidelberg). Collectively, both the PCR assay and PHASTER identified the presence of a large diversity of prophage integrase genes in the food-associated isolates compared to the clinical isolates, thus indicating a high degree of diversity in the food-associated isolates, and confirming the clonal nature of S . Enteritidis and S . Heidelberg. Moreover, PHASTER revealed a diversity of 29 different types of prophages and 23

Bacteriophage sensitivity patterns among bacteria isolated from marine waters

NASA Astrophysics Data System (ADS)

Moebus, K.; Nattkemper, H.

1981-09-01

Phage-host cross-reaction tests were performed with 774 bacterial strains and 298 bacteriophages. The bacteria (bacteriophages) were isolated at different times from water samples collected in the Atlantic Ocean between the European continental shelf and the Sargasso Sea: 733 (258) strains; in the North Sea near Helgoland: 31 (31) strains; and in the Bay of Biscay: 10 (9) strains. Of the Atlantic Ocean bacteria 326 were found to be susceptible to one or more Atlantic Ocean bacteriophage(s). The bacteriophage sensitivity patterns of these bacteria vary considerably, placing 225 of them in two large clusters of bacteriophage-host systems. Taking all into account, 250 of the 326 Atlantic Ocean bacteria are different from each other. This high degree of variation among the bacteria distinguishes microbial populations derived from widely separated eastern and western regions of the Atlantic Ocean. It also sets apart from each other the populations derived from samples collected at successive stations some 200 miles apart, although to a lesser degree. With bacterial populations found from samples collected on the way back and forth between Europe and the Sargasso Sea a gradual change was observed from "western" phage sensitivity patterns to "eastern" ones. Sixty-nine Atlantic Ocean bacteria are sensitive to bacteriophages isolated from the North Sea and the Bay of Biscay; of these only 26 strains are also susceptible to Atlantic Ocean phages. The interpretation of the results is based on the hydrographical conditions prevailing in the northern Atlantic Ocean including the North Sea, and on the assumption that the microbial populations investigated have undergone genetic changes while being transported within water masses from west to east.

Campylobacter bacteriophages and bacteriophage therapy.

PubMed

Connerton, P L; Timms, A R; Connerton, I F

2011-08-01

Members of the genus Campylobacter are frequently responsible for human enteric disease with occasionally very serious outcomes. Much of this disease burden is thought to arise from consumption of contaminated poultry products. More than 80% of poultry in the UK harbour Campylobacter as a part of their intestinal flora. To address this unacceptably high prevalence, various interventions have been suggested and evaluated. Among these is the novel approach of using Campylobacter-specific bacteriophages, which are natural predators of the pathogen. To optimize their use as therapeutic agents, it is important to have a comprehensive understanding of the bacteriophages that infect Campylobacter, and how they can affect their host bacteria. This review will focus on many aspects of Campylobacter-specific bacteriophages including: their first isolation in the 1960s, their use in bacteriophage typing schemes, their isolation from the different biological sources and genomic characterization. As well as their use as therapeutic agents to reduce Campylobacter in poultry their future potential, including their use in bio-sanitization of food, will be explored. The evolutionary consequences of naturally occurring bacteriophage infection that have come to light through investigations of bacteriophages in the poultry ecosystem will also be discussed. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Thalassiolins A-C: new marine-derived inhibitors of HIV cDNA integrase.

PubMed

Rowley, David C; Hansen, Mark S T; Rhodes, Denise; Sotriffer, Christoph A; Ni, Haihong; McCammon, J Andrew; Bushman, Frederic D; Fenical, William

2002-11-01

Human immunodeficiency virus (HIV) replication requires integration of viral cDNA into the host genome, a process mediated by the viral enzyme integrase. We describe a new series of HIV integrase inhibitors, thalassiolins A-C (1-3), isolated from the Caribbean sea grass Thalassia testudinum. The thalassiolins are distinguished from other flavones previously studied by the substitution of a sulfated beta-D-glucose at the 7-position, a substituent that imparts increased potency against integrase in biochemical assays. The most active of these molecules, thalassiolin A (1), displays in vitro inhibition of the integrase catalyzed strand transfer reaction (IC50=0.4 microM) and an antiviral IC50 of 30 microM. Molecular modeling studies indicate a favorable binding mode is probable at the catalytic core domain of HIV-1 integrase.

Bovine Lactoferrampin, Human Lactoferricin, and Lactoferrin 1-11 Inhibit Nuclear Translocation of HIV Integrase.

PubMed

Wang, Winston Yan; Wong, Jack Ho; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Cheung, Randy Chifai; Ng, Tzi Bun

2016-08-01

This study aimed to investigate fragments derived from human and bovine lactoferrins for ability to inhibit nuclear translocation of HIV-1 integrase. It was shown that human lactoferricin, human lactoferrin 1-11, and bovine lactoferrampin reduced nuclear distribution of HIV-1 integrase. Bovine lactoferrampin could inhibit both the activity and nuclear translocation of HIV-1 integrase. Human lactoferrampin, bovine lactoferricin, and bovine lactoferrin 1-11 had no effect on HIV-1 integrase nuclear translocation. Human lactoferrampin which inhibited the activity of integrase did not prevent its nuclear translocation. Human lactoferricin and lactoferrin 1-11 did not inhibit HIV-1 integrase nuclear translocation despite their ability to attenuate the enzyme activity. The discrepancy between the findings on reduction of HIV-1 activity and inhibition of nuclear translocation of HIV-1 integrase was due to the different mechanisms involved. A similar reasoning can also be applied to the different inhibitory potencies of the milk peptides on different HIV enzymes, i.e., nuclear translocation.

Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).

PubMed

Merrick, C A; Wardrope, C; Paget, J E; Colloms, S D; Rosser, S J

2016-01-01

Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways. © 2016 Elsevier Inc. All rights reserved.

Methods for Bacteriophage Preservation.

PubMed

Łobocka, Małgorzata B; Głowacka, Aleksandra; Golec, Piotr

2018-01-01

In a view of growing interest in bacteriophages as the most abundant members of microbial communities and as antibacterial agents, reliable methods for bacteriophage long-term preservation, that warrant the access to original or mutant stocks of unchanged properties, have become of crucial importance. A storage method that retains the infectivity of any kind of bacteriophage virions, either in a cell lysate or in a purified suspension, does not exist, due to the enormous diversity of bacteriophages and hence the differentiation of their sensitivity to various storage conditions. Here, we describe a method of long-term bacteriophage preservation, which is based on freezing of freshly infected susceptible bacteria at early stages of bacteriophage development. The infected bacteria release mature bacteriophages upon melting enabling the recovery of bacteriophage virions with high efficiency. The only limitation of this method is the sensitivity of bacteriophage host to deep-freezing, and thus it can be used for the long-term preservation of the vast majority of bacteriophages.

Molecular evolution of HIV-1 integrase during the 20 years prior to the first approval of integrase inhibitors.

PubMed

Meixenberger, Karolin; Yousef, Kaveh Pouran; Smith, Maureen Rebecca; Somogyi, Sybille; Fiedler, Stefan; Bartmeyer, Barbara; Hamouda, Osamah; Bannert, Norbert; von Kleist, Max; Kücherer, Claudia

2017-11-14

Detailed knowledge of the evolutionary potential of polymorphic sites in a viral protein is important for understanding the development of drug resistance in the presence of an inhibitor. We therefore set out to analyse the molecular evolution of the HIV-1 subtype B integrase at the inter-patient level in Germany during a 20-year period prior to the first introduction of integrase strand inhibitors (INSTIs). We determined 337 HIV-1 integrase subtype B sequences (amino acids 1-278) from stored plasma samples of antiretroviral treatment-naïve individuals newly diagnosed with HIV-1 between 1986 and 2006. Shannon entropy was calculated to determine the variability at each amino acid position. Time trends in the frequency of amino acid variants were identified by linear regression. Direct coupling analysis was applied to detect covarying sites. Twenty-two time trends in the frequency of amino acid variants demonstrated either single amino acid exchanges or variation in the degree of polymorphy. Covariation was observed for 17 amino acid variants with a temporal trend. Some minor INSTI resistance mutations (T124A, V151I, K156 N, T206S, S230 N) and some INSTI-selected mutations (M50I, L101I, T122I, T124 N, T125A, M154I, G193E, V201I) were identified at overall frequencies >5%. Among these, the frequencies of L101I, T122I, and V201I increased over time, whereas the frequency of M154I decreased. Moreover, L101I, T122I, T124A, T125A, M154I, and V201I covaried with non-resistance-associated variants. Time-trending, covarying polymorphisms indicate that long-term evolutionary changes of the HIV-1 integrase involve defined clusters of possibly structurally or functionally associated sites independent of selective pressure through INSTIs at the inter-patient level. Linkage between polymorphic resistance- and non-resistance-associated sites can impact the selection of INSTI resistance mutations in complex ways. Identification of these sites can help in improving genotypic

Incidence of class 1 and 2 integrases in clinical and commensal bacteria from livestock, companion animals, and exotics.

PubMed

Goldstein, C; Lee, M D; Sanchez, S; Hudson, C; Phillips, B; Register, B; Grady, M; Liebert, C; Summers, A O; White, D G; Maurer, J J

2001-03-01

Many pathogenic and commensal organisms are multidrug resistant due to exposure to various antibiotics. Often, this antimicrobial resistance is encoded by integrons that occur on plasmids or that are integrated into the bacterial chromosome. Integrons are commonly associated with bacterial genera in the family Enterobacteriaceae. We determined that class 1 integrases were present in approximately 46% of the isolates from the family Enterobacteriaceae; class 2 integrases were present only among Escherichia coli and Salmonella isolates. Seven percent of veterinary isolates were positive for class 3 integrase by DNA-DNA hybridization but could not be confirmed to be positive by PCR. None of the veterinary isolates possessed the class 4 integrase gene. The distribution of these integrase genes was variable within the members of the family Enterobacteriaceae when some or all integrase classes were absent from a particular genus. There was also considerable variability in the distribution of these integrases within a species, depending on the animal host. Unlike the class 1 integrases, the other integrase class, intI2, appears to be more restricted in its distribution among the members of the family Enterobacteriaceae. There is also considerable variability in the distribution of the class 1 integrases within E. coli strains isolated from different food animals. The class 1 integrases are the most widely disseminated of the four classes among the members of the family Enterobacteriaceae from both the clinical and normal flora of animals. This is the first report to closely examine the distribution of class 2 integrases in members of the family Enterobacteriaceae isolated in the United States.

Nucleic acid amplification of HIV-1 integrase sequence subtypes CRF01_AE and B for development of HIV anti-integrase drug resistance genotyping assay

NASA Astrophysics Data System (ADS)

Adlar, F. R.; Bela, B.

2017-08-01

To anticipate the potential use of anti-integrase drugs in Indonesia for treatment of HIV-1 infection, the development of a drug resistance genotyping assay for anti-integrase is crucial in identifying the genetic drug resistance profile of Indonesian HIV-1 strains. This experiment aimed to amplify a target region in the integrase gene of Indonesian HIV-1 subtypes CRF01_AE and B that contain genetic mutations known to confer resistance to anti-integrase drug. Eleven archived plasma samples from individuals living with HIV-1 were obtained from the Virology and Cancer Pathobiology Research Center for Health Service (VCPRC FKUI-RSCM) laboratory. One of the plasma samples contained HIV-1 subtype B, and the remaining plasma samples contained subtype CRF01_AE. The target regions for all samples were amplified through RT-PCR, with an annealing temperature of 55 °C, using the primer pair AE_POL 4086F and AE_POL 5232R that were designed by VCPRC FKUI-RSCM. The results of this experiment show that 18.2% (2/11) of the samples were successfully amplified using the one-step RT-PCR. While the primer pair was effective in amplifying the target region in the integrase gene sequence for subtype B (100%; 1/1), it had a low efficacy (10%, 1/10) for subtype CRF01_AE. In conclusion, the primer pair can be used to amplify the target region in Indonesian HIV-1 strain subtypes CRF01_AE and B. However, optimization of the PCR condition and an increased number of samples would help to determine an accurate representation of the efficacy of the primer pair.

Uncommon Pathways of Immune Escape Attenuate HIV-1 Integrase Replication Capacity

PubMed Central

Chopera, Denis R.; Olvera, Alex; Brumme, Chanson J.; Sela, Jennifer; Markle, Tristan J.; Martin, Eric; Carlson, Jonathan M.; Le, Anh Q.; McGovern, Rachel; Cheung, Peter K.; Kelleher, Anthony D.; Jessen, Heiko; Markowitz, Martin; Rosenberg, Eric; Frahm, Nicole; Sanchez, Jorge; Mallal, Simon; John, Mina; Harrigan, P. Richard; Heckerman, David; Brander, Christian; Walker, Bruce D.; Brumme, Zabrina L.

2012-01-01

An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = −0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF114–121) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ∼35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies. PMID:22496233

Uncommon pathways of immune escape attenuate HIV-1 integrase replication capacity.

PubMed

Brockman, Mark A; Chopera, Denis R; Olvera, Alex; Brumme, Chanson J; Sela, Jennifer; Markle, Tristan J; Martin, Eric; Carlson, Jonathan M; Le, Anh Q; McGovern, Rachel; Cheung, Peter K; Kelleher, Anthony D; Jessen, Heiko; Markowitz, Martin; Rosenberg, Eric; Frahm, Nicole; Sanchez, Jorge; Mallal, Simon; John, Mina; Harrigan, P Richard; Heckerman, David; Brander, Christian; Walker, Bruce D; Brumme, Zabrina L

2012-06-01

An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = -0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF(114-121)) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ~35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies.

Alternative Nucleophilic Substrates for the Endonuclease Activities of Human Immunodeficiency Virus Type 1 Integrase

PubMed Central

Ealy, Julie B.; Sudol, Malgorzata; Krzeminski, Jacek; Amin, Shantu; Katzman, Michael

2012-01-01

Retroviral integrase can use water or some small alcohols as the attacking nucleophile to nick DNA. To characterize the range of compounds that human immunodeficiency virus type 1 integrase can accommodate for its endonuclease activities, we tested 45 potential electron donors (having varied size and number or spacing of nucleophilic groups) as substrates during site-specific nicking at viral DNA ends and during nonspecific nicking reactions. We found that integrase used 22 of the 45 compounds to nick DNA, but not all active compounds were used for both activities. In particular, 13 compounds were used for site-specific and nonspecific nicking, 5 only for site-specific nicking, and 4 only for nonspecific nicking; 23 other compounds were not used for either activity. Thus, integrase can accommodate a large number of nucleophilic substrates but has selective requirements for its different activities, underscoring its dynamic properties and providing new information for modeling and understanding integrase. PMID:22910593

Hyperexpansion of RNA Bacteriophage Diversity

PubMed Central

Krishnamurthy, Siddharth R.; Janowski, Andrew B.; Zhao, Guoyan; Barouch, Dan; Wang, David

2016-01-01

Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent. PMID:27010970

Methods of the pharmacological imaging of the cannabinoid system (PhICS) study: towards understanding the role of the brain endocannabinoid system in human cognition.

PubMed

van Hell, Hendrika H; Bossong, Matthijs G; Jager, Gerry; Kahn, René S; Ramsey, Nick F

2011-03-01

Various lines of (pre)clinical research indicate that cannabinoid agents carry the potential for therapeutic application to reduce symptoms in several psychiatric disorders. However, direct testing of the involvement of cannabinoid brain systems in psychiatric syndromes is essential for further development. In the Pharmacological Imaging of the Cannabinoid System (PhICS) study, the involvement of the endocannabinoid system in cognitive brain function is assessed by comparing acute effects of the cannabinoid agonist Δ9-tetrahydrocannabinol (THC) on brain function between healthy controls and groups of psychiatric patients showing cognitive dysfunction. This article describes the objectives and methods of the PhICS study and presents preliminary results of the administration procedure on subjective and neurophysiological parameters. Core elements in the methodology of PhICS are the administration method (THC is administered by inhalation using a vaporizing device) and a comprehensive use of pharmacological magnetic resonance imaging (phMRI) combining several types of MRI scans including functional MRI (fMRI), Arterial Spin Labeling (ASL) to measure brain perfusion, and resting-state fMRI. Additional methods like neuropsychological testing further specify the exact role of the endocannabinoid system in regulating cognition. Preliminary results presented in this paper indicate robust behavioral and subjective effects of THC. In addition, fMRI paradigms demonstrate activation of expected networks of brain regions in the cognitive domains of interest. The presented administration and assessment protocol provides a basis for further research on the involvement of the endocannabionoid systems in behavior and in psychopathology, which in turn may lead to development of therapeutic opportunities of cannabinoid ligands. Copyright © 2011 John Wiley & Sons, Ltd.

Computational Determination of the Effects of Bacteriophage Bacteriophage Interactions in Human body.

PubMed

Mostafa, Marwa Mostafa; Nassef, Mohammad; Badr, Amr

2017-10-19

Chronic diseases are becoming more serious and widely spreading and this carries a heavy burden on doctors to deal with such patients. Although many of these diseases can be treated by bacteriophages, the situation is significantly dangerous in patients having concomitant more than one chronic disease, where conflicts between phages used in treating these diseases are very closer to happen. This research paper presents a method to detecting the Bacteriophage-Bacteriophage Interaction. This method is implemented based on Domain-Domain Interactions model and it was used to infer Domain-Domain Interactions between the bacteriophages injected in the human body at the same time. By testing the method over bacteriophages that are used to treat tuberculosis, salmonella and virulent E.coli, many interactions have been inferred and detected between these bacteriophages. Several effects were detected for the resulted interactions such as: playing a role in DNA repair such as non-homologous end joining, playing a role in DNA replication, playing a role in the interaction between the immune system and the tumor cells and playing a role in the stiff man syndrome. We revised all patents relating to bacteriophage bacteriophage interactions and phage therapy. The proposed method is developed to help doctors to realize the effect of simultaneously injecting different bacteriophages into the human body to treat different diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Bacteriophages and Biofilms

PubMed Central

Harper, David R.; Parracho, Helena M. R. T.; Walker, James; Sharp, Richard; Hughes, Gavin; Werthén, Maria; Lehman, Susan; Morales, Sandra

2014-01-01

Biofilms are an extremely common adaptation, allowing bacteria to colonize hostile environments. They present unique problems for antibiotics and biocides, both due to the nature of the extracellular matrix and to the presence within the biofilm of metabolically inactive persister cells. Such chemicals can be highly effective against planktonic bacterial cells, while being essentially ineffective against biofilms. By contrast, bacteriophages seem to have a greater ability to target this common form of bacterial growth. The high numbers of bacteria present within biofilms actually facilitate the action of bacteriophages by allowing rapid and efficient infection of the host and consequent amplification of the bacteriophage. Bacteriophages also have a number of properties that make biofilms susceptible to their action. They are known to produce (or to be able to induce) enzymes that degrade the extracellular matrix. They are also able to infect persister cells, remaining dormant within them, but re-activating when they become metabolically active. Some cultured biofilms also seem better able to support the replication of bacteriophages than comparable planktonic systems. It is perhaps unsurprising that bacteriophages, as the natural predators of bacteria, have the ability to target this common form of bacterial life.

Structure-Guided Optimization of HIV Integrase Strand Transfer Inhibitors | Center for Cancer Research

Cancer.gov

Integrase mutations can reduce the effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The second-generation agent, dolutegravir (DTG), has enjoyed considerable clinical success; however, resistancecausing mutations that diminish the efficacy of DTG have appeared. Our current findings

MOF-Bacteriophage Biosensor for Highly Sensitive and Specific Detection of Staphylococcus aureus.

PubMed

Bhardwaj, Neha; Bhardwaj, Sanjeev K; Mehta, Jyotsana; Kim, Ki-Hyun; Deep, Akash

2017-10-04

To produce a sensitive and specific biosensor for Staphylococcus aureus, bacteriophages have been interfaced with a water-dispersible and environmentally stable metal-organic framework (MOF), NH 2 -MIL-53(Fe). The conjugation of the MOF with bacteriophages has been achieved through the use of glutaraldehyde as cross-linker. Highly sensitive detection of S. aureus in both synthetic and real samples was realized by the proposed MOF-bacteriophage biosensor based on the photoluminescence quenching phenomena: limit of detection (31 CFU/mL) and range of detection (40 to 4 × 10 8 CFU/mL). This is the first report exploiting the use of an MOF-bacteriophage complex for the biosensing of S. aureus. The results of our study highlight that the proposed biosensor is more sensitive than most of the previous methods while exhibiting some advanced features like specificity, regenerability, extended range of linear detection, and stability for long-term storage (even at room temperature).

Diketo acids derivatives as integrase inhibitors: the war against the acquired immunodeficiency syndrome.

PubMed

Henao-Mejia, Jorge; Góez, Yenny; Patiño, Pablo; Rugeles, Maria T

2006-06-01

Since the human immunodeficiency virus was identified as etiological agent of the acquired immunodeficiency syndrome, great advances have been accomplished in the therapeutic field leading to reduced morbidity and mortality among infected patients. However, the high mutation rate of the viral genome generates strains resistant to multiple drugs, pointing to the importance of finding new therapeutic targets. Among the HIV structural genes, the POL gene codes for three essential enzymes: reverse transcriptase, protease, and integrase; nineteen of the twenty drugs currently approved by the Food and Drug Administration to treat this viral infection, inhibit the reverse transcriptase and the protease. Although intense research has been carried out in this area during the last 10 years, HIV integrase inhibitors are not yet approved for clinical use; however the fact that presence of this enzyme is a sine qua non for a productive HIV life cycle joined to its unique properties makes it a promissory target for anti-HIV therapy. Many compounds have been claimed to inhibit integrase in vitro; however, few of them have proven to have antiviral activity and low cytotoxicity in cell systems. Diketoacid derivatives are the most promising integrase inhibitors so far reported. Initially discovered independently by Shionogi & Co. and the Merck Research Laboratories, these compounds are highly specific for the integrase with potent antiviral activity in vitro and in vivo, and low cytotoxicity in cell cultures. Some of these compounds have recently entered clinical trials. Due to the high relevance of integrase inhibitors, and specifically of diketoacid derivatives, we review the latest findings and patents in this important field of research.

A historical sketch of the discovery and development of HIV-1 integrase inhibitors.

PubMed

Savarino, Andrea

2006-12-01

The long process of HIV-1 integrase inhibitor discovery and development can be attributed to both the complexity of HIV-1 integration and poor 'integration' of these researches into mainstream investigations on antiretroviral therapy in the mid-1990s. Of note, some fungal extracts investigated during this period contain the beta-hydroxyketo group, later recognised to be a key structural requirement for keto-enol acids (also referred to as diketo acids) and other integrase inhibitors. This review reconstructs (in the general context of the history of AIDS research) the principal steps that led to the integrase inhibitors currently in clinical trials, and discusses possible future directions.

Interaction of Bacteriophages with the Immune System: Induction of Bacteriophage-Specific Antibodies.

PubMed

Dąbrowska, Krystyna

2018-01-01

In all cases when a bacteriophage makes direct contact with a mammalian organism, it may challenge the mammalian immunological system. Its major consequence is production of antibodies specific to the bacteriophage. Here we present protocols applicable in studies of bacteriophage ability to induce specific antibodies. The protocols have been divided into three parts: purification, immunization, and detection (ELISA).

Isolation and characterization of glacier VMY22, a novel lytic cold-active bacteriophage of Bacillus cereus.

PubMed

Ji, Xiuling; Zhang, Chunjing; Fang, Yuan; Zhang, Qi; Lin, Lianbing; Tang, Bing; Wei, Yunlin

2015-02-01

As a unique ecological system with low temperature and low nutrient levels, glaciers are considered a "living fossil" for the research of evolution. In this work, a lytic cold-active bacteriophage designated VMY22 against Bacillus cereus MYB41-22 was isolated from Mingyong Glacier in China, and its characteristics were studied. Electron microscopy revealed that VMY22 has an icosahedral head (59.2 nm in length, 31.9 nm in width) and a tail (43.2 nm in length). Bacteriophage VMY22 was classified as a Podoviridae with an approximate genome size of 18 to 20 kb. A one-step growth curve revealed that the latent and the burst periods were 70 and 70 min, respectively, with an average burst size of 78 bacteriophage particles per infected cell. The pH and thermal stability of bacteriophage VMY22 were also investigated. The maximum stability of the bacteriophage was observed to be at pH 8.0 and it was comparatively stable at pH 5.0-9.0. As VMY22 is a cold-active bacteriophage with low production temperature, its characterization and the relationship between MYB41-22 and Bacillus cereus bacteriophage deserve further study.

Lytic bacteriophages

PubMed Central

Sharma, Manan

2013-01-01

Foodborne illnesses resulting from the consumption of produce commodities contaminated with enteric pathogens continue to be a significant public health issue. Lytic bacteriophages may provide an effective and natural intervention to reduce bacterial pathogens on fresh and fresh-cut produce commodities. The use of multi-phage cocktails specific for a single pathogen has been most frequently assessed on produce commodities to minimize the development of bacteriophage insensitive mutants (BIM) in target pathogen populations. Regulatory approval for the use of several lytic phage products specific for bacterial pathogens such as Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes in foods and on food processing surfaces has been granted by various agencies in the US and other countries, possibly allowing for the more widespread use of bacteriophages in the decontamination of fresh and minimally processed produce. Research studies have shown lytic bacteriophages specific for E. coli O157:H7, Salmonella spp. and Listeria monocytogenes have been effective in reducing pathogen populations on leafy greens, sprouts and tomatoes. PMID:24228223

[RATIONAL ASPECTS OF BACTERIOPHAGES USE].

PubMed

Vakarina, A A; Kataeva, L V; Karpukhina, N F

2015-01-01

Analysis of existing aspects of bacteriophage use and study features of their lytic activity by using various techniques. Effect of monophages and associated bacteriophages (staphylococci, piopolyvalent and piocombined, intestiphage, pneumonia klebsiella and polyvalent klebsiella produced by "Microgen") was studied with 380 strains of Staphylococcus aureus and 279 cultures of Klebsiella pneumoniae in liquid and solid nutrient media. From patients with intestinal disorder, sensitivity was analyzed to 184 strains of Salmonella genus bacteria 18 serological variants to salmonella bacteriophages, 137 strains of Escherichia coli (lactose-negative, hemolytic), as well as some members of OKA groups (21 serovars) to coli-proteic and piopolyvalent bacteriophages. Lytic ability of the piobacteriophage against Klebsiella and Proteus genus bacteria was determined. Staphylococcus aureus was sensitive to staphylococcus bacteriophage in 71.6% of cases and to piobacteriophage--in 86.15% of cases. A 100% lytic ability of salmonella bacteriophage against Salmonella spp. was established. Sensitivity of E. coli of various serogroups to coli-proteic and piobacteriophage was 66 - 100%. Klebsiella, Proteus genus bacteria were sensitive to piobacteriophage in only 35% and 43.15% of cases, respectively. A more rational use of bacteriophages is necessary: development of a technique, evaluation of sensitivity of bacteria to bacteriophage, introduction of corrections into their production (expansion of bacteriophage spectra, determination and indication of their concentration in accompanying documents).

Bacteriophages Infecting Propionibacterium acnes

PubMed Central

2013-01-01

Viruses specifically infecting bacteria, or bacteriophages, are the most common biological entity in the biosphere. As such, they greatly influence bacteria, both in terms of enhancing their virulence and in terms of killing them. Since the first identification of bacteriophages in the beginning of the 20th century, researchers have been fascinated by these microorganisms and their ability to eradicate bacteria. In this review, we will cover the history of the Propionibacterium acnes bacteriophage research and point out how bacteriophage research has been an important part of the research on P. acnes itself. We will further discuss recent findings from phage genome sequencing and the identification of phage sequence signatures in clustered regularly interspaced short palindromic repeats (CRISPRs). Finally, the potential to use P. acnes bacteriophages as a therapeutic strategy to combat P. acnes-associated diseases will be discussed. PMID:23691509

Toward modern inhalational bacteriophage therapy: nebulization of bacteriophages of Burkholderia cepacia complex.

PubMed

Golshahi, Laleh; Seed, Kimberley D; Dennis, Jonathan J; Finlay, Warren H

2008-12-01

Antibiotic-resistant bacterial infections have renewed interest in finding substitute methods of treatment. The purpose of the present in vitro study was to investigate the possibility of respiratory delivery of a Burkholderia cepacia complex (BCC) bacteriophage by nebulized aerosol administration. Bacteriophages in isotonic saline were aerosolized with Pari LC star and eFlow nebulizers, at titers with mean value (standard deviation) of 2.15 x 10(8) (1.63 x 10(8)) plaque-forming unit (PFU)/mL in 2.5-mL nebulizer fills. The breathing pattern of an adult was simulated using a pulmonary waveform generator. During breath simulation, the size distributions of the nebulized aerosol were measured using phase doppler anemometry (PDA). Efficiency of nebulizer delivery was subsequently determined by collection of aerosol on low resistance filters and measurement of bacteriophage titers. These filter titers were used as input data to a mathematical lung deposition model to predict regional deposition of bacteriophages in the lung and initial bacteriophage titers in the liquid surface layer of each conducting airway generation. The results suggest that BCC bacteriophages can be nebulized successfully within a reasonable delivery time and predicted titers in the lung indicate that this method may hold potential for treatment of bacterial lung infections common among cystic fibrosis patients.

von Hippel–Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

PubMed Central

Mousnier, Aurélie; Kubat, Nicole; Massias-Simon, Aurélie; Ségéral, Emmanuel; Rain, Jean-Christophe; Benarous, Richard; Emiliani, Stéphane; Dargemont, Catherine

2007-01-01

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin–proteasome pathway. Here, we identify von Hippel–Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel–Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin–VHL–proteasome pathway in the integration–transcription transition of the viral replication cycle. PMID:17698809

Towards β-globin gene-targeting with integrase-defective lentiviral vectors.

PubMed

Inanlou, Davoud Nouri; Yakhchali, Bagher; Khanahmad, Hossein; Gardaneh, Mossa; Movassagh, Hesam; Cohan, Reza Ahangari; Ardestani, Mehdi Shafiee; Mahdian, Reza; Zeinali, Sirous

2010-11-01

We have developed an integrase-defective lentiviral (LV) vector in combination with a gene-targeting approach for gene therapy of β-thalassemia. The β-globin gene-targeting construct has two homologous stems including sequence upstream and downstream of the β-globin gene, a β-globin gene positioned between hygromycin and neomycin resistant genes and a herpes simplex virus type 1 thymidine kinase (HSVtk) suicide gene. Utilization of integrase-defective LV as a vector for the β-globin gene increased the number of selected clones relative to non-viral methods. This method represents an important step toward the ultimate goal of a clinical gene therapy for β-thalassemia.

Bacteriophage in polar inland waters

USGS Publications Warehouse

Säwström, Christin; Lisle, John; Anesio, A.M.; Priscu, John C.; Laybourn-Parry, J.

2008-01-01

Bacteriophages are found wherever microbial life is present and play a significant role in aquatic ecosystems. They mediate microbial abundance, production, respiration, diversity, genetic transfer, nutrient cycling and particle size distribution. Most studies of bacteriophage ecology have been undertaken at temperate latitudes. Data on bacteriophages in polar inland waters are scant but the indications are that they play an active and dynamic role in these microbially dominated polar ecosystems. This review summarises what is presently known about polar inland bacteriophages, ranging from subglacial Antarctic lakes to glacial ecosystems in the Arctic. The review examines interactions between bacteriophages and their hosts and the abiotic and biotic variables that influence these interactions in polar inland waters. In addition, we consider the proportion of the bacteria in Arctic and Antarctic lake and glacial waters that are lysogenic and visibly infected with viruses. We assess the relevance of bacteriophages in the microbial loop in the extreme environments of Antarctic and Arctic inland waters with an emphasis on carbon cycling.

Nanoscale bacteriophage biosensors beyond phage display.

PubMed

Lee, Jong-Wook; Song, Jangwon; Hwang, Mintai P; Lee, Kwan Hyi

2013-01-01

Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology.

Nanoscale bacteriophage biosensors beyond phage display

PubMed Central

Lee, Jong-Wook; Song, Jangwon; Hwang, Mintai P; Lee, Kwan Hyi

2013-01-01

Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology. PMID:24143096

Differential bacteriophage mortality on exposure to copper.

PubMed

Li, Jinyu; Dennehy, John J

2011-10-01

Many studies report that copper can be used to control microbial growth, including that of viruses. We determined the rates of copper-mediated inactivation for a wide range of bacteriophages. We used two methods to test the effect of copper on bacteriophage survival. One method involved placing small volumes of bacteriophage lysate on copper and stainless steel coupons. Following exposure, metal coupons were rinsed with lysogeny broth, and the resulting fluid was serially diluted and plated on agar with the corresponding bacterial host. The second method involved adding copper sulfate (CuSO(4)) to bacteriophage lysates to a final concentration of 5 mM. Aliquots were removed from the mixture, serially diluted, and plated with the appropriate bacterial host. Significant mortality was observed among the double-stranded RNA (dsRNA) bacteriophages Φ6 and Φ8, the single-stranded RNA (ssRNA) bacteriophage PP7, the ssDNA bacteriophage ΦX174, and the dsDNA bacteriophage PM2. However, the dsDNA bacteriophages PRD1, T4, and λ were relatively unaffected by copper. Interestingly, lipid-containing bacteriophages were most susceptible to copper toxicity. In addition, in the first experimental method, the pattern of bacteriophage Φ6 survival over time showed a plateau in mortality after lysates dried out. This finding suggests that copper's effect on bacteriophage is mediated by the presence of water.

HIV‑1 Integrase Strand Transfer Inhibitors with Reduced Susceptibility to Drug Resistant Mutant Integrases | Center for Cancer Research

Cancer.gov

On the cover: Mutant forms of HIV-1 IN reduce the therapeutic effectiveness of integrase strand transfer inhibitors (INSTIs). The cover figure shows the IN of prototype foamy virus complexed to a novel INSTI (gold) that retains potency against resistant mutants of HIV-1 IN. Overlain are the host and viral DNA substrates (blue and green, respectively), showing substrate mimicry

[Strategies to prevent bacteriophage infection in industrial fermentation].

PubMed

Shen, Juntao; Xiu, Zhilong

2017-12-25

During the development of bacteria-based biotechnology, bacteriophage infection is one of the constant threats and troublesome problems in industrial fermentation. The core of puzzled bacteriophage infection is a complex arm race of coevolution between bacteriophages and their hosts where bacteriophage has evolved lots of escaped ways against bacterial resistance mechanisms. The strategies of rationally designing factories and rotation of starter strains could reduce the risk of bacteriophage infection, but often fail to avoid. Genetic engineering to increase bacterial resistance is one of the strategies to prevent bacteriophage infection and more knowledge about bacteriophage and its host is needed. Recently, there are some new findings on bacterial resistance mechanisms which provide new solutions for bacteriophage infection. For example, it is possible for a rational design of resistant strains to use CRISPR-Cas based technologies just based on the sequences of bacteriophages. Moreover, it is also possible to avoid the escape of bacteriophage by iteratively building up resistance levels to generate robust industrial starter cultures. Quorum-sensing signal molecules have recently been proved to be involved in the interactions between bacteria and bacteriophages, which provides a possible way to solve bacteriophage infection from a population level. Finally, the rapid development of bacteriophage genome editing and synthetic biology will bring some new cues for preventing bacteriophage infection in industrial fermentation.

Lack of integrase inhibitors associated resistance mutations among HIV-1C isolates.

PubMed

Mulu, Andargachew; Maier, Melanie; Liebert, Uwe Gerd

2015-12-01

Although biochemical analysis of HIV-1 integrase enzyme suggested the use of integrase inhibitors (INIs) against HIV-1C, different viral subtypes may favor different mutational pathways potentially leading to varying levels of drug resistance. Thus, the aim of this study was to search for the occurrence and natural evolution of integrase polymorphisms and/or resistance mutations in HIV-1C Ethiopian clinical isolates prior to the introduction of INIs. Plasma samples from chronically infected drug naïve patients (N = 45), of whom the PR and RT sequence was determined previously, were used to generate population based sequences of HIV-1 integrase. HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. Resistance mutations were interpreted according to the Stanford HIV drug resistance database ( http://hivdb.stanford.edu ) and the updated International Antiviral Society (IAS)-USA mutation lists. Moreover, rates of polymorphisms in the current isolates were compared with South African and global HIV-1C isolates. All subjects were infected with HIV-1C concordant to the protease (PR) and reverse transcriptase (RT) regions. Neither major resistance-associated IN mutations (T66I/A/K, E92Q/G, T97A, Y143HCR, S147G, Q148H/R/K, and N155H) nor silent mutations known to change the genetic barrier were observed. Moreover, the DDE-catalytic motif (D64G/D116G/E152 K) and signature HHCC zinc-binding motifs at codon 12, 16, 40 and 43 were found to be highly conserved. However, compared to other South African subtype C isolates, the rate of polymorphism was variable at various positions. Although the sample size is small, the findings suggest that this drug class could be effective in Ethiopia and other southern African countries where HIV-1C is predominantly circulating. The data will contribute to define the importance of integrase polymorphism and to improve resistance interpretation algorithms in HIV-1C isolates.

Incorporation of T4 bacteriophage in electrospun fibres.

PubMed

Korehei, R; Kadla, J

2013-05-01

Antibacterial food packaging materials, such as bacteriophage-activated electrospun fibrous mats, may address concerns triggered by waves of bacterial food contamination. To address this, we investigated several efficient methods for incorporating T4 bacteriophage into electrospun fibrous mats. The incorporation of T4 bacteriophage using simple suspension electrospinning led to more than five orders of magnitude decrease in bacteriophage activity. To better maintain bacteriophage viability, emulsion electrospinning was developed where the T4 bacteriophage was pre-encapsulated in an alginate reservoir via an emulsification process and subsequently electrospun into fibres. This resulted in an increase in bacteriophage viability, but there was still two orders of magnitude drop in activity. Using a coaxial electrospinning process, full bacteriophage activity could be maintained. In this process, a core/shell fibre structure was formed with the T4 bacteriophage being directly incorporated into the fibre core. The core/shell fibre encapsulated bacteriophage exhibited full bacteriophage viability after storing for several weeks at +4°C. Coaxial electrospinning was shown to be capable of encapsulating bacteriophages with high loading capacity, high viability and long storage time. These results are significant in the context of controlling and preventing bacterial infections in perishable foods during storage. © 2013 The Society for Applied Microbiology.

Bacteriophages as Potential Treatment for Urinary Tract Infections

PubMed Central

Sybesma, Wilbert; Zbinden, Reinhard; Chanishvili, Nino; Kutateladze, Mzia; Chkhotua, Archil; Ujmajuridze, Aleksandre; Mehnert, Ulrich; Kessler, Thomas M.

2016-01-01

Background: Urinary tract infections (UTIs) are among the most prevalent microbial diseases and their financial burden on society is substantial. The continuing increase of antibiotic resistance worldwide is alarming so that well-tolerated, highly effective therapeutic alternatives are urgently needed. Objective: To investigate the effect of bacteriophages on Escherichia coli and Klebsiella pneumoniae strains isolated from the urine of patients suffering from UTIs. Material and methods: Forty-one E. coli and 9 K. pneumoniae strains, isolated from the urine of patients suffering from UTIs, were tested in vitro for their susceptibility toward bacteriophages. The bacteriophages originated from either commercially available bacteriophage cocktails registered in Georgia or from the bacteriophage collection of the George Eliava Institute of Bacteriophage, Microbiology and Virology. In vitro screening of bacterial strains was performed by use of the spot-test method. The experiments were implemented three times by different groups of scientists. Results: The lytic activity of the commercial bacteriophage cocktails on the 41 E. coli strains varied between 66% (Pyo bacteriophage) and 93% (Enko bacteriophage). After bacteriophage adaptation of the Pyo bacteriophage cocktail, its lytic activity was increased from 66 to 93% and only one E. coli strain remained resistant. One bacteriophage of the Eliava collection could lyse all 9 K. pneumoniae strains. Conclusions: Based on the high lytic activity and the potential of resistance optimization by direct adaption of bacteriophages as reported in this study, and in view of the continuing increase of antibiotic resistance worldwide, bacteriophage therapy is a promising treatment option for UTIs highly warranting randomized controlled trials. PMID:27148173

Bacteriophages as Potential Treatment for Urinary Tract Infections.

PubMed

Sybesma, Wilbert; Zbinden, Reinhard; Chanishvili, Nino; Kutateladze, Mzia; Chkhotua, Archil; Ujmajuridze, Aleksandre; Mehnert, Ulrich; Kessler, Thomas M

2016-01-01

Urinary tract infections (UTIs) are among the most prevalent microbial diseases and their financial burden on society is substantial. The continuing increase of antibiotic resistance worldwide is alarming so that well-tolerated, highly effective therapeutic alternatives are urgently needed. To investigate the effect of bacteriophages on Escherichia coli and Klebsiella pneumoniae strains isolated from the urine of patients suffering from UTIs. Forty-one E. coli and 9 K. pneumoniae strains, isolated from the urine of patients suffering from UTIs, were tested in vitro for their susceptibility toward bacteriophages. The bacteriophages originated from either commercially available bacteriophage cocktails registered in Georgia or from the bacteriophage collection of the George Eliava Institute of Bacteriophage, Microbiology and Virology. In vitro screening of bacterial strains was performed by use of the spot-test method. The experiments were implemented three times by different groups of scientists. The lytic activity of the commercial bacteriophage cocktails on the 41 E. coli strains varied between 66% (Pyo bacteriophage) and 93% (Enko bacteriophage). After bacteriophage adaptation of the Pyo bacteriophage cocktail, its lytic activity was increased from 66 to 93% and only one E. coli strain remained resistant. One bacteriophage of the Eliava collection could lyse all 9 K. pneumoniae strains. Based on the high lytic activity and the potential of resistance optimization by direct adaption of bacteriophages as reported in this study, and in view of the continuing increase of antibiotic resistance worldwide, bacteriophage therapy is a promising treatment option for UTIs highly warranting randomized controlled trials.

Bacteriophages and bacteriophage-derived endolysins as potential therapeutics to combat Gram-positive spore forming bacteria.

PubMed

Nakonieczna, A; Cooper, C J; Gryko, R

2015-09-01

Since their discovery in 1915, bacteriophages have been routinely used within Eastern Europe to treat a variety of bacterial infections. Although initially ignored by the West due to the success of antibiotics, increasing levels and diversity of antibiotic resistance is driving a renaissance for bacteriophage-derived therapy, which is in part due to the highly specific nature of bacteriophages as well as their relative abundance. This review focuses on the bacteriophages and derived lysins of relevant Gram-positive spore formers within the Bacillus cereus group and Clostridium genus that could have applications within the medical, food and environmental sectors. © 2015 The Society for Applied Microbiology.

A Mos1 transposase in vivo assay to screen new HIV-1 integrase inhibitors.

PubMed

Cancian, Mariana; Loreto, Elgion L S

2018-04-01

The integrase and transposase enzymes of retrovirus and transposons, respectively, share the catalytic DDE domain. In vitro assays showed that inhibitors of HIV-1 integrase generally inhibit the mariner Mos1 transposase. Using a Drosophila strain in which the mobilisation of the mariner element can be quantified by mosaic eyes, we showed that flies maintained in medium containing 210 µM to 4 mM of raltegravir, or 1 or 2 mM of dolutegravir, which are HIV-1 integrase inhibitor used in AIDS treatment, have 23-33% less somatic mobilisation in mosaic eyes when treated with raltegravir and 28-32% when treated with dolutegravir. The gene expression of the mariner transposase gene, estimated by qPCR, is similar among treated and control flies. The results suggest that in vivo assays using Drosophila can be used as a primary screening of inhibitory drugs for transposase and retroviral integrase. The advantages of this assay are that it is easy, quick, cheap and is an in vivo test, meaning that the tested substance has to have been taken in by cells and has arrived at the target site, which is not the case when in vitro assays are applied.

Detection of drug resistance-associated mutations in human immunodeficiency virus type 1 integrase derived from drug-naive individuals in Surabaya, Indonesia.

PubMed

Kotaki, Tomohiro; Khairunisa, Siti Qamariyah; Sukartiningrum, Septhia Dwi; Witaningrum, Adiana Mutamsari; Rusli, Musofa; Diansyah, M Noor; Arfijanto, M Vitanata; Rahayu, Retno Pudji; Nasronudin; Kameoka, Masanori

2014-05-01

Although human immunodeficiency virus type 1 (HIV-1) infection causes serious health problems in Indonesia, information in regard to drug resistance is limited. We performed a genotypic study on HIV-1 integrase derived from drug-naive individuals in Surabaya, Indonesia. Sequencing analysis revealed that no primary mutations associated with drug resistance to integrase inhibitors were detected; however, secondary mutations, V72I, L74I/M, V165I, V201I, I203M, and S230N, were detected in more than 5% of samples. In addition, V201I was conserved among all samples. Most integrase genes were classified into CRF01_AE genes. Interestingly, 40% of the CRF01_AE genes had an unusual insertion in the C-terminus of integrase. These mutations and insertions were considered natural polymorphisms since these mutations coincided with previous reports, and integrase inhibitors have not been used in Indonesia. Our results indicated that further studies may be required to assess the impact of these mutations on integrase inhibitors prior to their introduction into Indonesia.

Development of Tricyclic Hydroxy-1H-pyrrolopyridine-trione Containing HIV-1 Integrase Inhibitors

PubMed Central

Zhao, Xue Zhi; Maddali, Kasthuraiah; Metifiot, Mathieu; Smith, Steven J.; Vu, B. Christie; Marchand, Christophe; Hughes, Stephen H.; Pommier, Yves; Burke, Terrence R.

2011-01-01

New tricyclic HIV-1 integrase (IN) inhibitors were prepared that combined structural features of bicyclic pyrimidinones with recently disclosed 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones. This combination resulted in the introduction of a nitrogen into the aryl ring and the addition of a fused third ring to our previously described inhibitors. The resulting analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays, with good selectivity for strand transfer relative to 3′-processing. PMID:21493066

Metal-dependent inhibition of HIV-1 integrase by 5CITEP inhibitor: A theoretical QM/MM approach

NASA Astrophysics Data System (ADS)

do Nascimento, Josenaide P.; Araújo Silva, José Rogério; Lameira, Jerônimo; Alves, Cláudio N.

2013-09-01

HIV-1 integrase (IN) is a potential target for developing drugs against AIDS. In this letter, QM/MM approach was used to study the inhibition of IN by 5CITEP inhibitor in presence of divalent cations (Mg2+ or Mn2+). In addition, the main interactions occurring in 5CITEP-IN complex and the influence of divalent cations (Mg2+ or Mn2+) in enzymatic inhibition were investigated using B3LYP/6-31+G(d,p)/MM. The results suggest that the Asp64, Asp116 and four crystal water molecules plays a crucial role in cation (Mg2+ or Mn2+) coordination sphere.

The evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water matrices

EPA Pesticide Factsheets

The data to support the evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water matricesThis dataset is associated with the following publication:Rhodes , E., E. Huff, D. Hamilton, and J. Jones. The evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water matrices. JOURNAL OF VIROLOGICAL METHODS. Elsevier Science Ltd, New York, NY, USA, 228(2): 31-38, (2016).

Developing a Dynamic Pharmacophore Model for HIV-1 Integrase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carlson, Heather A.; Masukawa, Keven M.; Rubins, Kathleen

2000-05-11

We present the first receptor-based pharmacophore model for HIV-1 integrase. The development of ''dynamic'' pharmacophore models is a new method that accounts for the inherent flexibility of the active site and aims to reduce the entropic penalties associated with binding a ligand. Furthermore, this new drug discovery method overcomes the limitation of an incomplete crystal structure of the target protein. A molecular dynamics (MD) simulation describes the flexibility of the uncomplexed protein. Many conformational models of the protein are saved from the MD simulations and used in a series of multi-unit search for interacting conformers (MUSIC) simulations. MUSIC is amore » multiple-copy minimization method, available in the BOSS program; it is used to determine binding regions for probe molecules containing functional groups that complement the active site. All protein conformations from the MD are overlaid, and conserved binding regions for the probe molecules are identified. Those conserved binding regions define the dynamic pharmacophore model. Here, the dynamic model is compared to known inhibitors of the integrase as well as a three-point, ligand-based pharmacophore model from the literature. Also, a ''static'' pharmacophore model was determined in the standard fashion, using a single crystal structure. Inhibitors thought to bind in the active site of HIV-1 integrase fit the dynamic model but not the static model. Finally, we have identified a set of compounds from the Available Chemicals Directory that fit the dynamic pharmacophore model, and experimental testing of the compounds has confirmed several new inhibitors.« less

[TL, the new bacteriophage of Pseudomonas aeruginosa and its application for the search of halo-producing bacteriophages].

PubMed

Pleteneva, E A; Burkal'tseva, M V; Shaburova, O V; Krylov, S V; Pechnikova, E V; Sokolova, O S; Krylov, V N

2011-01-01

The properties of new virulent bacteriophage TL of Pseudomonas aeruginosa belonging to the family Podoviridae (genome size of 46 kb) were investigated. This bacteriophage is capable of lysogenizing the bacterial lawn in halo zones around negative colonies (NC) of other bacteriophages. TL forms large NC, that are hardly distinguishable on the lawn of P. aeruginisa PAO1. At the same time, on the lawns of some phage-resistant PAO1 mutants, as well as on those produced by a number of clinical isolates, TL forms more transparent NC. It is suggested that more effective growth of the bacteriophage TL NC is associated with the differences in outer lipopolysaccharide (LPS) layer of the cell walls of different bacterial strains, as well as of the bacteria inside and outside of the halos. This TL property was used to optimize selection of bacteriophages producing halos around NC on the lawn of P. aeruginosa PAO1. As a result, a group of bacteriophages differing in the patterns of interaction between their halos and TL bacteriophage, as well as in some characters was identified. Taking into consideration the importance of cell-surfaced structures of P. aeruginosa in manifestation of virulence and pathogenicity, possible utilization of specific phage enzymes, polysacchadide depolymerases, for more effective treatment of P. aeruginosa infections is discussed.

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

PubMed

Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae; Chang, Hyo-Ihl; Ha, Chang Hoon

2016-04-22

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. Copyright © 2016 Elsevier Inc. All rights reserved.

High avidity anti-integrase antibodies discriminate recent and non-recent HIV infection: Implications for HIV incidence assay.

PubMed

Rikhtegaran Tehrani, Zahra; Azadmanesh, Kayhan; Mostafavi, Ehsan; Gharibzadeh, Safoora; Soori, Shahrzad; Azizi, Mohammad; Khabiri, Alireza

2018-03-01

Estimation of HIV incidence provides real-time information of HIV transmission trends for decision makers. Anti-integrase antibodies are the last ones produced during seroconversion and presence of high-avidity anti-integrase antibodies indicates the chronicity of HIV infection. This study aimed to evaluate the performance of these antibodies in discriminating of recent from non-recent HIV infection. For this purpose, different ELISA formats were developed to detect high-avidity anti-integrase antibodies in a commercially available performance panel, and the best assay was selected for further evaluation. The false recent rate of the selected assay was evaluated in a panel of Iranian patients and compared to two commercial assays, BED-EIA and LAg-Avidity. While the false recent rate of the developed assay was 3.8%, it was 14.1% and 1.3% for BED-EIA and LAg-Avidity, respectively. To our knowledge, this is the first report to study the performance of high-avidity anti-integrase antibodies for classification of HIV infection. The preliminary results showed that the specificity of the newly developed assay is markedly higher than BED-EIA and is comparable with LAg-Avidity. The promising results point to the potential use of anti-integrase antibodies as a biomarker in HIV incidence laboratory tests or algorithms. The developed assay needs further evaluation in future. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic evolution of bacteriophage. I. Hybrids between unrelated bacteriophages P22 and Fels 2.

PubMed

Yamamoto, N

1969-01-01

A new bacteriophage species, designated F22, was isolated from phage P22 stocks grown on Salmonella typhimurium Q1 lysogenic for Fels 2 at a frequency of less than 10(-11). P22 has a very short tail with a hexagonal base plate and six spikes. Phage Fels 2 is morphologically similar to E. coli T-even phages, having a long tail with a contractile sheath and carrying no genetic region related to P22. Phage F22 is morphologically and serologically indistinguishable from Fels 2, but carries the c(c(1), c(2), and c(3)) markers of P22. The color markers h(21), g, and m(3) of P22 do not appear in F22. Thus, F22 is evidently a recombinant between the unrelated bacteriophages P22 and Fels 2. The recombination between unrelated bacteriophages could play an important role in the evolution of bacteriophages.

The removal of RNA primers from DNA synthesized by the reverse transcriptase of the retrotransposon Tf1 is stimulated by Tf1 integrase.

PubMed

Herzig, Eytan; Voronin, Nickolay; Hizi, Amnon

2012-06-01

The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposon's reverse transcription process.

The Removal of RNA Primers from DNA Synthesized by the Reverse Transcriptase of the Retrotransposon Tf1 Is Stimulated by Tf1 Integrase

PubMed Central

Herzig, Eytan; Voronin, Nickolay

2012-01-01

The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposon's reverse transcription process. PMID:22491446

Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

PubMed

Yang, Fang; Lei, Yingying; Zhou, Meiling; Yao, Qili; Han, Yichao; Wu, Xiang; Zhong, Wanshun; Zhu, Chenghang; Xu, Weize; Tao, Ran; Chen, Xi; Lin, Da; Rahman, Khaista; Tyagi, Rohit; Habib, Zeshan; Xiao, Shaobo; Wang, Dang; Yu, Yang; Chen, Huanchun; Fu, Zhenfang; Cao, Gang

2018-02-16

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

GENETIC EVOLUTION OF BACTERIOPHAGE, I. HYBRIDS BETWEEN UNRELATED BACTERIOPHAGES P22 AND FELS 2*

PubMed Central

Yamamoto, Nobuto

1969-01-01

A new bacteriophage species, designated F22, was isolated from phage P22 stocks grown on Salmonella typhimurium Q1 lysogenic for Fels 2 at a frequency of less than 10-11. P22 has a very short tail with a hexagonal base plate and six spikes. Phage Fels 2 is morphologically similar to E. coli T-even phages, having a long tail with a contractile sheath and carrying no genetic region related to P22. Phage F22 is morphologically and serologically indistinguishable from Fels 2, but carries the c(c1, c2, and c3) markers of P22. The color markers h21, g, and m3 of P22 do not appear in F22. Thus, F22 is evidently a recombinant between the unrelated bacteriophages P22 and Fels 2. The recombination between unrelated bacteriophages could play an important role in the evolution of bacteriophages. Images PMID:4890254

[Bacteriophage λ: electrostatic properties of the genome and its elements].

PubMed

Krutinina, G G; Krutinin, E A; Kamzolova, S G; Osypov, A A

2015-01-01

Bacteriophage λ is a classical model object in molecular biology, but little is still known on the physical properties of its DNA and regulatory elements. A study was made of the electrostatic properties of phage λ DNA and regulatory elements. A global electrostatic potential distribution along the phage genome was found to be nonuniform with main regulatory elements being located in a limited region with a high potential. The RNA polymerase binding frequency on the linearized phage chromosome directly correlates with its local potential. Strong promoters of the phage and its host Escherichia coli have distinct electrostatic upstream elements, which differ in nucleotide sequence. Attachment and recombination sites of phage λ and its host have a higher potential, which possibly facilitates their recognition by integrase. Phage λ and host Rho-independent terminators have a symmetrical M-shaped potential profile, which only slightly depends on the annotated terminator palindrome length, and occur in a region with a substantially higher potential, which may cause polymerase retention, facilitating the formation of a terminator hairpin in RNA. It was concluded that virtually all elements of phage λ genome have potential distribution specifics, which are related to their structural properties and may play a role in their biological function. The global potential distribution along the phage genome reflects the architecture of the regulation of its transcription and integration in the host genome.

Plating Bacteriophage M13.

PubMed

Green, Michael R; Sambrook, Joseph

2017-10-03

A plaque of bacteriophage M13 derives from infection of a single bacterium by a single virus particle. The progeny particles infect neighboring bacteria, which, in turn, release another generation of daughter virus particles. If the bacteria are growing in semisolid medium (e.g., containing agar or agarose), then the diffusion of the progeny particles is limited. Cells infected with bacteriophage M13 are not killed, but have a longer generation time than uninfected Escherichia coli In consequence, plaques appear as areas of slower-growing cells on a faster-growing lawn of bacterial cells. This protocol describes plating of bacteriophage M13 stocks. Plaques are readily detectable on top agar after 4-8 h of incubation at 37°C. © 2017 Cold Spring Harbor Laboratory Press.

A first step toward liposome-mediated intracellular bacteriophage therapy.

PubMed

Nieth, Anita; Verseux, Cyprien; Barnert, Sabine; Süss, Regine; Römer, Winfried

2015-01-01

The emergence of antibiotic-resistant bacteria presents a severe challenge to medicine and public health. While bacteriophage therapy is a promising alternative to traditional antibiotics, the general inability of bacteriophages to penetrate eukaryotic cells limits their use against resistant bacteria, causing intracellular diseases like tuberculosis. Bacterial vectors show some promise in carrying therapeutic bacteriophages into cells, but also bring a number of risks like an overload of bacterial antigens or the acquisition of virulence genes from the pathogen. As a first step in the development of a non-bacterial vector for bacteriophage delivery into pathogen-infected cells, we attempted to encapsulate bacteriophages into liposomes. Here we report effective encapsulation of the model bacteriophage λeyfp and the mycobacteriophage TM4 into giant liposomes. Furthermore, we show that liposome-associated bacteriophages are taken up into eukaryotic cells more efficiently than free bacteriophages. These are important milestones in the development of an intracellular bacteriophage therapy that might be useful in the fight against multi-drug-resistant intracellular pathogens like Mycobacterium tuberculosis.

Taking Bacteriophage Therapy Seriously: A Moral Argument

PubMed Central

Verbeken, Gilbert; Huys, Isabelle; Jennes, Serge; Chanishvili, Nina; Górski, Andrzej; De Vos, Daniel

2014-01-01

The excessive and improper use of antibiotics has led to an increasing incidence of bacterial resistance. In Europe the yearly number of infections caused by multidrug resistant bacteria is more than 400.000, each year resulting in 25.000 attributable deaths. Few new antibiotics are in the pipeline of the pharmaceutical industry. Early in the 20th century, bacteriophages were described as entities that can control bacterial populations. Although bacteriophage therapy was developed and practiced in Europe and the former Soviet republics, the use of bacteriophages in clinical setting was neglected in Western Europe since the introduction of traditional antibiotics. Given the worldwide antibiotic crisis there is now a growing interest in making bacteriophage therapy available for use in modern western medicine. Despite the growing interest, access to bacteriophage therapy remains highly problematic. In this paper, we argue that the current state of affairs is morally unacceptable and that all stakeholders (pharmaceutical industry, competent authorities, lawmakers, regulators, and politicians) have the moral duty and the shared responsibility towards making bacteriophage therapy urgently available for all patients in need. PMID:24868534

Appearance of Drug Resistance-Associated Mutations in Human Immunodeficiency Virus Type 1 CRF01_AE Integrase Derived from Drug-Naive Thai Patients.

PubMed

Isarangkura-Na-Ayuthaya, Panasda; Kaewnoo, Wiyada; Auwanit, Wattana; de Silva, U Chandimal; Ikuta, Kazuyoshi; Sawanpanyalert, Pathom; Kameoka, Masanori

2010-12-01

CRF01_AE is a major subtype of human immunodeficiency virus type 1 (HIV-1) circulating in Southeast Asia, including Thailand. We performed genotypic studies on HIV-1 CRF01_AE integrase derived from plasma samples from drug-naive Thai patients. Direct sequencing of amplified CRF01_AE integrase genes revealed that although no primary mutations associated with drug resistance to integrase inhibitors were detected, at least one secondary mutation was found in 96% of samples. Our results indicate that the impact of these mutations on the baseline drug susceptibility of CRF01_AE viruses to integrase inhibitors may need to be addressed prior to the introduction of these drugs in Southeast Asian countries, including Thailand.

Cationic antimicrobial peptides inactivate Shiga toxin-encoding bacteriophages

NASA Astrophysics Data System (ADS)

Del Cogliano, Manuel E.; Hollmann, Axel; Martinez, Melina; Semorile, Liliana; Ghiringhelli, Pablo D.; Maffía, Paulo C.; Bentancor, Leticia V.

2017-12-01

Shiga toxin (Stx) is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC) infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs) are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: 1) direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, 2) cationic properties are necessary but not sufficient for bacteriophage inactivation, and 3) inactivation by cationic peptides could be sequence (or structure) specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.

[Isolation, idetification and anti-HIV-1 integrase activity of culturable endophytic fungi from Tibetan medicinal plant Phlomis younghusbandii Mukerjee].

PubMed

Zhang, Da-Wei; Zhao, Ming-Ming; Chen, Juan; Li, Chao; Guo, Shun-Xing

2013-05-01

A total of 52 endophytic fungi were isolated from roots and stems of Tibetan medicinal plant Phlomis younghusbandii Mukerjee. These fungal isolates were molecularly identified based on ITS sequnces and 28S sequences distributed to 12 genera, including Phoma, Chaetosphaeronema, Fusarium and Leptosphaeria, etc. Among them, the dominant genus was Phoma. Extracts of all strains were evaluated for anti-HIV-1 integrase activity by using soluable integrase expressed in E. coli BL21 (DE3). The results showed that seven samples from five fungal endophytes PHY-24, PHY-38, PHY-40, PHY-51, PHY-53, which belonged to genus Chaetosphaeronema, inhibited strand transfer reaction catalyzed by HIV-1 integrase with IC50 values, of 6.60, 5.20, 2.86, 7.86, 4.47, 4.56 and 3.23 microg x mL(-1) respectively. In conclusion, the endophytic fungi of Phlomis younghusbandii Mukerjee are valuable for further screening anti-HIV-1 integrase agents.

Bacteriophages in dairy products: pros and cons.

PubMed

Mc Grath, Stephen; Fitzgerald, Gerald F; van Sinderen, Douwe

2007-04-01

Since the time bacteriophages were first identified as a major cause of fermentation failure in the dairy industry, researchers have been struggling to develop strategies to exclude them from the dairy environment. Over 70 years of research has led to huge improvements in the consistency and quality of fermented dairy products, while also facilitating an appreciation of the beneficial properties of bacteriophages with respect to dairy product development. With specific reference to Lactococcus lactis and cheese production, this review outlines some recently reported novel methods aimed at limiting the bacteriophage infection as well as highlighting some beneficial aspects of bacteriophage activity.

Growing Bacteriophage M13 in Liquid Culture.

PubMed

Green, Michael R; Sambrook, Joseph

2017-11-01

Stocks of bacteriophage M13 are usually grown in liquid culture. The infected bacteria do not lyse but, instead, grow at a slower than normal rate to form a dilute suspension. The inoculum of bacteriophage is almost always a freshly picked plaque or a suspension of bacteriophage particles obtained from a single plaque, as described here. Infected cells contain up to 200 copies of double-stranded, replicative-form DNA and extrude several hundred bacteriophage particles per generation. Thus, a 1-mL culture of infected cells can produce enough double-stranded viral DNA (1-2 mg) for restriction mapping and recovery of cloned DNA inserts and sufficient single-stranded DNA (∼5-10 mg) for site-directed mutagenesis, DNA sequencing, or synthesis of radiolabeled probes. The titer of bacteriophages in the supernatant from infected cells is so high (∼10 12 pfu/mL) that a small aliquot serves as a permanent stock of the starting plaque. © 2017 Cold Spring Harbor Laboratory Press.

Novel Bifunctional Quinolonyl Diketo Acid Derivatives as HIV-1 Integrase Inhibitors: Design, Synthesis, Biological Activities and Mechanism of Action

PubMed Central

Di Santo, Roberto; Costi, Roberta; Roux, Alessandra; Artico, Marino; Lavecchia, Antonio; Marinelli, Luciana; Novellino, Ettore; Palmisano, Lucia; Andreotti, Mauro; Amici, Roberta; Galluzzo, Clementina Maria; Nencioni, Lucia; Palamara, Anna Teresa; Pommier, Yves; Marchand, Christophe

2008-01-01

The virally encoded integrase protein is an essential enzyme in the life cycle of the HIV-1 virus and represents an attractive and validated target in the development of therapeutics against HIV infection. Drugs that selectively inhibit this enzyme, when used in combination with inhibitors of reverse transcriptase and protease, are believed to be highly effective in suppressing the viral replication. Among the HIV-1 integrase inhibitors, the β-diketo acids (DKAs) represent a major lead for anti-HIV-1drug development. In this study, novel bifunctional quinolonyl diketo acid derivatives were designed, synthesized and tested for their inhibitory ability against HIV-1 integrase. The compounds are potent inhibitors of integrase activity. Particularly, derivative 8 is a potent IN inhibitor for both steps of the reaction (3′-processing and strand transfer) and exhibits both high antiviral activity against HIV-1 infected cells and low cytotoxicity. Molecular modeling studies provide a plausible mechanism of action, which is consistent with ligand SARs and enzyme photo-crosslinking experiments. PMID:16539381

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

PubMed

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-11-14

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

PubMed Central

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-01-01

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

Evidence for a lineage of virulent bacteriophages that target Campylobacter.

PubMed

Timms, Andrew R; Cambray-Young, Joanna; Scott, Andrew E; Petty, Nicola K; Connerton, Phillippa L; Clarke, Louise; Seeger, Kathy; Quail, Mike; Cummings, Nicola; Maskell, Duncan J; Thomson, Nicholas R; Connerton, Ian F

2010-03-30

Our understanding of the dynamics of genome stability versus gene flux within bacteriophage lineages is limited. Recently, there has been a renewed interest in the use of bacteriophages as 'therapeutic' agents; a prerequisite for their use in such therapies is a thorough understanding of their genetic complement, genome stability and their ecology to avoid the dissemination or mobilisation of phage or bacterial virulence and toxin genes. Campylobacter, a food-borne pathogen, is one of the organisms for which the use of bacteriophage is being considered to reduce human exposure to this organism. Sequencing and genome analysis was performed for two Campylobacter bacteriophages. The genomes were extremely similar at the nucleotide level (> or = 96%) with most differences accounted for by novel insertion sequences, DNA methylases and an approximately 10 kb contiguous region of metabolic genes that were dissimilar at the sequence level but similar in gene function between the two phages. Both bacteriophages contained a large number of radical S-adenosylmethionine (SAM) genes, presumably involved in boosting host metabolism during infection, as well as evidence that many genes had been acquired from a wide range of bacterial species. Further bacteriophages, from the UK Campylobacter typing set, were screened for the presence of bacteriophage structural genes, DNA methylases, mobile genetic elements and regulatory genes identified from the genome sequences. The results indicate that many of these bacteriophages are related, with 10 out of 15 showing some relationship to the sequenced genomes. Two large virulent Campylobacter bacteriophages were found to show very high levels of sequence conservation despite separation in time and place of isolation. The bacteriophages show adaptations to their host and possess genes that may enhance Campylobacter metabolism, potentially advantaging both the bacteriophage and its host. Genetic conservation has been shown to extend to other

Bacteriophages as indicators of faecal pollution and enteric ...

EPA Pesticide Factsheets

Bacteriophages are an attractive alternative to fecal indicator bacteria (FIB), particularly as surrogates of enteric virus fate and transport due to their closer morphological and biological properties compared to FIB. Based on a meta-analysis of published data, we summarize concentrations of coliphages (F+ and somatic), Bacteroides spp. and enterococci bacteriophages (phages) in human waste, non-human waste, fresh and marine waters as well as removal through wastewater treatment processes. We also provide comparisons with FIB and enteric viruses whenever possible. Lastly, we examine fate and transport characteristics in the environment and provide an overview of the methods available for detection and enumeration of bacteriophages. In summary, concentrations of FIB bacteriophages in various sources were consistently lower than FIB, but more reflective of infectious enteric virus levels. Our investigation supports use of bacteriophages as viral surrogates especially for wastewater treatment processes, while additional research is needed to clarify their utility as indicators of viral fate and transport in the ambient water. Describes concentrations and removal through environmental and engineered systems of bacteriophages, fecal indicator bacteria and viral pathogens.

Norovirus and FRNA bacteriophage determined by RT-qPCR and infectious FRNA bacteriophage in wastewater and oysters.

PubMed

Flannery, John; Keaveney, Sinéad; Rajko-Nenow, Paulina; O'Flaherty, Vincent; Doré, William

2013-09-15

Norovirus (NoV), the leading cause of adult non-bacterial gastroenteritis can be commonly detected in wastewater but the extent of NoV removal provided by wastewater treatment plants (WWTPs) is unclear. We monitored a newly commissioned WWTP with UV disinfection on a weekly basis over a six month period for NoV using RT-qPCR and for FRNA bacteriophage GA using both RT-qPCR (total concentration) and a plaque assay (infectious concentration). Mean concentrations of NoV GI and GII in influent wastewater were reduced by 0.25 and 0.41 log10 genome copies 100 ml(-1), respectively by the WWTP. The mean concentration of total FRNA bacteriophage GA was reduced by 0.35 log genome copies 100 ml(-1) compared to a reduction of infectious FRNA bacteriophage GA of 2.13 log PFU 100 ml(-1). A significant difference between concentrations of infectious and total FRNA bacteriophage GA was observed in treated, but not in untreated wastewaters. We conclude that RT-qPCR in isolation underestimates the reduction of infectious virus during wastewater treatment. We further compared the concentrations of infectious virus in combined sewer overflow (CSO) and UV treated effluents using FRNA bacteriophage GA. A greater percentage (98%) of infectious virus is released in CSO discharges than UV treated effluent (44%). Following a CSO discharge, concentrations of NoV GII and infectious FRNA bacteriophage GA in oysters from less than the limit of detection to 3150 genome copies 100 g(-1) and 1050 PFU 100 g(-1) respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

Call for a dedicated European legal framework for bacteriophage therapy.

PubMed

Verbeken, Gilbert; Pirnay, Jean-Paul; Lavigne, Rob; Jennes, Serge; De Vos, Daniel; Casteels, Minne; Huys, Isabelle

2014-04-01

The worldwide emergence of antibiotic resistances and the drying up of the antibiotic pipeline have spurred a search for alternative or complementary antibacterial therapies. Bacteriophages are bacterial viruses that have been used for almost a century to combat bacterial infections, particularly in Poland and the former Soviet Union. The antibiotic crisis has triggered a renewed clinical and agricultural interest in bacteriophages. This, combined with new scientific insights, has pushed bacteriophages to the forefront of the search for new approaches to fighting bacterial infections. But before bacteriophage therapy can be introduced into clinical practice in the European Union, several challenges must be overcome. One of these is the conceptualization and classification of bacteriophage therapy itself and the extent to which it constitutes a human medicinal product regulated under the European Human Code for Medicines (Directive 2001/83/EC). Can therapeutic products containing natural bacteriophages be categorized under the current European regulatory framework, or should this framework be adapted? Various actors in the field have discussed the need for an adapted (or entirely new) regulatory framework for the reintroduction of bacteriophage therapy in Europe. This led to the identification of several characteristics specific to natural bacteriophages that should be taken into consideration by regulators when evaluating bacteriophage therapy. One important consideration is whether bacteriophage therapy development occurs on an industrial scale or a hospital-based, patient-specific scale. More suitable regulatory standards may create opportunities to improve insights into this promising therapeutic approach. In light of this, we argue for the creation of a new, dedicated European regulatory framework for bacteriophage therapy.

Bacteriophage-based nanoprobes for rapid bacteria separation

NASA Astrophysics Data System (ADS)

Chen, Juhong; Duncan, Bradley; Wang, Ziyuan; Wang, Li-Sheng; Rotello, Vincent M.; Nugen, Sam R.

2015-10-01

The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying concentrations were determined. The results indicated a similar bacteria capture efficiency between the two nanoprobes.The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying

Isolation and characterization of bacteriophages of Salmonella enterica serovar Pullorum.

PubMed

Bao, H; Zhang, H; Wang, R

2011-10-01

In this study, 2 bacteriophages of Salmonella Pullorum were isolated using an enrichment protocol and the double agar layer method. They were named PSPu-95 and PSPu-4-116, respectively, against clinical isolates of Salmonella Pullorum SPu-95 and SPu-116. The host ranges of the 2 bacteriophages were determined by performing spot tests with 20 bacteria strains. Both bacteriophages had wide host ranges. Bacteriophage PSPu-95 had a lytic effect on 17 of the 20 isolates (85%), and PSPu-4-116 produced a lytic effect on 14 isolates (70%) and was the only bacteriophage that produced a clear plaque on enterotoxigenic Escherichia coli K88. Transmission electron microscopy revealed the bacteriophages belonged to the order Caudovirales. Bacteriophage PSPu-95 was a member of the family Siphoviridae, but bacteriophage PSPu-4-116 belonged to the family Myoviridae. Both had a double-stranded DNA, which was digested with HindIII or EcoRI, that was estimated to be 58.3 kbp (PSPu-95) and 45.2 kbp (PSPu-4-116) by 1% agar electrophoresis. One-step growth kinetics showed that the latent periods were all less than 20 min, and the burst size was 77.5 pfu/cell for PSPu-95 and 86 pfu/cell for PSPu-4-116. The bacteriophages were able to survive in a pH range between 4 and 10, and they were able to survive in a treatment of 70°C for 60 min. The characterizations of these 2 bacteriophages were helpful in establishing a basis for adopting the most effective bacteriophage to control bacteria in the poultry industry.

The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency.

PubMed

Feng, Lei; Dharmarajan, Venkatasubramanian; Serrao, Erik; Hoyte, Ashley; Larue, Ross C; Slaughter, Alison; Sharma, Amit; Plumb, Matthew R; Kessl, Jacques J; Fuchs, James R; Bushman, Frederic D; Engelman, Alan N; Griffin, Patrick R; Kvaratskhelia, Mamuka

2016-05-20

Allosteric HIV-1 integrase inhibitors (ALLINIs) have recently emerged as a promising class of antiretroviral agents and are currently in clinical trials. In infected cells, ALLINIs potently inhibit viral replication by impairing virus particle maturation but surprisingly exhibit a reduced EC50 for inhibiting HIV-1 integration in target cells. To better understand the reduced antiviral activity of ALLINIs during the early stage of HIV-1 replication, we investigated the competitive interplay between a potent representative ALLINI, BI/D, and LEDGF/p75 with HIV-1 integrase. While the principal binding sites of BI/D and LEDGF/p75 overlap at the integrase catalytic core domain dimer interface, we show that the inhibitor and the cellular cofactor induce markedly different multimerization patterns of full-length integrase. LEDGF/p75 stabilizes an integrase tetramer through the additional interactions with the integrase N-terminal domain, whereas BI/D induces protein-protein interactions in C-terminal segments that lead to aberrant, higher-order integrase multimerization. We demonstrate that LEDGF/p75 binds HIV-1 integrase with significantly higher affinity than BI/D and that the cellular protein is able to reverse the inhibitor induced aberrant, higher-order integrase multimerization in a dose-dependent manner in vitro. Consistent with these observations, alterations of the cellular levels of LEDGF/p75 markedly affected BI/D EC50 values during the early steps of HIV-1 replication. Furthermore, genome-wide sequencing of HIV-1 integration sites in infected cells demonstrate that LEDGF/p75-dependent integration site selection is adversely affected by BI/D treatment. Taken together, our studies elucidate structural and mechanistic details of the interplay between LEDGF/p75 and BI/D during the early stage of HIV-1 replication.

Polymer-based delivery systems for support and delivery of bacteriophages

NASA Astrophysics Data System (ADS)

Brown, Alyssa Marie

One of the most urgent problems in the fields of medicine and agriculture is the decreasing effectiveness of antibiotics. Once a miracle drug, antibiotics have recently become associated with the creation of antibiotic-resistant bacteria. The main limitations of these treatments include lack of both adaptability and specificity. To overcome these shortcomings of current antibiotic treatments, there has been a renewed interest in bacteriophage research. Bacteriophages are naturally-occurring viruses that lyse bacteria. They are highly specific, with each bacteriophage type lysing a narrow range of bacteria strains. Bacteriophages are also ubiquitous biological entities, populating environments where bacterial growth is supported. Just as humans are exposed to bacteria in their daily lives, we are exposed to bacteriophages as well. To use bacteriophages in practical applications, they must be delivered to the site of an infection in a controlled-release system. Two systems were studied to observe their support of bacteriophage lytic activity, as well as investigate the possibility of controlling bacteriophage release rates. First, hydrogels were studied, using crosslinking and blending techniques to achieve a range of release profiles. Second, polyanhydride microparticles were studied, evaluating release rates as a function of monomer chemistries.

Bacteriophage Applications for Food Production and Processing

PubMed Central

Moye, Zachary D.; Woolston, Joelle; Sulakvelidze, Alexander

2018-01-01

Foodborne illnesses remain a major cause of hospitalization and death worldwide despite many advances in food sanitation techniques and pathogen surveillance. Traditional antimicrobial methods, such as pasteurization, high pressure processing, irradiation, and chemical disinfectants are capable of reducing microbial populations in foods to varying degrees, but they also have considerable drawbacks, such as a large initial investment, potential damage to processing equipment due to their corrosive nature, and a deleterious impact on organoleptic qualities (and possibly the nutritional value) of foods. Perhaps most importantly, these decontamination strategies kill indiscriminately, including many—often beneficial—bacteria that are naturally present in foods. One promising technique that addresses several of these shortcomings is bacteriophage biocontrol, a green and natural method that uses lytic bacteriophages isolated from the environment to specifically target pathogenic bacteria and eliminate them from (or significantly reduce their levels in) foods. Since the initial conception of using bacteriophages on foods, a substantial number of research reports have described the use of bacteriophage biocontrol to target a variety of bacterial pathogens in various foods, ranging from ready-to-eat deli meats to fresh fruits and vegetables, and the number of commercially available products containing bacteriophages approved for use in food safety applications has also been steadily increasing. Though some challenges remain, bacteriophage biocontrol is increasingly recognized as an attractive modality in our arsenal of tools for safely and naturally eliminating pathogenic bacteria from foods. PMID:29671810

Comparative Genomics of Bacteriophage of the Genus Seuratvirus

PubMed Central

Sazinas, Pavelas; Redgwell, Tamsin; Rihtman, Branko; Grigonyte, Aurelija; Michniewski, Slawomir; Scanlan, David J; Hobman, Jon

2018-01-01

Abstract Despite being more abundant and having smaller genomes than their bacterial host, relatively few bacteriophages have had their genomes sequenced. Here, we isolated 14 bacteriophages from cattle slurry and performed de novo genome sequencing, assembly, and annotation. The commonly used marker genes polB and terL showed these bacteriophages to be closely related to members of the genus Seuratvirus. We performed a core-gene analysis using the 14 new and four closely related genomes. A total of 58 core genes were identified, the majority of which has no known function. These genes were used to construct a core-gene phylogeny, the results of which confirmed the new isolates to be part of the genus Seuratvirus and expanded the number of species within this genus to four. All bacteriophages within the genus contained the genes queCDE encoding enzymes involved in queuosine biosynthesis. We suggest these genes are carried as a mechanism to modify DNA in order to protect these bacteriophages against host endonucleases. PMID:29272407

Postexposure protection of macaques from vaginal SHIV infection by topical integrase inhibitors.

PubMed

Dobard, Charles; Sharma, Sunita; Parikh, Urvi M; West, Rolieria; Taylor, Andrew; Martin, Amy; Pau, Chou-Pong; Hanson, Debra L; Lipscomb, Jonathan; Smith, James; Novembre, Francis; Hazuda, Daria; Garcia-Lerma, J Gerardo; Heneine, Walid

2014-03-12

Coitally delivered microbicide gels containing antiretroviral drugs are important for HIV prevention. However, to date, microbicides have contained entry or reverse transcriptase inhibitors that block early steps in virus infection and thus need to be given as a preexposure dose that interferes with sexual practices and may limit compliance. Integrase inhibitors block late steps after virus infection and therefore are more suitable for post-coital dosing. We first determined the kinetics of strand transfer in vitro and confirmed that integration begins about 6 hours after infection. We then used a repeat-challenge macaque model to assess efficacy of vaginal gels containing integrase strand transfer inhibitors when applied before or after simian/human immunodeficiency virus (SHIV) challenge. We showed that gel containing the strand transfer inhibitor L-870812 protected two of three macaques when applied 30 min before SHIV challenge. We next evaluated the efficacy of 1% raltegravir gel and demonstrated its ability to protect macaques when applied 3 hours after SHIV exposure (five of six protected; P < 0.05, Fisher's exact test). Breakthrough infections showed no evidence of drug resistance in plasma or vaginal secretions despite continued gel dosing after infection. We documented rapid vaginal absorption reflecting a short pharmacological lag time and noted that vaginal, but not plasma, virus load was substantially reduced in the breakthrough infection after raltegravir gel treatment. We provide a proof of concept that topically applied integrase inhibitors protect against vaginal SHIV infection when administered shortly before or 3 hours after virus exposure.

Bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection.

PubMed

Khawaldeh, A; Morales, S; Dillon, B; Alavidze, Z; Ginn, A N; Thomas, L; Chapman, S J; Dublanchet, A; Smithyman, A; Iredell, J R

2011-11-01

We describe the success of adjunctive bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection in the context of bilateral ureteric stents and bladder ulceration, after repeated failure of antibiotics alone. No bacteriophage-resistant bacteria arose, and the kinetics of bacteriophage and bacteria in urine suggest self-sustaining and self-limiting infection.

Genomic diversity of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum.

PubMed

Castillo, Daniel; Middelboe, Mathias

2016-12-01

Bacteriophages infecting the fish pathogen Flavobacterium psychrophilum can potentially be used to prevent and control outbreaks of this bacterium in salmonid aquaculture. However, the application of bacteriophages in disease control requires detailed knowledge on their genetic composition. To explore the diversity of F. pyschrophilum bacteriophages, we have analyzed the complete genome sequences of 17 phages isolated from two distant geographic areas (Denmark and Chile), including the previously characterized temperate bacteriophage 6H. Phage genome size ranged from 39 302 to 89 010 bp with a G+C content of 27%-32%. None of the bacteriophages isolated in Denmark contained genes associated with lysogeny, whereas the Chilean isolates were all putative temperate phages and similar to bacteriophage 6H. Comparative genome analysis showed that phages grouped in three different genetic clusters based on genetic composition and gene content, indicating a limited genetic diversity of F. psychrophilum-specific bacteriophages. However, amino acid sequence dissimilarity (25%) was found in putative structural proteins, which could be related to the host specificity determinants. This study represents the first analysis of genomic diversity and composition among bacteriophages infecting the fish pathogen F. psychrophilum and discusses the implications for the application of phages in disease control. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Occurrence of Propionibacterium freudenreichii bacteriophages in swiss cheese.

PubMed Central

Gautier, M; Rouault, A; Sommer, P; Briandet, R

1995-01-01

We isolated bacteriophages active against Propionibacterium freudenreichii from 16 of 32 swiss cheese samples. Bacteriophage concentrations ranged from 14 to 7 x 10(5) PFU/g, depending on the sample and the sensitive strain used for detection. Only a few strains, 8 of the 44 strains of P. freudenreichii in our collection, were sensitive. We observed that multiplication of bacteriophages occurred in the cheese loaf during multiplication of propionibacteria in a warm curing room, but it seems that these bacteriophages have no adverse effect on the development of the propionic flora. We also found that sensitive cells, originating from either the starter or the cheese-making milk, were present at a high level (10(9) CFU/g) in the cheese. PMID:7618869

STUDIES ON THE PURIFICATION OF BACTERIOPHAGE

PubMed Central

Kalmanson, G.; Bronfenbrenner, J.

1939-01-01

A simple method of concentrating and purifying bacteriophage has been described. The procedure consisted essentially in collecting the active agent on a reinforced collodion membrane of a porosity that would just retain all the active agent and permit extraneous material to pass through. Advantage was taken of the fact that B. coli will proliferate and regenerate bacteriophage in a completely diffusible synthetic medium with ammonia as the only source of nitrogen, which permitted the purification of the bacteriophage by copious washing. The material thus obtained was concentrated by suction and after thorough washing possessed all the activity of the original filtrate. It was labile, losing its activity in a few days on standing, and was quickly and completely inactivated upon drying. This material contained approximately 15 per cent of nitrogen and with 2 or 3 mg. samples of inactive dry residue it was possible to obtain positive protein color tests. The concentrated and purified bacteriophage has about 10–14 mg. of nitrogen, or 6 x 10–17 gm. of protein per unit of lytic activity. Assuming that each unit of activity represents a molecule, the calculated maximum average molecular weight would be approximately 36,000,000, and on the assumption of a spherical shape of particles and a density of 1.3, the calculated radius would be about 22 millimicra. By measurement of the diffusion rate, the average radius of particle of the fraction of the purified bacteriophage which diffuses most readily through a porous plate was found to be of the order of magnitude of 9 millimicra, or of a calculated molecular weight of 2,250,000. Furthermore, when this purified bacteriophage was fractionated by forcing it through a thin collodion membrane, which permits the passage of only the smaller particles, it was possible to demonstrate in the ultrafiltrate active particles of about 2 millimicra in radius, and of a calculated molecular weight of 25,000. It was of interest to apply

Reduction of Salmonella in ground chicken using a bacteriophage.

PubMed

Grant, Ar'Quette; Parveen, Salina; Schwarz, Jurgen; Hashem, Fawzy; Vimini, Bob

2017-08-01

This study's goal was to ascertain the effectiveness of a commercially available Salmonella bacteriophage during ground chicken production focusing on: water source, different Salmonella serovars, and time. Salmonella-free boneless, skinless chicken meat was inoculated with 4.0 Log CFU/cm2 of either a cocktail of 3 Salmonella isolates derived from ground chicken (GC) or a cocktail of 3 Salmonella strains not isolated from ground chicken (non-GC). Bacteriophages were spread onto the chicken using sterile tap or filtered water for 30 min or 8 h. Salmonella was recovered using standard plating method. Greater Salmonella reduction was observed when the bacteriophage was diluted in sterile tap water than in sterile filtered water: 0.39 Log CFU/cm2 and 0.23 Log CFU/cm2 reduction after 30 min, respectively (P < 0.05). The non-GC isolates showed reductions of 0.71 Log CFU/cm2 and 0.90 Log CFU/cm2 after 30 min and 8 h, respectively (P < 0.05). The GC isolates were less sensitive to the bacteriophage: 0.39 Log CFU/cm2 and 0.67 Log CFU/cm2 reductions after 30 min and 8 h, respectively (P < 0.05). In conclusion, bacteriophage reduction was dependent on water used to dilute the bacteriophage, Salmonella's susceptibility to the bacteriophage, and treatment time. © 2017 Poultry Science Association Inc.

Susceptibility of Escherichia coli isolated from uteri of postpartum dairy cows to antibiotic and environmental bacteriophages. Part I: Isolation and lytic activity estimation of bacteriophages.

PubMed

Bicalho, R C; Santos, T M A; Gilbert, R O; Caixeta, L S; Teixeira, L M; Bicalho, M L S; Machado, V S

2010-01-01

The objective of this study was to isolate bacteriophages from environmental samples of 2 large commercial dairy farms using Escherichia coli isolated from the uteri of postpartum Holstein dairy cows as hosts. A total of 11 bacteriophage preparations were isolated from manure systems of commercial dairy farms and characterized for in vitro antimicrobial activity. In addition, a total of 57 E. coli uterine isolates from 5 dairy cows were phylogenetically grouped by triplex PCR. Each E. coli bacterial host from the uterus was inoculated with their respective bacteriophage preparation at several different multiplicities of infections (MOI) to determine minimum inhibitory MOI. The effect of a single dose (MOI=10(2)) of bacteriophage on the growth curve of all 57 E. coli isolates was assessed using a microplate technique. Furthermore, genetic diversity within and between the different bacteriophage preparations was assessed by bacteriophage purification followed by DNA extraction, restriction, and agarose gel electrophoresis. Phylogenetic grouping based on triplex PCR showed that all isolates of E. coli belonged to phylogroup B1. Bacterial growth was completely inhibited at considerably low MOI, and the effect of a single dose (MOI=10(2)) of bacteriophage preparations on the growth curve of all 57 E. coli isolates showed that all bacteriophage preparations significantly decreased the growth rate of the isolates. Bacteriophage preparation 1230-10 had the greatest antimicrobial activity and completely inhibited the growth of 71.7% (n=57) of the isolates. The combined action of bacteriophage preparations 1230-10, 6375-10, 2540-4, and 6547-2, each at MOI=10(2), had the broadest spectrum of action and completely inhibited the growth (final optical density at 600 nm

Genomic Diversity of Type B3 Bacteriophages of Caulobacter crescentus.

PubMed

Ash, Kurt T; Drake, Kristina M; Gibbs, Whitney S; Ely, Bert

2017-07-01

The genomes of the type B3 bacteriophages that infect Caulobacter crescentus are among the largest phage genomes thus far deposited into GenBank with sizes over 200 kb. In this study, we introduce six new bacteriophage genomes which were obtained from phage collected from various water systems in the southeastern United States and from tropical locations across the globe. A comparative analysis of the 12 available genomes revealed a "core genome" which accounts for roughly 1/3 of these bacteriophage genomes and is predominately localized to the head, tail, and lysis gene regions. Despite being isolated from geographically distinct locations, the genomes of these bacteriophages are highly conserved in both genome sequence and gene order. We also identified the insertions, deletions, translocations, and horizontal gene transfer events which are responsible for the genomic diversity of this group of bacteriophages and demonstrated that these changes are not consistent with the idea that modular reassortment of genomes occurs in this group of bacteriophages.

[Isolation and identification of the temperate bacteriophage from isolated strains of Streptococcus suis serotype 2].

PubMed

Ma, Yuling; Lu, Chengping; Fan, Hongjie

2008-04-01

A PCR assay was developed to study the distributional characteristics of phage integrase gene in Streptococcus suis serotype 2 (SS2). A 323bp distinct DNA target can be amplified in 25 strains of virulent SS2, while can not be amplified in avirulent strain T15, 5 strains of other serotypes (SS1, SS7, SS9) and strains of group C Streptococcus strains from pigs, which suggested that the phage integrase gene may be related to the pathogenicity of SS2 and can be consider as a detection factor of the virulent gene of SS2. The sequencing and restriction endonuclease analysis of the PCR products were also done. Comparisons between the sequences of phage integrase gene with that of SS2 strain, showed a high homology with SS2 China strains 98HAH33, 05ZYH33 and North American strain 89-1591. Complete cell lysis was observed with SS2 virulent strains but not with avirulent strain T15 after the induction by mitomycin C. Electron microscopy analysis of the lysate from SS2 virulent strains HA9801 and ZY05719 revealed the presence of phage particles. The induced phage, named SS2-HA and SS2-ZY, both have a small isometric nucleocapsid approximately 50 nm in diameter and have no tail and is therefore a member of the Tectiviridae family. The phage integrase gene sequence of phage SS2-HA and SS2-ZY shared high homologue identities with virulent SS2 strains, which suggested that the phage integrase gene of SS2 has high specify. The temperate phage and phage integrase gene can only detected from SS2 virulent strains but not from avirulent strain, and the detection of phage integrase gene was related to the virulence-associate factors of SS2, such as the muramidase-released protein gene (mrp), which suggested that the temperate phage of SS2 may be related to the pathogenicity of SS2.

Bacteriophages as indicators of faecal pollution and enteric virus removal.

PubMed

McMinn, B R; Ashbolt, N J; Korajkic, A

2017-07-01

Bacteriophages are an attractive alternative to faecal indicator bacteria (FIB), particularly as surrogates of enteric virus fate and transport, due to their closer morphological and biological properties. Based on a review of published data, we summarize densities of coliphages (F+ and somatic), Bacteroides spp. and enterococci bacteriophages (phages) in individual human waste, raw wastewater, ambient fresh and marine waters and removal through wastewater treatment processes utilizing traditional treatments. We also provide comparisons with FIB and enteric viruses whenever possible. Lastly, we examine fate and transport characteristics in the aquatic environment and provide an overview of the environmental factors affecting their survival. In summary, concentrations of bacteriophages in various sources were consistently lower than FIB, but more reflective of infectious enteric virus levels. Overall, our investigation indicates that bacteriophages may be adequate viral surrogates, especially in built systems, such as wastewater treatment plants. Bacteriophage are alternative fecal indicators that may be better surrogates for viral pathogens than fecal indicator bacteria (FIB). This report offers a summary of the existing literature concerning the utility of bacteriophage as indicators of viral presence (fecal sources and surface waters) and persistence (in built infrastructure and aquatic environments). Our findings indicate that bacteriophage levels in all matrices examined are consistently lower than FIB, but similar to viral pathogens. Furthermore, in built infrastructure (e.g. wastewater treatment systems) bacteriophage closely mimic viral pathogen persistence suggesting they may be adequate sentinels of enteric virus removal. © 2017 The Society for Applied Microbiology.

Convection-enhanced delivery of M13 bacteriophage to the brain

PubMed Central

Ksendzovsky, Alexander; Walbridge, Stuart; Saunders, Richard C.; Asthagiri, Ashok R.; Heiss, John D.; Lonser, Russell R.

2013-01-01

Object Recent studies indicate that M13 bacteriophage, a very large nanoparticle, binds to β-amyloid and α-synuclein proteins, leading to plaque disaggregation in models of Alzheimer and Parkinson disease. To determine the feasibility, safety, and characteristics of convection-enhanced delivery (CED) of M13 bacteriophage to the brain, the authors perfused primate brains with bacteriophage. Methods Four nonhuman primates underwent CED of M13 bacteriophage (900 nm) to thalamic gray matter (4 infusions) and frontal white matter (3 infusions). Bacteriophage was coinfused with Gd-DTPA (1 mM), and serial MRI studies were performed during infusion. Animals were monitored for neurological deficits and were killed 3 days after infusion. Tissues were analyzed for bacteriophage distribution. Results Real-time T1-weighted MRI studies of coinfused Gd-DTPA during infusion demonstrated a discrete region of perfusion in both thalamic gray and frontal white matter. An MRI-volumetric analysis revealed that the mean volume of distribution (Vd) to volume of infusion (Vi) ratio of M13 bacteriophage was 2.3 ± 0.2 in gray matter and 1.9 ± 0.3 in white matter. The mean values are expressed ± SD. Immunohistochemical analysis demonstrated mean Vd:Vi ratios of 2.9 ± 0.2 in gray matter and 2.1 ± 0.3 in white matter. The Gd-DTPA accurately tracked M13 bacteriophage distribution (the mean difference between imaging and actual bacteriophage Vd was insignificant [p > 0.05], and was −2.2% ± 9.9% in thalamic gray matter and 9.1% ± 9.5% in frontal white matter). Immunohistochemical analysis revealed evidence of additional spread from the initial delivery site in white matter (mean Vd:Vi, 16.1 ± 9.1). All animals remained neurologically intact after infusion during the observation period, and histological studies revealed no evidence of toxicity. Conclusions The CED method can be used successfully and safely to distribute M13 bacteriophage in the brain. Furthermore, additional white

Convection-enhanced delivery of M13 bacteriophage to the brain.

PubMed

Ksendzovsky, Alexander; Walbridge, Stuart; Saunders, Richard C; Asthagiri, Ashok R; Heiss, John D; Lonser, Russell R

2012-08-01

Recent studies indicate that M13 bacteriophage, a very large nanoparticle, binds to β-amyloid and α-synuclein proteins, leading to plaque disaggregation in models of Alzheimer and Parkinson disease. To determine the feasibility, safety, and characteristics of convection-enhanced delivery (CED) of M13 bacteriophage to the brain, the authors perfused primate brains with bacteriophage. Four nonhuman primates underwent CED of M13 bacteriophage (900 nm) to thalamic gray matter (4 infusions) and frontal white matter (3 infusions). Bacteriophage was coinfused with Gd-DTPA (1 mM), and serial MRI studies were performed during infusion. Animals were monitored for neurological deficits and were killed 3 days after infusion. Tissues were analyzed for bacteriophage distribution. Real-time T1-weighted MRI studies of coinfused Gd-DTPA during infusion demonstrated a discrete region of perfusion in both thalamic gray and frontal white matter. An MRI-volumetric analysis revealed that the mean volume of distribution (Vd) to volume of infusion (Vi) ratio of M13 bacteriophage was 2.3 ± 0.2 in gray matter and 1.9 ± 0.3 in white matter. The mean values are expressed ± SD. Immunohistochemical analysis demonstrated mean Vd:Vi ratios of 2.9 ± 0.2 in gray matter and 2.1 ± 0.3 in white matter. The Gd-DTPA accurately tracked M13 bacteriophage distribution (the mean difference between imaging and actual bacteriophage Vd was insignificant [p > 0.05], and was -2.2% ± 9.9% in thalamic gray matter and 9.1% ± 9.5% in frontal white matter). Immunohistochemical analysis revealed evidence of additional spread from the initial delivery site in white matter (mean Vd:Vi, 16.1 ± 9.1). All animals remained neurologically intact after infusion during the observation period, and histological studies revealed no evidence of toxicity. The CED method can be used successfully and safely to distribute M13 bacteriophage in the brain. Furthermore, additional white matter spread after infusion cessation

The effects of bacteriophage and nanoparticles on microbial processes

NASA Astrophysics Data System (ADS)

Moody, Austin L.

There are approximately 1031 tailed phages in the biosphere, making them the most abundant organism. Bacteriophages are viruses that infect bacteria. Due to the large diversity and abundance, no two bacteriophages that have been isolated are genetically the same. Phage products have potential in disease therapy to solve bacteria-related problems, such as infections resulting from resistant strains of Staphylococcus aureus. A bacteriophage capable of infecting methicillin-resistant S. aureus (MRSA) was isolated from bovine hair. The bacteriophage, named JB phage, was characterized using purification, amplification, cesium chloride banding, scanning electron microscopy, and transmission electron microscopy. JB phage and nanoparticles were used in various in vitro and in vivo models to test their effects on microbial processes. Scanning and transmission electron microscopy studies revealed strong interactions between JB phage and nanoparticles, which resulted in increased bacteriophage infectivity. JB phage and nanoparticle cocktails were used as a therapeutic to treat skin and systemic infections in mice caused by MRSA.

Bacteriophage Infectivity Against Pseudomonas aeruginosa in Saline Conditions

PubMed Central

Scarascia, Giantommaso; Yap, Scott A.; Kaksonen, Anna H.; Hong, Pei-Ying

2018-01-01

Pseudomonas aeruginosa is a ubiquitous member of marine biofilm, and reduces thiosulfate to produce toxic hydrogen sulfide gas. In this study, lytic bacteriophages were isolated and applied to inhibit the growth of P. aeruginosa in planktonic mode at different temperature, pH, and salinity. Bacteriophages showed optimal infectivity at a multiplicity of infection of 10 in saline conditions, and demonstrated lytic abilities over all tested temperature (25, 30, 37, and 45°C) and pH 6–9. Planktonic P. aeruginosa exhibited significantly longer lag phase and lower specific growth rates upon exposure to bacteriophages. Bacteriophages were subsequently applied to P. aeruginosa-enriched biofilm and were determined to lower the relative abundance of Pseudomonas-related taxa from 0.17 to 5.58% in controls to 0.01–0.61% in treated microbial communities. The relative abundance of Alphaproteobacteria, Pseudoalteromonas, and Planococcaceae decreased, possibly due to the phage-induced disruption of the biofilm matrix. Lastly, when applied to mitigate biofouling of ultrafiltration membranes, bacteriophages were determined to reduce the transmembrane pressure increase by 18% when utilized alone, and by 49% when used in combination with citric acid. The combined treatment was more effective compared with the citric acid treatment alone, which reported ca. 30% transmembrane pressure reduction. Collectively, the findings demonstrated that bacteriophages can be used as a biocidal agent to mitigate undesirable P. aeruginosa-associated problems in seawater applications. PMID:29770130

Microneedle-mediated transdermal bacteriophage delivery

PubMed Central

Ryan, Elizabeth; Garland, Martin J.; Singh, Thakur Raghu Raj; Bambury, Eoin; O’Dea, John; Migalska, Katarzyna; Gorman, Sean P.; McCarthy, Helen O.; Gilmore, Brendan F.; Donnelly, Ryan F.

2012-01-01

Interest in bacteriophages as therapeutic agents has recently been reawakened. Parenteral delivery is the most routinely-employed method of administration. However, injection of phages has numerous disadvantages, such as the requirement of a health professional for administration and the possibility of cross-contamination. Transdermal delivery offers one potential means of overcoming many of these problems. The present study utilized a novel poly (carbonate) (PC) hollow microneedle (MN) device for the transdermal delivery of Escherichia coli-specific T4 bacteriophages both in vitro and in vivo. MN successfully achieved bacteriophage delivery in vitro across dermatomed and full thickness skin. A concentration of 2.67 × 106 PFU/ml (plaque forming units per ml) was detected in the receiver compartment when delivered across dermatomed skin and 4.0 × 103 PFU/ml was detected in the receiver compartment when delivered across full thickness skin. An in vivo study resulted in 4.13 × 103 PFU/ml being detected in blood 30 min following initial MN-mediated phage administration. Clearance occurred rapidly, with phages being completely cleared from the systemic circulation within 24 h, which was expected in the absence of infection. We have shown here that MN-mediated delivery allows successful systemic phage absorption. Accordingly, bacteriophage-based therapeutics may now have an alternative route for systemic delivery. Once fully-investigated, this could lead to more widespread investigation of these interesting therapeutic viruses. PMID:22750416

Predicting bacteriophage proteins located in host cell with feature selection technique.

PubMed

Ding, Hui; Liang, Zhi-Yong; Guo, Feng-Biao; Huang, Jian; Chen, Wei; Lin, Hao

2016-04-01

A bacteriophage is a virus that can infect a bacterium. The fate of an infected bacterium is determined by the bacteriophage proteins located in the host cell. Thus, reliably identifying bacteriophage proteins located in the host cell is extremely important to understand their functions and discover potential anti-bacterial drugs. Thus, in this paper, a computational method was developed to recognize bacteriophage proteins located in host cells based only on their amino acid sequences. The analysis of variance (ANOVA) combined with incremental feature selection (IFS) was proposed to optimize the feature set. Using a jackknife cross-validation, our method can discriminate between bacteriophage proteins located in a host cell and the bacteriophage proteins not located in a host cell with a maximum overall accuracy of 84.2%, and can further classify bacteriophage proteins located in host cell cytoplasm and in host cell membranes with a maximum overall accuracy of 92.4%. To enhance the value of the practical applications of the method, we built a web server called PHPred (〈http://lin.uestc.edu.cn/server/PHPred〉). We believe that the PHPred will become a powerful tool to study bacteriophage proteins located in host cells and to guide related drug discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stabilization of T4 bacteriophage at acidic and basic pH by adsorption on paper.

PubMed

Meyer, Abigail; Greene, Melissa; Kimmelshue, Chad; Cademartiri, Rebecca

2017-12-01

Bacteriophages find applications in agriculture, medicine, and food safety. Many of these applications can expose bacteriophages to stresses that inactivate them including acidic and basic pH. Bacteriophages can be stabilized against these stresses by materials including paper, a common material in packaging and consumer products. Combining paper and bacteriophages creates antibacterial materials, which can reduce the use of antibiotics. Here we show that adsorption on paper protects T4, T5, and T7 bacteriophage from acidic and basic pH. We added bacteriophages to filter paper functionalized with carboxylic acid (carboxyl methyl cellulose) or amine (chitosan) groups, and exposed them to pH from 5.6 to 14. We determined the number of infective bacteriophages after exposure directly on the paper. All papers extended the lifetime of infective bacteriophage by at least a factor of four with some papers stabilizing bacteriophages for up to one week. The degree of stabilization depended on five main factors (i) the family of the bacteriophage, (ii) the charge of the paper and bacteriophages, (iii) the location of the bacteriophages within the paper, (iv) the ability of the paper to prevent bacteriophage-bacteriophage aggregation, and (v) the sensitivity of the bacteriophage proteins to the tested pH. Even when adsorbed on paper the bacteriophages were able to remove E. coli in milk. Choosing the right paper modification or material will protect bacteriophages adsorbed on that material against detrimental pH and other environmental challenges increasing the range of applications of bacteriophages on materials. Copyright © 2017 Elsevier B.V. All rights reserved.

A computational model for predicting integrase catalytic domain of retrovirus.

PubMed

Wu, Sijia; Han, Jiuqiang; Zhang, Xinman; Zhong, Dexing; Liu, Ruiling

2017-06-21

Integrase catalytic domain (ICD) is an essential part in the retrovirus for integration reaction, which enables its newly synthesized DNA to be incorporated into the DNA of infected cells. Owing to the crucial role of ICD for the retroviral replication and the absence of an equivalent of integrase in host cells, it is comprehensible that ICD is a promising drug target for therapeutic intervention. However, annotated ICDs in UniProtKB database have still been insufficient for a good understanding of their statistical characteristics so far. Accordingly, it is of great importance to put forward a computational ICD model in this work to annotate these domains in the retroviruses. The proposed model then discovered 11,660 new putative ICDs after scanning sequences without ICD annotations. Subsequently in order to provide much confidence in ICD prediction, it was tested under different cross-validation methods, compared with other database search tools, and verified on independent datasets. Furthermore, an evolutionary analysis performed on the annotated ICDs of retroviruses revealed a tight connection between ICD and retroviral classification. All the datasets involved in this paper and the application software tool of this model can be available for free download at https://sourceforge.net/projects/icdtool/files/?source=navbar. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bacteriophages of Yersinia pestis.

PubMed

Zhao, Xiangna; Skurnik, Mikael

2016-01-01

Bacteriophage play many varied roles in microbial ecology and evolution. This chapter collates a vast body of knowledge and expertise on Yersinia pestis phages, including the history of their isolation and classical methods for their isolation and identification. The genomic diversity of Y. pestis phage and bacteriophage islands in the Y. pestis genome are also discussed because all phage research represents a branch of genetics. In addition, our knowledge of the receptors that are recognized by Y. pestis phage, advances in phage therapy for Y. pestis infections, the application of phage in the detection of Y. pestis, and clustered regularly interspaced short palindromic repeats (CRISPRs) sequences of Y. pestis from prophage DNA are all reviewed here.

Bacteriophage-based synthetic biology for the study of infectious diseases

PubMed Central

Lu, Timothy K.

2014-01-01

Since their discovery, bacteriophages have contributed enormously to our understanding of molecular biology as model systems. Furthermore, bacteriophages have provided many tools that have advanced the fields of genetic engineering and synthetic biology. Here, we discuss bacteriophage-based technologies and their application to the study of infectious diseases. New strategies for engineering genomes have the potential to accelerate the design of novel phages as therapies, diagnostics, and tools. Though almost a century has elapsed since their discovery, bacteriophages continue to have a major impact on modern biological sciences, especially with the growth of multidrug-resistant bacteria and interest in the microbiome. PMID:24997401

A novel family of integrases associated with prophages and genomic islands integrated within the tRNA-dihydrouridine synthase A (dusA) gene

PubMed Central

Farrugia, Daniel N.; Elbourne, Liam D. H.; Mabbutt, Bridget C.; Paulsen, Ian T.

2015-01-01

Genomic islands play a key role in prokaryotic genome plasticity. Genomic islands integrate into chromosomal loci such as transfer RNA genes and protein coding genes, whilst retaining various cargo genes that potentially bestow novel functions on the host organism. A gene encoding a putative integrase was identified at a single site within the 5′ end of the dusA gene in the genomes of over 200 bacteria. This integrase was discovered to be a component of numerous genomic islands, which appear to share a target site within the dusA gene. dusA encodes the tRNA-dihydrouridine synthase A enzyme, which catalyses the post-transcriptional reduction of uridine to dihydrouridine in tRNA. Genomic islands encoding homologous dusA-associated integrases were found at a much lower frequency within the related dusB and dusC genes, and non-dus genes. Excision of these dusA-associated islands from the chromosome as circularized intermediates was confirmed by polymerase chain reaction. Analysis of the dusA-associated islands indicated that they were highly diverse, with the integrase gene representing the only universal common feature. PMID:25883135

Evolution and the complexity of bacteriophages.

PubMed

Serwer, Philip

2007-03-13

The genomes of both long-genome (> 200 Kb) bacteriophages and long-genome eukaryotic viruses have cellular gene homologs whose selective advantage is not explained. These homologs add genomic and possibly biochemical complexity. Understanding their significance requires a definition of complexity that is more biochemically oriented than past empirically based definitions. Initially, I propose two biochemistry-oriented definitions of complexity: either decreased randomness or increased encoded information that does not serve immediate needs. Then, I make the assumption that these two definitions are equivalent. This assumption and recent data lead to the following four-part hypothesis that explains the presence of cellular gene homologs in long bacteriophage genomes and also provides a pathway for complexity increases in prokaryotic cells: (1) Prokaryotes underwent evolutionary increases in biochemical complexity after the eukaryote/prokaryote splits. (2) Some of the complexity increases occurred via multi-step, weak selection that was both protected from strong selection and accelerated by embedding evolving cellular genes in the genomes of bacteriophages and, presumably, also archaeal viruses (first tier selection). (3) The mechanisms for retaining cellular genes in viral genomes evolved under additional, longer-term selection that was stronger (second tier selection). (4) The second tier selection was based on increased access by prokaryotic cells to improved biochemical systems. This access was achieved when DNA transfer moved to prokaryotic cells both the more evolved genes and their more competitive and complex biochemical systems. I propose testing this hypothesis by controlled evolution in microbial communities to (1) determine the effects of deleting individual cellular gene homologs on the growth and evolution of long genome bacteriophages and hosts, (2) find the environmental conditions that select for the presence of cellular gene homologs, (3) determine

Substrate mimicry—overcoming HIV-1 integrase resistance mutations | Center for Cancer Research

Cancer.gov

HIV integrase (IN) strand transfer inhibitors (INSTIs) are among the newest anti-AIDS drugs; however, mutant forms of IN can confer resistance. We developed noncytotoxic naphthyridine-containing INSTIs that retain low nanomolar IC50 values against HIV-1 variants harboring all of the major INSTI-resistant mutations. We found by analyzing crystal structures of inhibitors bound

Lysogenic bacteriophage isolated from acidophilium

DOEpatents

Ward, Thomas W.; Bruhn, Debby F.; Bulmer, Deborah K.

1992-01-01

A bacteriophage identified as .phi.Ac1 capable of infecting acidophilic heterotropic bacteria (such as Acidiphilium sp.) and processes for genetically engineering acidophilic bacteria for biomining or sulfur removal from coal are disclosed. The bacteriophage is capable of growth in cells existing at pH at or below 3.0. Lytic forms of the phage introduced into areas experiencing acid drainage kill the bacteria causing such drainage. Lysogenic forms of the phase having genes for selective removal of metallic or nonmetallic elements can be introduced into acidophilic bacteria to effect removal of the desired element form ore or coal.

Performance of the Abbott RealTime HIV-1 Viral Load Assay Is Not Impacted by Integrase Inhibitor Resistance-Associated Mutations▿

PubMed Central

Young, Thomas P.; Cloherty, Gavin; Fransen, Signe; Napolitano, Laura; Swanson, Priscilla; Herman, Christine; Parkin, Neil T.; Hackett, John

2011-01-01

The Abbott RealTime HIV-1 viral load assay uses primers and probes targeted to integrase, which is also the target of integrase inhibitors such as raltegravir. Viral loads of 42 raltegravir-susceptible and 40 raltegravir-resistant specimens were determined using RealTime HIV-1 and Roche Monitor (v1.5). The differences in viral load measurements between assays were comparable in the two groups, demonstrating that the RealTime HIV-1 assay can tolerate raltegravir-selected mutations. PMID:21289145

T4 bacteriophage conjugated magnetic particles for E. coli capturing: Influence of bacteriophage loading, temperature and tryptone.

PubMed

Liana, Ayu Ekajayanthi; Marquis, Christopher P; Gunawan, Cindy; Gooding, J Justin; Amal, Rose

2017-03-01

This work demonstrates the use of bacteriophage conjugated magnetic particles (Fe 3 O 4 ) for the rapid capturing and isolation of Escherichia coli. The investigation of T4 bacteriophage adsorption to silane functionalised Fe 3 O 4 with amine (NH 2 ), carboxylic (COOH) and methyl (CH 3 ) surface functional groups reveals the domination of net electrostatic and hydrophobic interactions in governing bacteriophage adsorption. The bare Fe 3 O 4 and Fe 3 O 4 -NH 2 with high T4 loading captured 3-fold more E. coli (∼70% capturing efficiency) compared to the low loading T4 on Fe 3 O 4 -COOH, suggesting the significance of T4 loading in E. coli capturing efficiency. Importantly, it is further revealed that E. coli capture is highly dependent on the incubation temperature and the presence of tryptone in the media. Effective E. coli capturing only occurs at 37°C in tryptone-containing media with the absence of either conditions resulted in poor bacteria capture. The incubation temperature dictates the capturing ability of Fe 3 O 4 /T4, whereby T4 and E. coli need to establish an irreversible binding that occurred at 37°C. The presence of tryptophan-rich tryptone in the suspending media was also critical, as shown by a 3-fold increase in E. coli capture efficiency of Fe 3 O 4 /T4 in tryptone-containing media compared to that in tryptone-free media. This highlights for the first time that successful bacteria capturing requires not only an optimum tailoring of the particle's surface physicochemical properties for favourable bacteriophage loading, but also an in-depth understanding of how factors, such as temperature and solution chemistry influence the subsequent bacteriophage-bacteria interactions. Copyright © 2016 Elsevier B.V. All rights reserved.

Multiplex PCR to detect bacteriophages from natural whey cultures of buffalo milk and characterisation of two phages active against Lactococcus lactis, ΦApr-1 and ΦApr-2.

PubMed

Aprea, Giuseppe; Mullan, William Michael; Murru, Nicoletta; Fitzgerald, Gerald; Buonanno, Marialuisa; Cortesi, Maria Luisa; Prencipe, Vincenza Annunziata; Migliorati, Giacomo

2017-09-30

This work investigated bacteriophage induced starter failures in artisanal buffalo Mozzarella production plants in Southern Italy. Two hundred and ten samples of whey starter cultures were screened for bacteriophage infection. Multiplex polymerase chain reaction (PCR) revealed phage infection in 28.56% of samples, all showing acidification problems during cheese making. Based on DNA sequences, bacteriophages for Lactococcus lactis (L. lactis), Lactobacillus delbruekii (L. delbruekii) and Streptococcus thermophilus (S. thermophilus) were detected. Two phages active against L. lactis, ΦApr-1 and ΦApr-2, were isolated and characterised. The genomes, approximately 31.4 kb and 31 kb for ΦApr-1 and ΦApr-2 respectively, consisted of double-stranded linear DNA with pac-type system. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‑PAGE) showed one major structural protein of approximately 32.5 kDa and several minor proteins. This is the first report of phage isolation in buffalo milk and of the use of multiplex PCR to screen and study the diversity of phages against Lactic Acid Bacteria (LAB) strains in artisanal Water Buffalo Mozzarella starters.

Control of Salmonella on sprouting mung bean and alfalfa seeds by using a biocontrol preparation based on antagonistic bacteria and lytic bacteriophages.

PubMed

Ye, Jianxiong; Kostrzynska, Magdalaena; Dunfield, Kari; Warriner, Keith

2010-01-01

The following reports on the application of a combination of antagonistic bacteria and lytic bacteriophages to control the growth of Salmonella on sprouting mung beans and alfalfa seeds. Antagonistic bacteria were isolated from mung bean sprouts and tomatoes by using the deferred plate assay to assess anti-Salmonella activity. From the isolates screened, an Enterobacter asburiae strain (labeled "JX1") exhibited stable antagonistic activity against a broad range of Salmonella serovars (Agona, Berta, Enteritidis, Hadar, Heidelberg, Javiana, Montevideo, Muenchen, Newport, Saint Paul, and Typhimurium). Lytic bacteriophages against Salmonella were isolated from pig or cattle manure effluent. A bacteriophage cocktail prepared from six isolates was coinoculated with E. asburiae JX1 along with Salmonella in broth culture. The combination of E. asburiae JX1 and bacteriophage cocktail reduced the levels of Salmonella by 5.7 to 6.4 log CFU/ml. Mung beans inoculated with Salmonella and sprouted over a 4-day period attained levels of 6.72 + or - 0.78 log CFU/g. In contrast, levels of Salmonella were reduced to 3.31 + or - 2.48 or 1.16 + or - 2.14 log CFU/g when the pathogen was coinoculated with bacteriophages or E. asburiae JX1, respectively. However, by using a combination of E. asburiae JX1 and bacteriophages, the levels of Salmonella associated with mung bean sprouts were only detected by enrichment. The biocontrol preparation was effective at controlling the growth of Salmonella under a range of sprouting temperatures (20 to 30 degrees Celsius) and was equally effective at suppressing the growth of Salmonella on sprouting alfalfa seeds. The combination of E. asburiae JX1 and bacteriophages represents a promising, chemical-free approach for controlling the growth of Salmonella on sprouting seeds.

Integrase inhibitor versus protease inhibitor based regimen for HIV-1 infected women (WAVES): a randomised, controlled, double-blind, phase 3 study

PubMed Central

Squires, Kathleen; Kityo, Cissy; Hodder, Sally; Johnson, Margaret; Voronin, Evgeny; Hagins, Debbie; Avihingsanon, Anchalee; Koenig, Ellen; Jiang, Shuping; White, Kirsten; Cheng, Andrew; Szwarcberg, Javier; Cao, Huyen

2018-01-01

Summary Background Women are under-represented in HIV antiretroviral therapy (ART) studies. Guidelines for selection of ART as initial therapy in patients with HIV-1 infection do not contain sex-specific treatment. We aimed to assess the safety and efficacy of the single tablet integrase inhibitor regimen containing elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate compared with a boosted protease inhibitor regimen of ritonavir-boosted atazanavir with emtricitabine and tenofovir disoproxil fumarate. Methods In this international, randomised, controlled, double-blind, phase 3 study (Women AntiretroViral Efficacy and Safety study [WAVES]), we recruited treatment-naive HIV-infected women with an estimated creatinine clearance of 70 mL/min or higher from 80 centres in 11 countries. Women were randomly assigned (1:1) to receive elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate (integrase inhibitor regimen) or ritonavir-boosted atazanavir with emtricitabine and tenofovir disoproxil fumarate (protease inhibitor based regimen); regimens were masked with matching placebos. Randomisation was done by a computer-generated allocation sequence (block size four) and was stratified by HIV-1 RNA viral load and race. Investigators, patients, study staff, and those assessing outcomes were masked to treatment group. All participants who received one dose of study drug were included in the primary efficacy and safety analyses. The main outcome was the proportion of patients with plasma HIV-1 RNA less than 50 copies per mL at week 48 as defined by US Food and Drug Administration snapshot algorithm (prespecified non-inferiority margin of 12%). This study is registered with ClinicalTrials.gov, number NCT01705574. Findings Between Nov 28, 2012, and March 12, 2014, 575 women were enrolled. 289 were randomly assigned to receive the integrase inhibitor regimen and 286 to receive the protease inhibitor based regimen. 252 (87%) women in the

[Biological properties of bacteriophages, active to Yersinia enterocolitica].

PubMed

Darsavelidze, M A; Kapanadze, Zh S; Chanishvili, T G

2004-01-01

The biological properties of 16 clones of Y. enterolitica bacteriophages were tested to select the most active for subsequent use. For the first time Y. enterocolitica virulent phages belonging to the family of Podoviridae were described and 7 serological groups of phages with no cross reactions were registered. The technology for the production of new therapeutic and prophylactic Y. enterocolitica polyvalent bacteriophage under laboratory conditions was developed. The effective multiplicity of contamination ensuring the maximum release of phages from bacterial cells, the optimum incubation temperature and the time of exposure were established. The experimental batches of therapeutic and prophylactic Y. enterocolitica polyvalent bacteriophage thus obtained met the requirements for antibacterial preparations.

Real-time monitoring of disintegration activity of catalytic core domain of HIV-1 integrase using molecular beacon.

PubMed

Zhang, Da-wei; Zhao, Ming-ming; He, Hong-qiu; Guo, Shun-xing

2013-09-15

HIV-1 integrase, an essential enzyme for retroviral replication, is a validated target for anti-HIV therapy development. The catalytic core domain of integrase (IN-CCD) is capable of catalyzing disintegration reaction. In this work, a hairpin-shaped disintegration substrate was designed and validated by enzyme-linked immunosorbent assay; a molecular beacon-based assay was developed for disintegration reaction of IN-CCD. Results showed that the disintegration substrate could be recognized and catalyzed by IN-CCD, and the disintegration reaction can be monitored according to the increase of fluorescent signal. The assay can be applied to real-time detection of disintegration with advantages of simplicity, high sensitivity, and excellent specificity. Copyright © 2013 Elsevier Inc. All rights reserved.

Genome Sequences of 19 Novel Erwinia amylovora Bacteriophages

PubMed Central

Esplin, Ian N. D.; Berg, Jordan A.; Sharma, Ruchira; Allen, Robert C.; Arens, Daniel K.; Ashcroft, Cody R.; Bairett, Shannon R.; Beatty, Nolan J.; Bickmore, Madeline; Bloomfield, Travis J.; Brady, T. Scott; Bybee, Rachel N.; Carter, John L.; Choi, Minsey C.; Duncan, Steven; Fajardo, Christopher P.; Foy, Brayden B.; Fuhriman, David A.; Gibby, Paul D.; Grossarth, Savannah E.; Harbaugh, Kala; Harris, Natalie; Hilton, Jared A.; Hurst, Emily; Hyde, Jonathan R.; Ingersoll, Kayleigh; Jacobson, Caitlin M.; James, Brady D.; Jarvis, Todd M.; Jaen-Anieves, Daniella; Jensen, Garrett L.; Knabe, Bradley K.; Kruger, Jared L.; Merrill, Bryan D.; Pape, Jenny A.; Payne Anderson, Ashley M.; Payne, David E.; Peck, Malia D.; Pollock, Samuel V.; Putnam, Micah J.; Ransom, Ethan K.; Ririe, Devin B.; Robinson, David M.; Rogers, Spencer L.; Russell, Kerri A.; Schoenhals, Jonathan E.; Shurtleff, Christopher A.; Simister, Austin R.; Smith, Hunter G.; Stephenson, Michael B.; Staley, Lyndsay A.; Stettler, Jason M.; Stratton, Mallorie L.; Tateoka, Olivia B.; Tatlow, P. J.; Taylor, Alexander S.; Thompson, Suzanne E.; Townsend, Michelle H.; Thurgood, Trever L.; Usher, Brittian K.; Whitley, Kiara V.; Ward, Andrew T.; Ward, Megan E. H.; Webb, Charles J.; Wienclaw, Trevor M.; Williamson, Taryn L.; Wells, Michael J.; Wright, Cole K.; Breakwell, Donald P.; Hope, Sandra

2017-01-01

ABSTRACT Erwinia amylovora is the causal agent of fire blight, a devastating disease affecting some plants of the Rosaceae family. We isolated bacteriophages from samples collected from infected apple and pear trees along the Wasatch Front in Utah. We announce 19 high-quality complete genome sequences of E. amylovora bacteriophages. PMID:29146842

Seven Bacteriophages Isolated from the Female Urinary Microbiota

PubMed Central

Malki, Kema; Sible, Emily; Cooper, Alexandria; Garretto, Andrea; Bruder, Katherine; Watkins, Siobhan C.

2016-01-01

Recent research has debunked the myth that urine is sterile, having uncovered bacteria within the bladders of healthy individuals. However, the identity, diversity, and putative roles of bacteriophages in the bladder are unknown. We report the draft genome sequences of seven bacteriophages isolated from microbial communities from adult female bladders. PMID:27881533

Methods of expanding bacteriophage host-range and bacteriophage produced by the methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crown, Kevin K.; Santarpia, Joshua

A method of producing novel bacteriophages with expanded host-range and bacteriophages with expanded host ranges are disclosed. The method produces mutant phage strains which are infectious to a second host and can be more infectious to their natural host than in their natural state. The method includes repeatedly passaging a selected phage strain into bacterial cultures that contain varied ratios of its natural host bacterial strain with a bacterial strain that the phage of interest is unable to infect; the target-host. After each passage the resulting phage are purified and screened for activity against the target-host via double-overlay assays. Whenmore » mutant phages that are shown to infect the target-host are discovered, they are further propagated in culture that contains only the target-host to produce a stock of the resulting mutant phage.« less

Bacteriophage cocktail for biocontrol of Salmonella in dried pet food.

PubMed

Heyse, Serena; Hanna, Leigh Farris; Woolston, Joelle; Sulakvelidze, Alexander; Charbonneau, Duane

2015-01-01

Human salmonellosis has been associated with contaminated pet foods and treats. Therefore, there is interest in identifying novel approaches for reducing the risk of Salmonella contamination within pet food manufacturing environments. The use of lytic bacteriophages shows promise as a safe and effective way to mitigate Salmonella contamination in various food products. Bacteriophages are safe, natural, highly targeted antibacterial agents that specifically kill bacteria and can be targeted to kill food pathogens without affecting other microbiota. In this study, we show that a cocktail containing six bacteriophages had a broadspectrum activity in vitro against a library of 930 Salmonella enterica strains representing 44 known serovars. The cocktail was effective against 95% of the strains in this tested library. In liquid culture dose-ranging experiments, bacteriophage cocktail concentrations of ≥10(8) PFU/ml inactivated more than 90% of the Salmonella population (10(1) to 10(3) CFU/ml). Dried pet food inoculated with a mixture containing equal proportions of Salmonella serovars Enteritidis (ATCC 4931), Montevideo (ATCC 8387), Senftenberg (ATCC 8400), and Typhimurium (ATCC 13311) and then surface treated with the six-bacteriophage cocktail (≥2.5 ± 1.5 × 10(6) PFU/g) achieved a greater than 1-log (P < 0.001) reduction compared with the phosphate-buffered saline-treated control in measured viable Salmonella within 60 min. Moreover, this bacteriophage cocktail reduced natural contamination in samples taken from an undistributed lot of commercial dried dog food that tested positive for Salmonella. Our results indicate that bacteriophage biocontrol of S. enterica in dried pet food is technically feasible.

Optimizing the European regulatory framework for sustainable bacteriophage therapy in human medicine.

PubMed

Verbeken, Gilbert; Pirnay, Jean-Paul; De Vos, Daniel; Jennes, Serge; Zizi, Martin; Lavigne, Rob; Casteels, Minne; Huys, Isabelle

2012-06-01

For practitioners at hospitals seeking to use natural (not genetically modified, as appearing in nature) bacteriophages for treatment of antibiotic-resistant bacterial infections (bacteriophage therapy), Europe's current regulatory framework for medicinal products hinders more than it facilitates. Although many experts consider bacteriophage therapy to be a promising complementary (or alternative) treatment to antibiotic therapy, no bacteriophage-specific framework for documentation exists to date. Decades worth of historical clinical data on bacteriophage therapy (from Eastern Europe, particularly Poland, and the former Soviet republics, particularly Georgia and Russia, as well as from today's 27 EU member states and the US) have not been taken into account by European regulators because these data have not been validated under current Western regulatory standards. Consequently, applicants carrying out standard clinical trials on bacteriophages in Europe are obliged to initiate clinical work from scratch. This paper argues for a reduced documentation threshold for Phase 1 clinical trials of bacteriophages and maintains that bacteriophages should not be categorized as classical medicinal products for at least two reasons: (1) such a categorization is scientifically inappropriate for this specific therapy and (2) such a categorization limits the marketing authorization process to industry, the only stakeholder with sufficient financial resources to prepare a complete dossier for the competent authorities. This paper reflects on the current regulatory framework for medicines in Europe and assesses possible regulatory pathways for the (re-)introduction of bacteriophage therapy in a way that maintains its effectiveness and safety as well as its inherent characteristics of sustainability and in situ self-amplification and limitation.

Dehydration of bacteriophages in electrospun nanofibers: effect of excipients in polymeric solutions

NASA Astrophysics Data System (ADS)

Koo, Charmaine K. W.; Senecal, Kris; Senecal, Andre; Nugen, Sam R.

2016-12-01

Bacteriophages are viruses capable of infecting and lysing target bacterial cells; as such they have potential applications in agriculture for decontamination of foods, food contact surfaces and food rinse water. Although bacteriophages can retain infectivity long-term using lyophilized storage, the process of freeze-drying can be time consuming and expensive. In this study, electrospinning was used for dehydrating bacteriophages in polyvinylpyrrolidone polymer solutions with addition of excipients (sodium chloride, magnesium sulfate, Tris-HCl, sucrose) in deionized water. The high voltage dehydration reduced the infectivity of bacteriophages following electrospinning, with the damaging effect abated with addition of storage media (SM) buffer and sucrose. SM buffer and sucrose also provided the most protection over extended storage (8 weeks; 20 °C 1% relative humidity) by mitigating environmental effects on the dried bacteriophages. Magnesium sulfate however provided the least protection due to coagulation effects of the ion, which can disrupt the native conformation of the bacteriophage protein coat. Storage temperatures (20 °C, 4 °C and -20 °C 1% relative humidity) had a minimal effect while relative humidity had substantial effect on the infectivity of bacteriophages. Nanofibers stored in higher relative humidity (33% and 75%) underwent considerable damage due to extensive water absorption and disruption of the fibers. Overall, following storage of nanofiber mats for eight weeks at ambient temperatures, high infective phage concentrations (106-107 PFU ml-1) were retained. Therefore, this study provided valuable insights on preservation and dehydration of bacteriophages by electrospinning in comparison to freeze drying and liquid storage, and the influence of excipients on the viability of bacteriophages.

A bacteriophage for Myxococcus xanthus: isolation, characterization and relation of infectivity to host morphogenesis.

PubMed

Burchard, R P; Dworkin, M

1966-03-01

Burchard, Robert P. (University of Minnesota, Minneapolis), and M. Dworkin. A bacteriophage for Myxococcus xanthus: isolation, characterization and relation of infectivity to host morphogenesis. J. Bacteriol. 91:1305-1313. 1966.-A bacteriophage (MX-1) infecting Myxococcus xanthus FB(t) has been isolated from cow dung. The bacteriophage particle is approximately 175 mmu long. A tail about 100 mmu in length is encased in a contractile sheath and terminates in a tail plate. The head is polyhedral with a width of about 75 mmu. The nucleic acid of the bacteriophage is deoxyribonucleic acid and has a guanine plus cytosine content of 55.5%. The bacteriophage requires 10(-3)m Ca(++) and 10(-2)m monovalent cation for optimal adsorption. Grown on vegetative cells of M. xanthus FB(t) at 30 C in 2% Casitone medium, the bacteriophage has a latent period of 120 min and a burst size of approximately 100. Host range studies indicate that three strains of M. xanthus including a morphogenetic mutant are sensitive to the bacteriophage, whereas M. fulvus, Cytophaga, Sporocytophaga myxococcoides, and a fourth strain of M. xanthus are not. Of the two cellular forms characteristic of the Myxococcus life cycle, the bacteriophage infect only the vegetative cells; they do not adsorb to microcysts. Ability to adsorb bacteriophage is lost between 65 and 75 min after initiation of the relatively synchronous conversion of vegetative cells to microcysts. The bacteriophage does not adsorb to spheroplasts. After the appearance of visible morphogenesis and before the loss of bacteriophage receptor sites, addition of bacteriophage results in the formation of microcysts which give rise to infective centers only upon germination. The possibility that the infected microcysts are harboring intact bacteriophages has been eliminated.

Potential of a lytic bacteriophage to disrupt Acinetobacter baumannii biofilms in vitro.

PubMed

Liu, Yannan; Mi, Zhiqiang; Niu, Wenkai; An, Xiaoping; Yuan, Xin; Liu, Huiying; Wang, Yong; Feng, Yuzhong; Huang, Yong; Zhang, Xianglilan; Zhang, Zhiyi; Fan, Hang; Peng, Fan; Li, Puyuan; Tong, Yigang; Bai, Changqing

2016-10-01

The ability of Acinetobacter baumannii to form biofilms and develop antibiotic resistance makes it difficult to control infections caused by this bacterium. In this study, we explored the potential of a lytic bacteriophage to disrupt A. baumannii biofilms. The potential of the lytic bacteriophage to disrupt A. baumannii biofilms was assessed by performing electron microscopy, live/dead bacterial staining, crystal violet staining and by determining adenosine triphosphate release. The bacteriophage inhibited the formation of and disrupted preformed A. baumannii biofilms. Results of disinfection assay showed that the lytic bacteriophage lysed A. baumannii cells suspended in blood or grown on metal surfaces. These results suggest the potential of the lytic bacteriophage to disrupt A. baumannii biofilms.

Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice.

PubMed

Maura, Damien; Morello, Eric; du Merle, Laurence; Bomme, Perrine; Le Bouguénec, Chantal; Debarbieux, Laurent

2012-08-01

Bacteriophages have been known to be present in the gut for many years, but studies of relationships between these viruses and their hosts in the intestine are still in their infancy. We isolated three bacteriophages specific for an enteroaggregative O104:H4 Escherichia coli (EAEC) strain responsible for diarrhoeal diseases in humans. We studied the replication of these bacteriophages in vitro and in vivo in a mouse model of gut colonization. Each bacteriophage was able to replicate in vitro in both aerobic and anaerobic conditions. Each bacteriophage individually reduced biofilms formed on plastic pegs and a cocktail of the three bacteriophages was found to be more efficient. The cocktail was also able to infect bacterial aggregates formed on the surface of epithelial cells. In the mouse intestine, bacteriophages replicated for at least 3 weeks, provided the host was present, with no change in host levels in the faeces. This model of stable and continuous viral replication provides opportunities for studying the long-term coevolution of virulent bacteriophages with their hosts within a mammalian polymicrobial ecosystem. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Evaluation of aerosol spray and intramuscular injection of bacteriophage to treat an Escherichia coli respiratory infection.

PubMed

Huff, W E; Huff, G R; Rath, N C; Balog, J M; Donoghue, A M

2003-07-01

Two studies were conducted to determine the efficacy of either aerosol or i.m. injection of bacteriophage to treat an Escherichia coli respiratory infection in broiler chickens. An additional two studies were conducted to enumerate the bacteriophage in the blood of birds at 1, 2, 3, 4, 5, 6, 24, and 48 h after being sprayed or injected i.m. with bacteriophage. Five birds were bled at each period. In study 1, there were 10 treatments with three replicate pens of 10 birds. The treatments consisted of an untreated control, heat-killed bacteriophage spray, active bacteriophage spray, E. coli challenge at 7 d of age, and E. coli challenge followed by spraying the birds with heat-killed bacteriophage or active bacteriophage at 2, 24, or 48 h after challenge. In study 2 there were 11 treatments with three replicate pens of 10 birds per pen. The treatments were untreated controls, birds injected i.m. in the thigh with heat-killed or active bacteriophage, E. coli challenge at 7 d of age, PBS challenge, E. coli challenge followed by injection of heat-killed or active bacteriophage immediately after challenge or at 24 or 48 h after challenge. In both studies the E. coli challenge consisted of injecting 10(4) cfu into the thoracic air sac. Treatment of this severe E. coli infection with the bacteriophage aerosol spray significantly reduced mortality from 50 to 20% when given immediately after the challenge but had little treatment efficacy when administered 24 or 48 h after challenge. The i.m. injection of bacteriophage significantly reduced mortality from 53 to 17%, 46 to 10%, and 44 to 20% when given immediately, 24, or 48 h after challenge, respectively. Only a few birds sprayed with bacteriophage had detectable bacteriophage in their blood with an average of 96 pfu/mL 1 h after bacteriophage administration, and no bacteriophage was detected 24 and 48 h after bacteriophage administration. All birds injected i.m. with bacteriophage had detectable levels of bacteriophage in

A novel site-specific recombination system derived from bacteriophage phiMR11.

PubMed

Rashel, Mohammad; Uchiyama, Jumpei; Ujihara, Takako; Takemura, Iyo; Hoshiba, Hiroshi; Matsuzaki, Shigenobu

2008-04-04

We report identification of a novel site-specific DNA recombination system that functions in both in vivo and in vitro, derived from lysogenic Staphylococcus aureus phage phiMR11. In silico analysis of the phiMR11 genome indicated orf1 as a putative integrase gene. Phage and bacterial attachment sites (attP and attB, respectively) and attachment junctions were determined and their nucleotide sequences decoded. Sequences of attP and attB were mostly different to each other except for a two bp common core that was the crossover point. We found several inverted repeats adjacent to the core sequence of attP as potential protein binding sites. The precise and efficient integration properties of phiMR11 integrase were shown on attP and attB in Escherichia coli and the minimum size of attP was found to be 34bp. In in vitro assays using crude or purified integrase, only buffer and substrate DNAs were required for the recombination reaction, indicating that other bacterially encoded factors are not essential for activity.

Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

PubMed Central

Costa, Liliana; Faustino, Maria Amparo F.; Neves, Maria Graça P. M. S.; Cunha, Ângela; Almeida, Adelaide

2012-01-01

Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process. PMID:22852040

Genome Sequences of 19 Novel Erwinia amylovora Bacteriophages.

PubMed

Esplin, Ian N D; Berg, Jordan A; Sharma, Ruchira; Allen, Robert C; Arens, Daniel K; Ashcroft, Cody R; Bairett, Shannon R; Beatty, Nolan J; Bickmore, Madeline; Bloomfield, Travis J; Brady, T Scott; Bybee, Rachel N; Carter, John L; Choi, Minsey C; Duncan, Steven; Fajardo, Christopher P; Foy, Brayden B; Fuhriman, David A; Gibby, Paul D; Grossarth, Savannah E; Harbaugh, Kala; Harris, Natalie; Hilton, Jared A; Hurst, Emily; Hyde, Jonathan R; Ingersoll, Kayleigh; Jacobson, Caitlin M; James, Brady D; Jarvis, Todd M; Jaen-Anieves, Daniella; Jensen, Garrett L; Knabe, Bradley K; Kruger, Jared L; Merrill, Bryan D; Pape, Jenny A; Payne Anderson, Ashley M; Payne, David E; Peck, Malia D; Pollock, Samuel V; Putnam, Micah J; Ransom, Ethan K; Ririe, Devin B; Robinson, David M; Rogers, Spencer L; Russell, Kerri A; Schoenhals, Jonathan E; Shurtleff, Christopher A; Simister, Austin R; Smith, Hunter G; Stephenson, Michael B; Staley, Lyndsay A; Stettler, Jason M; Stratton, Mallorie L; Tateoka, Olivia B; Tatlow, P J; Taylor, Alexander S; Thompson, Suzanne E; Townsend, Michelle H; Thurgood, Trever L; Usher, Brittian K; Whitley, Kiara V; Ward, Andrew T; Ward, Megan E H; Webb, Charles J; Wienclaw, Trevor M; Williamson, Taryn L; Wells, Michael J; Wright, Cole K; Breakwell, Donald P; Hope, Sandra; Grose, Julianne H

2017-11-16

Erwinia amylovora is the causal agent of fire blight, a devastating disease affecting some plants of the Rosaceae family. We isolated bacteriophages from samples collected from infected apple and pear trees along the Wasatch Front in Utah. We announce 19 high-quality complete genome sequences of E. amylovora bacteriophages. Copyright © 2017 Esplin et al.

Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni

PubMed Central

Siringan, Patcharin; Connerton, Phillippa L.; Cummings, Nicola J.; Connerton, Ian F.

2014-01-01

Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage. PMID:24671947

Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni.

PubMed

Siringan, Patcharin; Connerton, Phillippa L; Cummings, Nicola J; Connerton, Ian F

2014-03-26

Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage.

Development of a novel and highly efficient method of isolating bacteriophages from water.

PubMed

Liu, Weili; Li, Chao; Qiu, Zhi-Gang; Jin, Min; Wang, Jing-Feng; Yang, Dong; Xiao, Zhong-Hai; Yuan, Zhao-Kang; Li, Jun-Wen; Xu, Qun-Ying; Shen, Zhi-Qiang

2017-08-01

Bacteriophages are widely used to the treatment of drug-resistant bacteria and the improvement of food safety through bacterial lysis. However, the limited investigations on bacteriophage restrict their further application. In this study, a novel and highly efficient method was developed for isolating bacteriophage from water based on the electropositive silica gel particles (ESPs) method. To optimize the ESPs method, we evaluated the eluent type, flow rate, pH, temperature, and inoculation concentration of bacteriophage using bacteriophage f2. The quantitative detection reported that the recovery of the ESPs method reached over 90%. The qualitative detection demonstrated that the ESPs method effectively isolated 70% of extremely low-concentration bacteriophage (10 0 PFU/100L). Based on the host bacteria composed of 33 standard strains and 10 isolated strains, the bacteriophages in 18 water samples collected from the three sites in the Tianjin Haihe River Basin were isolated by the ESPs and traditional methods. Results showed that the ESPs method was significantly superior to the traditional method. The ESPs method isolated 32 strains of bacteriophage, whereas the traditional method isolated 15 strains. The sample isolation efficiency and bacteriophage isolation efficiency of the ESPs method were 3.28 and 2.13 times higher than those of the traditional method. The developed ESPs method was characterized by high isolation efficiency, efficient handling of large water sample size and low requirement on water quality. Copyright © 2017. Published by Elsevier B.V.

Cytoplasmic bacteriophage display system

DOEpatents

Studier, F.W.; Rosenberg, A.H.

1998-06-16

Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest. 1 fig.

Cytoplasmic bacteriophage display system

DOEpatents

Studier, F. William; Rosenberg, Alan H.

1998-06-16

Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest.

Predicting In Vivo Efficacy of Therapeutic Bacteriophages Used To Treat Pulmonary Infections

PubMed Central

Henry, Marine; Lavigne, Rob

2013-01-01

The potential of bacteriophage therapy to treat infections caused by antibiotic-resistant bacteria has now been well established using various animal models. While numerous newly isolated bacteriophages have been claimed to be potential therapeutic candidates on the basis of in vitro observations, the parameters used to guide their choice among billions of available bacteriophages are still not clearly defined. We made use of a mouse lung infection model and a bioluminescent strain of Pseudomonas aeruginosa to compare the activities in vitro and in vivo of a set of nine different bacteriophages (PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5, CHA_P1, LBL3, LUZ19, and PhiKZ). For seven bacteriophages, a good correlation was found between in vitro and in vivo activity. While the remaining two bacteriophages were active in vitro, they were not sufficiently active in vivo under similar conditions to rescue infected animals. Based on the bioluminescence recorded at 2 and 8 h postinfection, we also define for the first time a reliable index to predict treatment efficacy. Our results showed that the bacteriophages isolated directly on the targeted host were the most efficient in vivo, supporting a personalized approach favoring an optimal treatment. PMID:24041900

Arthrobacter globiformis and its bacteriophage in soil

NASA Technical Reports Server (NTRS)

Casida, L. E., Jr.; Liu, K.-C.

1974-01-01

An attempt was made to correlate bacteriophages for Arthrobacter globiformis with soils containing that bacterium. The phages were not detected unless the soil was nutritionally amended (with glucose or sucrose) and incubated for several days. Phage was continuously produced after amendment without the addition of host Arthrobacter. These results indicate that the bacteriophage is present in a masked state and that the bacteria are present in an insensitive form which becomes sensitive after addition of nutrient.

Aligning the unalignable: bacteriophage whole genome alignments.

PubMed

Bérard, Sèverine; Chateau, Annie; Pompidor, Nicolas; Guertin, Paul; Bergeron, Anne; Swenson, Krister M

2016-01-13

In recent years, many studies focused on the description and comparison of large sets of related bacteriophage genomes. Due to the peculiar mosaic structure of these genomes, few informative approaches for comparing whole genomes exist: dot plots diagrams give a mostly qualitative assessment of the similarity/dissimilarity between two or more genomes, and clustering techniques are used to classify genomes. Multiple alignments are conspicuously absent from this scene. Indeed, whole genome aligners interpret lack of similarity between sequences as an indication of rearrangements, insertions, or losses. This behavior makes them ill-prepared to align bacteriophage genomes, where even closely related strains can accomplish the same biological function with highly dissimilar sequences. In this paper, we propose a multiple alignment strategy that exploits functional collinearity shared by related strains of bacteriophages, and uses partial orders to capture mosaicism of sets of genomes. As classical alignments do, the computed alignments can be used to predict that genes have the same biological function, even in the absence of detectable similarity. The Alpha aligner implements these ideas in visual interactive displays, and is used to compute several examples of alignments of Staphylococcus aureus and Mycobacterium bacteriophages, involving up to 29 genomes. Using these datasets, we prove that Alpha alignments are at least as good as those computed by standard aligners. Comparison with the progressive Mauve aligner - which implements a partial order strategy, but whose alignments are linearized - shows a greatly improved interactive graphic display, while avoiding misalignments. Multiple alignments of whole bacteriophage genomes work, and will become an important conceptual and visual tool in comparative genomics of sets of related strains. A python implementation of Alpha, along with installation instructions for Ubuntu and OSX, is available on bitbucket (https://bitbucket.org/thekswenson/alpha).

Reduction of Escherichia coli O157:H7 in Biofilms Using Bacteriophage BPECO 19.

PubMed

Sadekuzzaman, Mohammad; Yang, Sungdae; Mizan, Md Furkanur Rahaman; Ha, Sang-Do

2017-06-01

Biofilm formation is a growing concern in the food industry. Escherichia coli O157:H7 is one of the most important foodborne pathogens that can persists in food and food-related environments and subsequently produce biofilms. The efficacy of bacteriophage BPECO 19 was evaluated against three E. coli O157:H7 strains in biofilms. Biofilms of the three E. coli O157:H7 strains were grown on abiotic (stainless steel, rubber, and minimum biofilm eradication concentration [MBEC TM ] device) and biotic (lettuce) surfaces at different temperatures. The effectiveness of bacteriophage BPECO 19 in reducing preformed biofilms on these surfaces was further evaluated by treating the surfaces with a phage suspension (10 8 PFU/mL) for 2 h. The results indicated that the phage treatment significantly reduced (P  < 0.05) the number of adhered cells in all the surfaces. Following phage treatment, the viability of adhered cells was reduced by ≥3 log CFU/cm 2 , 2.4 log CFU/cm 2 , and 3.1 log CFU/peg in biofilms grown on stainless steel, rubber, and the MBEC TM device, respectively. Likewise, the phage treatment reduced cell viability by ≥2 log CFU/cm 2 in biofilms grown on lettuce. Overall, these results suggested that bacteriophages such as BPECO 19 could be effective in reducing the viability of biofilm-adhered cells. © 2017 Institute of Food Technologists®.

The role of bacteriophages in periodontal health and disease.

PubMed

Pinto, Graça; Silva, Maria Daniela; Peddey, Mark; Sillankorva, Sanna; Azeredo, Joana

2016-10-01

The human periodontium health is commonly compromised by chronic inflammatory conditions and has become a major public health concern. Dental plaque, the precursor of periodontal disease, is a complex biofilm consisting mainly of bacteria, but also archaea, protozoa, fungi and viruses. Viruses that specifically infect bacteria - bacteriophages - are most common in the oral cavity. Despite this, their role in the progression of periodontal disease remains poorly explored. This review aims to summarize how bacteriophages interact with the oral microbiota, their ability to increase bacterial virulence and mediate the transfer of resistance genes and suggests how bacteriophages can be used as an alternative to the current periodontal disease therapies.

HIV-2 Integrase Polymorphisms and Longitudinal Genotypic Analysis of HIV-2 Infected Patients Failing a Raltegravir-Containing Regimen

PubMed Central

Cavaco-Silva, Joana; Abecasis, Ana; Miranda, Ana Cláudia; Poças, José; Narciso, Jorge; Águas, Maria João; Maltez, Fernando; Almeida, Isabel; Germano, Isabel; Diniz, António; Gonçalves, Maria de Fátima; Gomes, Perpétua; Cunha, Celso; Camacho, Ricardo Jorge

2014-01-01

To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients. PMID:24681625

HIV-2 integrase polymorphisms and longitudinal genotypic analysis of HIV-2 infected patients failing a raltegravir-containing regimen.

PubMed

Cavaco-Silva, Joana; Abecasis, Ana; Miranda, Ana Cláudia; Poças, José; Narciso, Jorge; Águas, Maria João; Maltez, Fernando; Almeida, Isabel; Germano, Isabel; Diniz, António; Gonçalves, Maria de Fátima; Gomes, Perpétua; Cunha, Celso; Camacho, Ricardo Jorge

2014-01-01

To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients.

Potential impact of environmental bacteriophages in spreading antibiotic resistance genes.

PubMed

Muniesa, Maite; Colomer-Lluch, Marta; Jofre, Juan

2013-06-01

The idea that bacteriophage transduction plays a role in the horizontal transfer of antibiotic resistance genes is gaining momentum. Such transduction might be vital in horizontal transfer from environmental to human body-associated biomes and here we review many lines of evidence supporting this notion. It is well accepted that bacteriophages are the most abundant entities in most environments, where they have been shown to be quite persistent. This fact, together with the ability of many phages to infect bacteria belonging to different taxa, makes them suitable vehicles for gene transfer. Metagenomic studies confirm that substantial percentages of the bacteriophage particles present in most environments contain bacterial genes, including mobile genetic elements and antibiotic resistance genes. When specific genes of resistance to antibiotics are detected by real-time PCR in the bacteriophage populations of different environments, only tenfold lower numbers of these genes are observed, compared with those found in the corresponding bacterial populations. In addition, the antibiotic resistance genes from these bacteriophages are functional and generate resistance to the bacteria when these genes are transfected. Finally, reports about the transduction of antibiotic resistance genes are on the increase.

Bacteriophage-based Probiotic Preparation for Managing Shigella Infections

DTIC Science & Technology

2015-04-16

for a probiotic preparation – based on naturally occurring bacteriophages – as a way to condition the GI tract’s microflora gently and favorably...10-Apr-2013 Approved for Public Release; Distribution Unlimited Final Report: Bacteriophage-based Probiotic Preparation for Managing Shigella...Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 Phage, Shigella, probiotics REPORT DOCUMENTATION PAGE 11. SPONSOR/MONITOR’S

40 CFR 180.1307 - Bacteriophage of Clavibacter michiganensis subspecies michiganensis; exemption from the...

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacteriophage of Clavibacter... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1307 Bacteriophage of... exemption from the requirement of a tolerance is established for residues of lytic bacteriophage of...

40 CFR 180.1307 - Bacteriophage of Clavibacter michiganensis subspecies michiganensis; exemption from the...

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacteriophage of Clavibacter... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1307 Bacteriophage of... exemption from the requirement of a tolerance is established for residues of lytic bacteriophage of...

40 CFR 180.1307 - Bacteriophage of Clavibacter michiganensis subspecies michiganensis; exemption from the...

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacteriophage of Clavibacter... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1307 Bacteriophage of... exemption from the requirement of a tolerance is established for residues of lytic bacteriophage of...

Template reporter bacteriophage platform and multiple bacterial detection assays based thereon

NASA Technical Reports Server (NTRS)

Goodridge, Lawrence (Inventor)

2007-01-01

The invention is a method for the development of assays for the simultaneous detection of multiple bacteria. A bacteria of interest is selected. A host bacteria containing plasmid DNA from a T even bacteriophage that infects the bacteria of interest is infected with T4 reporter bacteriophage. After infection, the progeny bacteriophage are plating onto the bacteria of interest. The invention also includes single-tube, fast and sensitive assays which utilize the novel method.

Newly Isolated Bacteriophages from the Podoviridae, Siphoviridae, and Myoviridae Families Have Variable Effects on Putative Novel Dickeya spp.

PubMed

Alič, Špela; Naglič, Tina; Tušek-Žnidarič, Magda; Ravnikar, Maja; Rački, Nejc; Peterka, Matjaž; Dreo, Tanja

2017-01-01

Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This study reports the isolation of three bacteriophages active against putative novel Dickeya spp. isolates from commercially produced infected orchids that show variable host-range profiles. Bacteriophages were isolated through enrichment from Dickeya -infected orchid tissue. Convective interaction media monolith chromatography was used to isolate bacteriophages from wastewaters, demonstrating its suitability for the isolation of infective bacteriophages from natural sources. Based on bacteriophage morphology, all isolated bacteriophages were classified as being in the order Caudovirales , belonging to three different families, Podoviridae , Myoviridae , and Siphoviridae . The presence of three different groups of bacteriophages was confirmed by analyzing the bacteriophage specificity of bacterial hosts, restriction fragment length polymorphism and plaque morphology. Bacteriophage BF25/12, the first reported Podoviridae bacteriophage effective against Dickeya spp., was selected for further characterization. Its genome sequence determined by next-generation sequencing showed limited similarity to other characterized Podoviridae bacteriophages. Interactions among the bacteriophages and Dickeya spp. were examined using transmission electron microscopy, which revealed degradation of electron-dense granules in response to bacteriophage infection in some Dickeya strains. The temperature stability of the chosen Podoviridae bacteriophage monitored over 1 year showed a substantial decrease in the survival of bacteriophages stored at -20°C over longer periods. It showed susceptibility to low pH and UV radiation but was stable in neutral

Newly Isolated Bacteriophages from the Podoviridae, Siphoviridae, and Myoviridae Families Have Variable Effects on Putative Novel Dickeya spp.

PubMed Central

Alič, Špela; Naglič, Tina; Tušek-Žnidarič, Magda; Ravnikar, Maja; Rački, Nejc; Peterka, Matjaž; Dreo, Tanja

2017-01-01

Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This study reports the isolation of three bacteriophages active against putative novel Dickeya spp. isolates from commercially produced infected orchids that show variable host-range profiles. Bacteriophages were isolated through enrichment from Dickeya-infected orchid tissue. Convective interaction media monolith chromatography was used to isolate bacteriophages from wastewaters, demonstrating its suitability for the isolation of infective bacteriophages from natural sources. Based on bacteriophage morphology, all isolated bacteriophages were classified as being in the order Caudovirales, belonging to three different families, Podoviridae, Myoviridae, and Siphoviridae. The presence of three different groups of bacteriophages was confirmed by analyzing the bacteriophage specificity of bacterial hosts, restriction fragment length polymorphism and plaque morphology. Bacteriophage BF25/12, the first reported Podoviridae bacteriophage effective against Dickeya spp., was selected for further characterization. Its genome sequence determined by next-generation sequencing showed limited similarity to other characterized Podoviridae bacteriophages. Interactions among the bacteriophages and Dickeya spp. were examined using transmission electron microscopy, which revealed degradation of electron-dense granules in response to bacteriophage infection in some Dickeya strains. The temperature stability of the chosen Podoviridae bacteriophage monitored over 1 year showed a substantial decrease in the survival of bacteriophages stored at -20°C over longer periods. It showed susceptibility to low pH and UV radiation but was stable in neutral and

Expression of a bioactive bacteriophage endolysin in Nicotiana benthamiana plants

USDA-ARS?s Scientific Manuscript database

The emergence and spread of antibiotic-resistant pathogens has led to an increased interest in alternative antimicrobial treatments, such as bacteriophage, bacteriophage-encoded peptidoglycan hydrolases (endolysins) and antimicrobial peptides. In our study, the antimicrobial activity of the CP933 en...

Metavirome Sequencing of the Termite Gut Reveals the Presence of an Unexplored Bacteriophage Community

PubMed Central

Tikhe, Chinmay V.; Husseneder, Claudia

2018-01-01

The Formosan subterranean termite; Coptotermes formosanus is nutritionally dependent on the complex and diverse community of bacteria and protozoa in their gut. Although, there have been many studies to decipher the taxonomic and functional diversity of bacterial communities in the guts of termites, their bacteriophages remain unstudied. We sequenced the metavirome of the guts of Formosan subterranean termite workers to study the diversity of bacteriophages and other associated viruses. Results showed that the termites harbor a virome in their gut comprised of varied and previously unknown bacteriophages. Between 87–90% of the predicted dsDNA virus genes by Metavir showed similarity to the tailed bacteriophages (Caudovirales). Many predicted genes from the virome matched to bacterial prophage regions. These data are suggestive of a virome dominated by temperate bacteriophages. We predicted the genomes of seven novel Caudovirales bacteriophages from the termite gut. Three of these predicted bacteriophage genomes were found in high proportions in all the three termite colonies tested. Two bacteriophages are predicted to infect endosymbiotic bacteria of the gut protozoa. The presence of these putative bacteriophages infecting endosymbionts of the gut protozoa, suggests a quadripartite relationship between the termites their symbiotic protozoa, endosymbiotic bacteria of the protozoa and their bacteriophages. Other than Caudovirales, ss-DNA virus related genes were also present in the termite gut. We predicted the genomes of 12 novel Microviridae phages from the termite gut and seven of those possibly represent a new proposed subfamily. Circovirus like genomes were also assembled from the termite gut at lower relative abundance. We predicted 10 novel circovirus genomes in this study. Whether these circoviruses infect the termites remains elusive at the moment. The functional and taxonomical annotations suggest that the termites may harbor a core virome comprised of

Genomics of Three New Bacteriophages Useful in the Biocontrol of Salmonella

PubMed Central

Bardina, Carlota; Colom, Joan; Spricigo, Denis A.; Otero, Jennifer; Sánchez-Osuna, Miquel; Cortés, Pilar; Llagostera, Montserrat

2016-01-01

Non-typhoid Salmonella is the principal pathogen related to food-borne diseases throughout the world. Widespread antibiotic resistance has adversely affected human health and has encouraged the search for alternative antimicrobial agents. The advances in bacteriophage therapy highlight their use in controlling a broad spectrum of food-borne pathogens. One requirement for the use of bacteriophages as antibacterials is the characterization of their genomes. In this work, complete genome sequencing and molecular analyses were carried out for three new virulent Salmonella-specific bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) able to infect a broad range of Salmonella strains. Sequence analysis of the genomes of UAB_Phi20, UAB_Phi78, and UAB_Phi87 bacteriophages did not evidence the presence of known virulence-associated and antibiotic resistance genes, and potential immunoreactive food allergens. The UAB_Phi20 genome comprised 41,809 base pairs with 80 open reading frames (ORFs); 24 of them with assigned function. Genome sequence showed a high homology of UAB_Phi20 with Salmonella bacteriophage P22 and other P22likeviruses genus of the Podoviridae family, including ST64T and ST104. The DNA of UAB_Phi78 contained 44,110 bp including direct terminal repeats (DTR) of 179 bp and 58 putative ORFs were predicted and 20 were assigned function. This bacteriophage was assigned to the SP6likeviruses genus of the Podoviridae family based on its high similarity not only with SP6 but also with the K1-5, K1E, and K1F bacteriophages, all of which infect Escherichia coli. The UAB_Phi87 genome sequence consisted of 87,669 bp with terminal direct repeats of 608 bp; although 148 ORFs were identified, putative functions could be assigned to only 29 of them. Sequence comparisons revealed the mosaic structure of UAB_Phi87 and its high similarity with bacteriophages Felix O1 and wV8 of E. coli with respect to genetic content and functional organization. Phylogenetic analysis of large

[Advance on genome research of Yersinia pestis bacteriophage].

PubMed

Tan, H L; Wang, P; Li, W

2017-04-10

Completion of the genome sequences on Yersinia pestis bacteriophage offered unprecedented opportunity for researchers to carry out related genomic studies. This review was based on the genomic sequences and provided a genomic perspective in describing the essential features of genome on Yersinia pestis bacteriophage. Based on the comparative genomics, genetic evolutionary relationship was discussed. Description of functions from the gene prediction and protein annotation provided evidence for further related studies.

Bacteriophage immobilized graphene electrodes for impedimetric sensing of bacteria (Staphylococcus arlettae).

PubMed

Bhardwaj, Neha; Bhardwaj, Sanjeev K; Mehta, Jyotsana; Mohanta, Girish C; Deep, Akash

2016-07-15

Bacteriophages are a class of viruses that specifically infect and replicate within a bacterium. They possess inherent affinity and specificity to the particular bacterial cells. This property of bacteriophages makes them an attractive biorecognition element in the field of biosensor development. In this work, we report the use of an immobilized bacteriophage for the development of a highly sensitive electrochemical sensor for Staphylococcus arlettae, bacteria from the pathogenic family of coagulase-negative staphylococci (CNS). The specific bacteriophages were covalently immobilized on the screen-printed graphene electrodes. Thus, the fabricated bacteriophage biosensor displayed quantitative response for the target bacteria (S. arlettae) for a broad detection range (2.0-2.0 × 10(6) cfu). A fast response time (2 min), low limit of detection (2 cfu), specificity, and stability over a prolonged period (3 months) are some of the important highlights of the proposed sensor. The practical utility of the developed sensor has been demonstrated by the analysis of S. arlettae in spiked water and apple juice samples. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential Effects of the G118R, H51Y, and E138K Resistance Substitutions in Different Subtypes of HIV Integrase

PubMed Central

Quashie, Peter K.; Oliviera, Maureen; Veres, Tamar; Osman, Nathan; Han, Ying-Shan; Hassounah, Said; Lie, Yolanda; Huang, Wei; Mesplède, Thibault

2014-01-01

ABSTRACT Dolutegravir (DTG) is the latest antiretroviral (ARV) approved for the treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir, may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a low enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrases were severely restricted in their viral replication capacity, and G118R/E138K-bearing viruses had various levels of resistance to dolutegravir, raltegravir, and elvitegravir. In cell-free experiments, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which significantly affect native protein activity, structure, or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in the interpretation of resistance patterns in dolutegravir-treated patients. IMPORTANCE Recognized drug resistance mutations have never been reported for naive patients treated with dolutegravir. Additionally, in integrase inhibitor-experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how “minor” polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This work also

Differential effects of the G118R, H51Y, and E138K resistance substitutions in different subtypes of HIV integrase.

PubMed

Quashie, Peter K; Oliviera, Maureen; Veres, Tamar; Osman, Nathan; Han, Ying-Shan; Hassounah, Said; Lie, Yolanda; Huang, Wei; Mesplède, Thibault; Wainberg, Mark A

2015-03-01

Dolutegravir (DTG) is the latest antiretroviral (ARV) approved for the treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir, may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a low enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrases were severely restricted in their viral replication capacity, and G118R/E138K-bearing viruses had various levels of resistance to dolutegravir, raltegravir, and elvitegravir. In cell-free experiments, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which significantly affect native protein activity, structure, or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in the interpretation of resistance patterns in dolutegravir-treated patients. Recognized drug resistance mutations have never been reported for naive patients treated with dolutegravir. Additionally, in integrase inhibitor-experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how "minor" polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This work also highlights the importance

Bacteriophage-nanocomposites: an easy and reproducible method for the construction, handling, storage and transport of conjugates for deployment of bacteriophages active against Pseudomonas aeruginosa.

PubMed

Cooper, Ian R; Illsley, Matthew; Korobeinyk, Alina V; Whitby, Raymond L D

2015-04-01

The purpose of this work was proof of concept to develop a novel, cost effective protocol for the binding of bacteriophages to a surface without loss of function, after storage in various media. The technology platform involved covalently bonding bacteriophage 13 (a Pseudomonas aeruginosa bacteriophage) to two magnetised multiwalled carbon nanotube scaffolds using a series of buffers; bacteriophage-nanotube (B-N) conjugates were efficacious after storage at 20 °C for six weeks. B-N conjugates were added to human cell culture in vitro for 9 days without causing necrosis and apoptosis. B-N conjugates were frozen (-20 °C) in cell culture media for several weeks, after which recovery from the human cell culture medium was possible using a simple magnetic separation technique. The retention of viral infective potential was demonstrated by subsequent spread plating onto lawns of susceptible P. aeruginosa. Analysis of the human cell culture medium revealed the production of interleukins by the human fibroblasts upon exposure to the bacteriophage. One day after exposure, IL-8 levels transitorily increased between 60 and 100 pg/mL, but this level was not found on any subsequent days, suggesting an initial but not long lasting response. This paper outlines the development of a method to deliver antimicrobial activity to a surface that is small enough to be combined with other materials. To our knowledge at time of publication, this is the first report of magnetically coupled bacteriophages specific to human pathogens which can be recovered from test systems, and could represent a novel means to conditionally deploy antibacterial agents into living eukaryotic systems without the risks of some antibiotic therapies. Copyright © 2015. Published by Elsevier B.V.

Two New Lytic Bacteriophages of the Myoviridae Family Against Carbapenem-Resistant Acinetobacter baumannii

PubMed Central

Zhou, Weilong; Feng, Yu; Zong, Zhiyong

2018-01-01

Two lytic bacteriophages, WCHABP1 and WCHABP12, were recovered from hospital sewage and were able to infect 9 and 12 out of 18 carbapenem-resistant Acinetobacter baumannii clinical strains, which belonged to different clones. Electron microscopy scan showed that both bacteriophages had the similar morphology as those of the Myoviridae family. Whole genomic sequencing revealed 45.4- or 45.8-kb genome with a 37.6% GC content for WCHABP1 and WCHABP12, both of which showed significant DNA sequence similarity with bacteriophages of the Ap22virus genus within the Myoviridae family. Taxonomic analysis was therefore performed following the proposal approved by the International Committee on Taxonomy of Viruses, which confirmed that WCHABP1 and WCHABP12 represented two new species of the Ap22virus genus. No tRNAs but 88 and 89 open reading frames (ORFs) were predicted for the two bacteriophages, among which 22 and 21 had known function and encoded proteins for morphogenesis, packaging, lysis, and nucleiotide metabolism. The C-terminal amino acids of the large unit of fiber tail proteins varied between the bacteriophages, which may explain their different host ranges. For most lytic bacteriophages, a set of holin and endolysin are required for lysis. However, no known holin-encoding genes were identified in WCHABP1 and WCHABP12, suggesting that they may use alternative, yet-to-be-identified, novel holins for host cell membrane lysis. To test the efficacy of the bacteriophages in protecting against A. baumannii infection, a Galleria mellonella larva model was used. Only <20% G. mellonella larvae survived at 96 h after being infected by carbapenem-resistant A. baumannii strains, from which the two bacteriophages were recovered. With the administration of WCHABP1 and WCHABP12, the survival of larvae increased to 75%, while the treatment of polymyxin B only slightly increased the survival rate to 25%. The isolation of two new lytic bacteriophages in this study could expand our

Role of the His-Cys finger of Moloney murine leukemia virus integrase protein in integration and disintegration.

PubMed Central

Jonsson, C B; Roth, M J

1993-01-01

Retroviral integrases mediate site-specific endonuclease and transesterification reactions in the absence of exogenous energy. The basis for the sequence specificity in these integrase-viral DNA recognition processes is unknown. Structural analogs of the disintegration substrate were made to analyze the disintegration reaction mechanism for the Moloney murine leukemia virus (M-MuLV) integrase (IN). Modifications in the target DNA portion of the disintegration substrate decreased enzymatic activity, while substitution of the highly conserved CA in the viral long terminal repeat portion had no effect on activity. The role of the His-Cys finger region in catalysis was addressed by N-ethylmaleimide (NEM) modification of the cysteine residues of M-MuLV IN as well as by mutations. Both integration activities, 3' processing, and strand transfer, were completely inhibited by NEM modification of M-MuLV IN, while disintegration activity was only partially sensitive. However, structural analogs of the disintegration substrates that were modified in the target DNA and had the conserved CA removed were not active with NEM-treated M-MuLV IN. In addition, mutants made in the His-Cys region of M-MuLV IN were examined and found to also be completely blocked in integration but not disintegration activity. These data suggest that the domains of M-MuLV IN that are required for the forward integration reaction substrate differ from those required for the reverse disintegration reaction substrate. Images PMID:8350412

Improved bacteriophage genome data is necessary for integrating viral and bacterial ecology.

PubMed

Bibby, Kyle

2014-02-01

The recent rise in "omics"-enabled approaches has lead to improved understanding in many areas of microbial ecology. However, despite the importance that viruses play in a broad microbial ecology context, viral ecology remains largely not integrated into high-throughput microbial ecology studies. A fundamental hindrance to the integration of viral ecology into omics-enabled microbial ecology studies is the lack of suitable reference bacteriophage genomes in reference databases-currently, only 0.001% of bacteriophage diversity is represented in genome sequence databases. This commentary serves to highlight this issue and to promote bacteriophage genome sequencing as a valuable scientific undertaking to both better understand bacteriophage diversity and move towards a more holistic view of microbial ecology.

Application of bacteriophages in post-harvest control of human pathogenic and food spoiling bacteria.

PubMed

Pérez Pulido, Rubén; Grande Burgos, Maria José; Gálvez, Antonio; Lucas López, Rosario

2016-10-01

Bacteriophages have attracted great attention for application in food biopreservation. Lytic bacteriophages specific for human pathogenic bacteria can be isolated from natural sources such as animal feces or industrial wastes where the target bacteria inhabit. Lytic bacteriophages have been tested in different food systems for inactivation of main food-borne pathogens including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, Salmonella enterica, Shigella spp., Campylobacter jejuni and Cronobacter sakazkii, and also for control of spoilage bacteria. Application of lytic bacteriophages could selectively control host populations of concern without interfering with the remaining food microbiota. Bacteriophages could also be applied for inactivation of bacteria attached to food contact surfaces or grown as biofilms. Bacteriophages may receive a generally recognized as safe status based on their lack of toxicity and other detrimental effects to human health. Phage preparations specific for L. monocytogenes, E. coli O157:H7 and S. enterica serotypes have been commercialized and approved for application in foods or as part of surface decontamination protocols. Phage endolysins have a broader host specificity compared to lytic bacteriophages. Cloned endolysins could be used as natural preservatives, singly or in combination with other antimicrobials such as bacteriocins.

Detection of a Bacteriophage Gene Encoding a Mu-like Portal Protein in Haemophilus parasuis Reference Strains and Field Isolates by Nested Polymerase Chain Reaction

USDA-ARS?s Scientific Manuscript database

A nested PCR assay was developed to determine the presence of a gene encoding a bacteriophage Mu-like portal protein, gp29, in 15 reference strains and 31 field isolates of Haemophilus parasuis. Specific primers, based on the gene’s sequence, were utilized. A majority of the virulent reference strai...

Application of bacteriophages in sensor development.

PubMed

Peltomaa, Riikka; López-Perolio, Irene; Benito-Peña, Elena; Barderas, Rodrigo; Moreno-Bondi, María Cruz

2016-03-01

Bacteriophage-based bioassays are a promising alternative to traditional antibody-based immunoassays. Bacteriophages, shortened to phages, can be easily conjugated or genetically engineered. Phages are robust, ubiquitous in nature, and harmless to humans. Notably, phages do not usually require inoculation and killing of animals; and thus, the production of phages is simple and economical. In recent years, phage-based biosensors have been developed featuring excellent robustness, sensitivity, and selectivity in combination with the ease of integration into transduction devices. This review provides a critical overview of phage-based bioassays and biosensors developed in the last few years using different interrogation methods such as colorimetric, enzymatic, fluorescence, surface plasmon resonance, quartz crystal microbalance, magnetoelastic, Raman, or electrochemical techniques.

Isolation, characterization, and application of bacteriophages for Salmonella spp. biocontrol in pigs.

PubMed

Albino, Luiz A A; Rostagno, Marcos H; Húngaro, Humberto M; Mendonça, Regina C S

2014-08-01

Foodborne illness due to Salmonella-contaminated pork products is an important public health problem, causing significant economic losses worldwide. The use of bacteriophages is a potential intervention tool that has attracted interest for the control of foodborne pathogens. The objective of this study was to detect the presence of Salmonella in commercial pig farms and to isolate specific autochthonous bacteriophages against Salmonella Typhimurium, to characterize them and to evaluate their lytic capacity against Salmonella Typhimurium in vivo and in vitro. Salmonella was isolated on 50% (4/8) of the farms, with serotype Typhimurium being the most prevalent, detected in 48.2% of samples (13/27). The isolated Salmonella Typhimurium bacteriophages belong to the Podoviridae family, were active against serotypes Abony, Enteritidis, Typhi, and Typhimurium, but not against serotypes Arizonae, Cholerasuis, Gallinarum, and Pullorum. In in vitro tests, bacteriophage at 10(7) PFU/mL and 10(9) PFU/mL significantly reduced (p<0.05) Salmonella Typhimurium counts in 1.6 and 2.5 log10 colony-forming units (CFU)/mL, respectively, after 24 h. Before the in vivo treatment with bacteriophages, Salmonella was identified in 93.3% (28/30) of the fecal samples from the pigs inoculated with 10(6) CFU/mL, and only in 56.6% (17/30) after the treatment consisting of oral administration of the pool of the bacteriophages after the fasting period, simulating a common preslaughter practice. These results indicate that the pool of bacteriophages administered was capable of reducing the colonization of Salmonella in pigs.

Significance of the Bacteriophage Treatment Schedule in Reducing Salmonella Colonization of Poultry

PubMed Central

Bardina, Carlota; Spricigo, Denis A.; Cortés, Pilar

2012-01-01

Salmonella remains the major cause of food-borne diseases worldwide, with chickens known to be the main reservoir for this zoonotic pathogen. Among the many approaches to reducing Salmonella colonization of broilers, bacteriophage offers several advantages. In this study, three bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) obtained from our collection that exhibited a broad host range against Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium were characterized with respect to morphology, genome size, and restriction patterns. A cocktail composed of the three bacteriophages was more effective in promoting the lysis of S. Enteritidis and S. Typhimurium cultures than any of the three bacteriophages alone. In addition, the cocktail was able to lyse the Salmonella enterica serovars Virchow, Hadar, and Infantis. The effectiveness of the bacteriophage cocktail in reducing the concentration of S. Typhimurium was tested in two animal models using different treatment schedules. In the mouse model, 50% survival was obtained when the cocktail was administered simultaneously with bacterial infection and again at 6, 24, and 30 h postinfection. Likewise, in the White Leghorn chicken specific-pathogen-free (SPF) model, the best results, defined as a reduction of Salmonella concentration in the chicken cecum, were obtained when the bacteriophage cocktail was administered 1 day before or just after bacterial infection and then again on different days postinfection. Our results show that frequent treatment of the chickens with bacteriophage, and especially prior to colonization of the intestinal tract by Salmonella, is required to achieve effective bacterial reduction over time. PMID:22773654

Characterization of the endolysin from the Enterococcus faecalis bacteriophage VD13

USDA-ARS?s Scientific Manuscript database

Bacteriophage infecting bacteria produce endolysins (peptidoglycan hydrolases) to lyse the host cell from within and release nascent bacteriophage particles. Recombinant endolysins can also lyse Gram-positive bacteria when added exogenously. As a potential alternative to antibiotics, we cloned and...

Hyperparasitism by the bacteriophage (Caudovirales) infecting Candidatus Xenohaliotis californiensis (Rickettsiales-like prokaryote) parasite of wild abalone Haliotis fulgens and Haliotis corrugata from the Peninsula of Baja California, Mexico.

PubMed

Cruz-Flores, Roberto; Cáceres-Martínez, Jorge; Muñoz-Flores, Monserrat; Vásquez-Yeomans, Rebeca; Hernández Rodriguez, Mónica; Ángel Del Río-Portilla, Miguel; Rocha-Olivares, Axayácatl; Castro-Longoria, Ernestina

2016-10-01

Candidatus Xenohaliotis californiensis (CXc) is a Rickettsiales-like prokaryote that is considered the causal agent of Withering Syndrome (WS), a chronic disease of abalone, from the west coast of North America and it is listed by the International Organization for Animal Health (OIE) as a reportable agent due to its pathogenicity. This bacterium in red abalone Haliotis rufescens, black abalone Haliotis cracherodii, and yellow abalone Haliotis corrugata from California, US and Baja California, Mexico has been found to be infected by a bacteriophage. To date, there is no information on the epizootiology of CXc and its bacteriophage in natural populations of abalone; furthermore, it is unknown if the bacteriophage was also present in CXc infecting blue abalone Haliotis fulgens. The objective of this study was to determine the distribution, prevalence and intensity of CXc, as well as to determine the distribution and prevalence of the bacteriophage and to study interactions between host sex and hyperparasitism in blue abalone and yellow abalone. Tissue samples were obtained from seven localities where the commercial capture of wild abalone is carried out. Samplings were conducted throughout the 2012-2013 capture seasons and a total of 182 blue abalone and 170 yellow abalone were obtained. The prevalence and intensity of CXc and the prevalence of the bacteriophage were determined by histology. The identity of CXc was confirmed by PCR, product sequence analysis and in situ hybridization while the identity of the bacteriophage was corroborated by TEM. The prevalence of CXc infected and uninfected by the bacteriophage was 80% in blue abalone and 62% in yellow abalone. Low infection intensities were found in 86% of blue abalone and 82% of yellow abalone. Infection intensity was significantly higher in undifferentiated yellow abalone. The bacteriophage in CXc showed a prevalence of 22% and 31% in blue abalone and yellow abalone respectively. These results show that CXc and

[Genetic study of bacteriophage phi81. I. Isolation, study of complementation and preliminary mapping of amber-mutants of bacteriophage phi81].

PubMed

Sineokiĭ, S P; Pogosov, V Z; Iankovskiĭ, N K; Krylov, V N

1976-01-01

123 Amber mutants of lambdoid bacteriophage phi81 are isolated and distributed into 19 complementation groups. Deletion mapping made possible to locate 5 gene groups on the genetic map of bacteriophage phi81 and to determine a region of possible location of mm' sticky ends on the prophage genetic map. A gene of phage phi81 is localized, which controls the adsorption specificity, and which functional similarity to a respective gene of phage phi80 is demonstrated.

Adsorption of bacteriophages on clay minerals

USGS Publications Warehouse

Chattopadhyay, Sandip; Puls, Robert W.

1999-01-01

The ability to predict the fate of microorganisms in soil is dependent on an understanding of the process of their sorption on soil and subsurface materials. Presently, we have focused on studying the thermodynamics of sorption of bacteriophages (T-2, MS-2, and φX-174) on clays (hectorite, saponite, kaolinite, and clay fraction of samples collected from a landfill site). The thermodynamic study not only determines the feasibility of the process but also provides information on the relative magnitudes of the different forces under a particular set of conditions. The total free energy of interaction during sorption of bacteriophages on clays (ΔG) has been assumed to be the summation of ΔGH (ΔG due to hydrophobic interactions) and ΔGEL (ΔG due to electrostatic interactions). The magnitude of ΔGH was determined from the different interfacial tensions (γ) present in the system, while ΔGEL was calculated from ζ-potentials of the colloidal particles. Calculated results show that surface hydrophobicities of the selected sorbents and sorbates dictate sorption. Among the selected bacteriophages, maximum sorption was observed with T-2, while hectorite has the maximum sorption capacity. Experimental results obtained from the batch adsorption studies also corroborated those obtained from the theoretical study.

Novel bacteriophages containing a genome of another bacteriophage within their genomes.

PubMed

Swanson, Maud M; Reavy, Brian; Makarova, Kira S; Cock, Peter J; Hopkins, David W; Torrance, Lesley; Koonin, Eugene V; Taliansky, Michael

2012-01-01

A novel bacteriophage infecting Staphylococus pasteuri was isolated during a screen for phages in Antarctic soils. The phage named SpaA1 is morphologically similar to phages of the family Siphoviridae. The 42,784 bp genome of SpaA1 is a linear, double-stranded DNA molecule with 3' protruding cohesive ends. The SpaA1 genome encompasses 63 predicted protein-coding genes which cluster within three regions of the genome, each of apparently different origin, in a mosaic pattern. In two of these regions, the gene sets resemble those in prophages of Bacillus thuringiensis kurstaki str. T03a001 (genes involved in DNA replication/transcription, cell entry and exit) and B. cereus AH676 (additional regulatory and recombination genes), respectively. The third region represents an almost complete genome (except for the short terminal segments) of a distinct bacteriophage, MZTP02. Nearly the same gene module was identified in prophages of B. thuringiensis serovar monterrey BGSC 4AJ1 and B. cereus Rock4-2. These findings suggest that MZTP02 can be shuttled between genomes of other bacteriophages and prophages, leading to the formation of chimeric genomes. The presence of a complete phage genome in the genome of other phages apparently has not been described previously and might represent a 'fast track' route of virus evolution and horizontal gene transfer. Another phage (BceA1) nearly identical in sequence to SpaA1, and also including the almost complete MZTP02 genome within its own genome, was isolated from a bacterium of the B. cereus/B. thuringiensis group. Remarkably, both SpaA1 and BceA1 phages can infect B. cereus and B. thuringiensis, but only one of them, SpaA1, can infect S. pasteuri. This finding is best compatible with a scenario in which MZTP02 was originally contained in BceA1 infecting Bacillus spp, the common hosts for these two phages, followed by emergence of SpaA1 infecting S. pasteuri.

Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains.

PubMed

Urban-Chmiel, Renata; Wernicki, Andrzej; Stęgierska, Diana; Dec, Marta; Dudzic, Anna; Puchalski, Andrzej

2015-01-01

The objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves. The material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC®) BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR). Eight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101. The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages

[Determination of Azospirillum Brasilense Cells With Bacteriophages via Electrooptical Analysis of Microbial Suspensions].

PubMed

Gulii, O I; Karavayeva, O A; Pavlii, S A; Sokolov, O I; Bunin, V D; Ignatov, O V

2015-01-01

The dependence-of changes in the electrooptical properties of Azospirillum brasilense cell suspension Sp7 during interaction with bacteriophage ΦAb-Sp7 on the number and time of interactions was studied. Incubation of cells with bacteriophage significantly changed the electrooptical signal within one minute. The selective effect of bacteriophage ΦAb on 18 strains of bacteria of the genus Azospirillum was studied: A. amazonense Ami4, A. brasilense Sp7, Cd, Sp107, Sp245, Jm6B2, Brl4, KR77, S17, S27, SR55, SR75, A. halopraeferans Au4, A. irakense KBC1, K A3, A. lipoferum Sp59b, SR65 and RG20a. We determined the limit of reliable determination of microbial cells infected with bacteriophage: - 10(4) cells/mL. The presence of foreign cell cultures of E. coli B-878 and E. coli XL-1 did not complicate the detection of A brasilense Sp7 cells with the use of bacteriophage ΦAb-Sp7. The results demonstrated that bacteriophage (ΦAb-Sp7 can be used for the detection of Azospirillum microbial cells via t electrooptical analysis of cell suspensions.

Discovery of 2-Pyridinone Aminals: A Prodrug Strategy to Advance a Second Generation of HIV-1 Integrase Strand Transfer Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raheem, Izzat T.; Walji, Abbas M.; Klein, Daniel

The search for new molecular constructs that resemble the critical two-metal binding pharmacophore required for HIV integrase strand transfer inhibition represents a vibrant area of research within drug discovery. Here we present the discovery of a new class of HIV integrase strand transfer inhibitors based on the 2-pyridinone core of MK-0536. These efforts led to the identification of two lead compounds with excellent antiviral activity and preclinical pharmacokinetic profiles to support a once-daily human dose prediction. Dose escalating PK studies in dog revealed significant issues with limited oral absorption and required an innovative prodrug strategy to enhance the high-dose plasmamore » exposures of the parent molecules.« less

The Genome Sequence of Bacteriophage CPV1 Virulent for Clostridium perfringens

USDA-ARS?s Scientific Manuscript database

Application of bacteriophages and their lytic enzymes to control Clostridium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. Bacteriophages lytic for C. perfringens were isolated from sewage, feces and broiler intestinal contents. P...

M13 Bacteriophage-Based Self-Assembly Structures and Their Functional Capabilities.

PubMed

Moon, Jong-Sik; Kim, Won-Geun; Kim, Chuntae; Park, Geun-Tae; Heo, Jeong; Yoo, So Y; Oh, Jin-Woo

2015-06-01

Controlling the assembly of basic structural building blocks in a systematic and orderly fashion is an emerging issue in various areas of science and engineering such as physics, chemistry, material science, biological engineering, and electrical engineering. The self-assembly technique, among many other kinds of ordering techniques, has several unique advantages and the M13 bacteriophage can be utilized as part of this technique. The M13 bacteriophage (Phage) can easily be modified genetically and chemically to demonstrate specific functions. This allows for its use as a template to determine the homogeneous distribution and percolated network structures of inorganic nanostructures under ambient conditions. Inexpensive and environmentally friendly synthesis can be achieved by using the M13 bacteriophage as a novel functional building block. Here, we discuss recent advances in the application of M13 bacteriophage self-assembly structures and the future of this technology.

M13 Bacteriophage-Based Self-Assembly Structures and Their Functional Capabilities

PubMed Central

Moon, Jong-Sik; Kim, Won-Geun; Kim, Chuntae; Park, Geun-Tae; Heo, Jeong; Yoo, So Y; Oh, Jin-Woo

2015-01-01

Controlling the assembly of basic structural building blocks in a systematic and orderly fashion is an emerging issue in various areas of science and engineering such as physics, chemistry, material science, biological engineering, and electrical engineering. The self-assembly technique, among many other kinds of ordering techniques, has several unique advantages and the M13 bacteriophage can be utilized as part of this technique. The M13 bacteriophage (Phage) can easily be modified genetically and chemically to demonstrate specific functions. This allows for its use as a template to determine the homogeneous distribution and percolated network structures of inorganic nanostructures under ambient conditions. Inexpensive and environmentally friendly synthesis can be achieved by using the M13 bacteriophage as a novel functional building block. Here, we discuss recent advances in the application of M13 bacteriophage self-assembly structures and the future of this technology. PMID:26146494

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site | Center for Cancer Research

Cancer.gov

The lowest energy-binding conformation of an inhibitor bound to the dimeric interface of HIV-1 integrase core domain. The yellow region represents a unique allosteric binding site identified by affinity labeling and mass spectrometry and validated through mutagenesis. This site can provide a potential platform for the rational design of inhibitors selective for disruption of

Discovery of Novel HIV-1 Integrase Inhibitors Using QSAR-Based Virtual Screening of the NCI Open Database.

PubMed

Ko, Gene M; Garg, Rajni; Bailey, Barbara A; Kumar, Sunil

2016-01-01

Quantitative structure-activity relationship (QSAR) models can be used as a predictive tool for virtual screening of chemical libraries to identify novel drug candidates. The aims of this paper were to report the results of a study performed for descriptor selection, QSAR model development, and virtual screening for identifying novel HIV-1 integrase inhibitor drug candidates. First, three evolutionary algorithms were compared for descriptor selection: differential evolution-binary particle swarm optimization (DE-BPSO), binary particle swarm optimization, and genetic algorithms. Next, three QSAR models were developed from an ensemble of multiple linear regression, partial least squares, and extremely randomized trees models. A comparison of the performances of three evolutionary algorithms showed that DE-BPSO has a significant improvement over the other two algorithms. QSAR models developed in this study were used in consensus as a predictive tool for virtual screening of the NCI Open Database containing 265,242 compounds to identify potential novel HIV-1 integrase inhibitors. Six compounds were predicted to be highly active (plC50 > 6) by each of the three models. The use of a hybrid evolutionary algorithm (DE-BPSO) for descriptor selection and QSAR model development in drug design is a novel approach. Consensus modeling may provide better predictivity by taking into account a broader range of chemical properties within the data set conducive for inhibition that may be missed by an individual model. The six compounds identified provide novel drug candidate leads in the design of next generation HIV- 1 integrase inhibitors targeting drug resistant mutant viruses.

Application of bacteriophages to reduce Salmonella contamination on workers' boots in rendering-processing environment.

PubMed

Gong, C; Jiang, X; Wang, J

2017-10-01

Workers' boots are considered one of the re-contamination routes of Salmonella for rendered meals in the rendering-processing environment. This study was conducted to evaluate the efficacy of a bacteriophage cocktail for reducing Salmonella on workers' boots and ultimately for preventing Salmonella re-contamination of rendered meals. Under laboratory conditions, biofilms of Salmonella Typhimurium avirulent strain 8243 formed on rubber templates or boots were treated with a bacteriophage cocktail of 6 strains (ca. 9 log PFU/mL) for 6 h at room temperature. Bacteriophage treatments combined with sodium hypochlorite (400 ppm) or 30-second brush scrubbing also were investigated for a synergistic effect on reducing Salmonella biofilms. Sodium magnesium (SM) buffer and sodium hypochlorite (400 ppm) were used as controls. To reduce indigenous Salmonella on workers' boots, a field study was conducted to apply a bacteriophage cocktail and other combined treatments 3 times within one wk in a rendering-processing environment. Prior to and after bacteriophage treatments, Salmonella populations on the soles of rubber boots were swabbed and enumerated on XLT-4, Miller-Mallinson or CHROMagar™ plates. Under laboratory conditions, Salmonella biofilms formed on rubber templates and boots were reduced by 95.1 to 99.999% and 91.5 to 99.2%, respectively. In a rendering-processing environment (ave. temperature: 19.3°C; ave. relative humidity: 48%), indigenous Salmonella populations on workers' boots were reduced by 84.2, 92.9, and 93.2% after being treated with bacteriophages alone, bacteriophages + sodium hypochlorite, and bacteriophages + scrubbing for one wk, respectively. Our results demonstrated the effectiveness of bacteriophage treatments in reducing Salmonella contamination on the boots in both laboratory and the rendering-processing environment. © 2017 Poultry Science Association Inc.

Bacteriophage ecology in environmental biotechnology processes.

PubMed

Shapiro, Orr H; Kushmaro, Ariel

2011-06-01

Heterotrophic bacteria are an integral part of any environmental biotechnology process (EBP). Therefore, factors controlling bacterial abundance, activity, and community composition are central to the understanding of such processes. Among these factors, top-down control by bacteriophage predation has so far received very limited attention. With over 10(8) particles per ml, phage appear to be the most numerous biological entities in EBP. Phage populations in EBP appear to be highly dynamic and to correlate with the population dynamics of their hosts and genomic evidence suggests bacteria evolve to avoid phage predation. Clearly, there is much to learn regarding bacteriophage in EBP before we can truly understand the microbial ecology of these globally important systems. Copyright © 2011 Elsevier Ltd. All rights reserved.

Natural mummification of the human gut preserves bacteriophage DNA.

PubMed

Santiago-Rodriguez, Tasha M; Fornaciari, Gino; Luciani, Stefania; Dowd, Scot E; Toranzos, Gary A; Marota, Isolina; Cano, Raul J

2016-01-01

The natural mummification process of the human gut represents a unique opportunity to study the resulting microbial community structure and composition. While results are providing insights into the preservation of bacteria, fungi, pathogenic eukaryotes and eukaryotic viruses, no studies have demonstrated that the process of natural mummification also results in the preservation of bacteriophage DNA. We characterized the gut microbiome of three pre-Columbian Andean mummies, namely FI3, FI9 and FI12, and found sequences homologous to viruses. From the sequences attributable to viruses, 50.4% (mummy FI3), 1.0% (mummy FI9) and 84.4% (mummy FI12) were homologous to bacteriophages. Sequences corresponding to the Siphoviridae, Myoviridae, Podoviridae and Microviridae families were identified. Predicted putative bacterial hosts corresponded mainly to the Firmicutes and Proteobacteria, and included Bacillus, Staphylococcus, Clostridium, Escherichia, Vibrio, Klebsiella, Pseudomonas and Yersinia. Predicted functional categories associated with bacteriophages showed a representation of structural, replication, integration and entry and lysis genes. The present study suggests that the natural mummification of the human gut results in the preservation of bacteriophage DNA, representing an opportunity to elucidate the ancient phageome and to hypothesize possible mechanisms of preservation. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Comparison of Polymerase Subunits from Double-Stranded RNA Bacteriophages

PubMed Central

Yang, Hongyan; Makeyev, Eugene V.; Bamford, Dennis H.

2001-01-01

The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage φ6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3′ termini from the φ6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from φ6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the φ6-related bacteriophages φ8 and φ13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from φ6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3′-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription in Cystoviridae is controlled by the template specificity of the polymerase subunit. PMID:11602748

40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

Code of Federal Regulations, 2014 CFR

2014-07-01

... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3, 2009] ...

40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

Code of Federal Regulations, 2012 CFR

2012-07-01

... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3, 2009] ...

40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

Code of Federal Regulations, 2011 CFR

2011-07-01

... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3, 2009] ...

40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

Code of Federal Regulations, 2013 CFR

2013-07-01

... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3, 2009] ...

40 CFR 180.1261 - Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages.

Code of Federal Regulations, 2010 CFR

2010-07-01

... and Pseudomonas syringae pv. tomato specific Bacteriophages. 180.1261 Section 180.1261 Protection of.... vesicatoria and Pseudomonas syringae pv. tomato specific Bacteriophages. An exemption from the requirement of... syringae pv. tomato specific bacteriophages in or on pepper and tomato. [74 FR 26536, June 3, 2009] ...

Development of a bacteriophage displayed peptide library and biosensor

NASA Astrophysics Data System (ADS)

Chin, Robert C.; Salazar, Noe; Mayo, Michael W.; Villavicencio, Victor I.; Taylor, Richard B.; Chambers, James P.; Valdes, James J.

1996-04-01

A miniaturized, handheld biosensor for identification of hazardous biowarfare agents with high specificity is being developed. An innovative biological recognition system based on bacteriophage displayed peptide receptors will be utilized in conjunction with the miniature biosensor technology being developed. A bacteriophage library has been constructed to provide the artificial receptors. The library can contain millions of bacteriophage with randomly displayed peptide sequences in the phage outer protein coat which act as binding sites for the agents of interest. This library will be used to 'bio-pan' for phages that bind to a number of toxins and infectious agents and can, thus, provide an endless supply of low cost, reliable, specific, and stable artificial receptors. The biosensor instrument will utilize evanescent wave, planar waveguide, far-red dyes, diode laser and miniature circuit technologies for performance and portability.

Bacteriophage T5 DNA ejection under pressure.

PubMed

Leforestier, A; Brasilès, S; de Frutos, M; Raspaud, E; Letellier, L; Tavares, P; Livolant, F

2008-12-19

The transfer of the bacteriophage genome from the capsid into the host cell is a key step of the infectious process. In bacteriophage T5, DNA ejection can be triggered in vitro by simple binding of the phage to its purified Escherichia coli receptor FhuA. Using electrophoresis and cryo-electron microscopy, we measure the extent of DNA ejection as a function of the external osmotic pressure. In the high pressure range (7-16 atm), the amount of DNA ejected decreases with increasing pressure, as theoretically predicted and observed for lambda and SPP1 bacteriophages. In the low and moderate pressure range (2-7 atm), T5 exhibits an unexpected behavior. Instead of a unique ejected length, multiple populations coexist. Some phages eject their complete genome, whereas others stop at some nonrandom states that do not depend on the applied pressure. We show that contrarily to what is observed for the phages SPP1 and lambda, T5 ejection cannot be explained as resulting from a simple pressure equilibrium between the inside and outside of the capsid. Kinetics parameters and/or structural characteristics of the ejection machinery could play a determinant role in T5 DNA ejection.

Multiple roles of genome-attached bacteriophage terminal proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Redrejo-Rodríguez, Modesto; Salas, Margarita, E-mail: msalas@cbm.csic.es

2014-11-15

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid.more » Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer. - Highlights: • Protein-primed genome replication constitutes a strategy to initiate DNA or RNA synthesis in linear genomes. • Bacteriophage terminal proteins (TPs) are covalently attached to viral genomes by their primary function priming DNA replication. • TPs are also DNA-binding proteins and target phage genomes to the host nucleoid. • TPs can also localize in the eukaryotic nucleus and may have a role in phage-mediated interkingdom gene transfer.« less

Isolation of Dickeya dadantii strains from potato disease and biocontrol by their bacteriophages.

PubMed

Soleimani-Delfan, Abbas; Etemadifar, Zahra; Emtiazi, Giti; Bouzari, Majid

2015-01-01

One of the most economically important bacterial pathogens of plants and plant products is Dickeya dadantii. This bacterium causes soft rot disease in tubers and other parts of the potato and other plants of the Solanaceae family. The application of restricted host range bacteriophages as biocontrol agents has recently gained widespread interest. This study purposed to isolate the infectious agent of the potato and evaluate its biocontrol by bacteriophages. Two phytopathogenic strains were isolated from infected potatoes, identified based on biochemical and 16S rRNA gene sequencing, and submitted to GenBank as D. dadantii strain pis3 (accession no. HQ423668) and D. dadantii strain sip4 (accession no. HQ423669). Their bacteriophages were isolated from Caspian Sea water by enriching the water filtrate with D. dadantii strains as hosts using spot or overlay methods. On the basis of morphotypes, the isolated bacteriophages were identified as members of the Myoviridae and Siphoviridae families and could inhibit the growth of antibiotic resistant D. dadantii strains in culture medium. Moreover, in Dickeya infected plants treated with bacteriophage, no disease progression was detected. No significant difference was seen between phage-treated and control plants. Thus, isolated bacteriophages can be suggested for the biocontrol of plant disease caused by Dickeya strains.

Genetically engineered acidophilic heterotrophic bacteria by bacteriophage transduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, T.E.; Bruhn, D.F.; Bulmer, D.F.

1989-05-10

A bacteriophage capable of infecting acidophilic heterotrophic bacteria and processes for genetically engineering acidophilic bacteria for biomining or sulfur removal from coal are disclosed. The bacteriophage is capable of growth in cells existing at pH at or below 3.0. Lytic forms of the phage introduced into areas experiencing acid drainage kill the bacteria causing such drainage. Lysogenic forms of the phage having genes for selective removal of metallic or nonmetallic elements can be introduced into acidophilic bacteria to effect removal of the desired element from ore or coal. 1 fig., 1 tab.

Analysis of the site-specific integration system of the Streptomyces aureofaciens phage μ1/6.

PubMed

Farkašovská, Jarmila; Godány, Andrej

2012-03-01

The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3' end of a putative tRNA(Thr) gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.

Antigenic properties of HCMV peptides displayed by filamentous bacteriophages vs. synthetic peptides.

PubMed

Ulivieri, Cristina; Citro, Alessandra; Ivaldi, Federico; Mascolo, Dina; Ghittoni, Raffaella; Fanigliulo, Daniela; Manca, Fabrizio; Baldari, Cosima Tatiana; Li Pira, Giuseppina; Del Pozzo, Giovanna

2008-08-15

Several efforts have been invested in the identification of CTL and Th epitopes, as well as in the characterization of their immunodominance and MHC restriction, for the generation of a peptide-based HCMV vaccine. Small synthetic peptides are, however, poor antigens and carrier proteins are important for improving the efficacy of synthetic peptide vaccines. Recombinant bacteriophages appear as promising tools in the design of subunit vaccines. To investigate the antigenicity of peptides carried by recombinant bacteriophages we displayed different HCMV MHCII restricted peptides on the capsid of filamentous bacteriophage (fd) and found that hybrid bacteriophages are processed by human APC and activate HCMV-specific CD4 T-cells. Furthermore we constructed a reporter T-cell hybridoma expressing a chimeric TCR comprising murine alphabeta constant regions and human variable regions specific for the HLA-A2 restricted immunodominant NLV peptide of HCMV. Using the filamentous bacteriophage as an epitope carrier, we detected a more robust and long lasting response of the reporter T-cell hybridoma compared to peptide stimulation. Our results show a general enhancement of T-cell responses when antigenic peptides are carried by phages.

Removal of MS2, Qβ and GA bacteriophages during drinking water treatment at pilot scale.

PubMed

Boudaud, Nicolas; Machinal, Claire; David, Fabienne; Fréval-Le Bourdonnec, Armelle; Jossent, Jérôme; Bakanga, Fanny; Arnal, Charlotte; Jaffrezic, Marie Pierre; Oberti, Sandrine; Gantzer, Christophe

2012-05-15

The removal of MS2, Qβ and GA, F-specific RNA bacteriophages, potential surrogates for pathogenic waterborne viruses, was investigated during a conventional drinking water treatment at pilot scale by using river water, artificially and independently spiked with these bacteriophages. The objective of this work is to develop a standard system for assessing the effectiveness of drinking water plants with respect to the removal of MS2, Qβ and GA bacteriophages by a conventional pre-treatment process (coagulation-flocculation-settling-sand filtration) followed or not by an ultrafiltration (UF) membrane (complete treatment process). The specific performances of three UF membranes alone were assessed by using (i) pre-treated water and (ii) 0.1 mM sterile phosphate buffer solution (PBS), spiked with bacteriophages. These UF membranes tested in this work were designed for drinking water treatment market and were also selected for research purpose. The hypothesis serving as base for this study was that the interfacial properties for these three bacteriophages, in terms of electrostatic charge and the degree of hydrophobicity, could induce variations in the removal performances achieved by drinking water treatments. The comparison of the results showed a similar behaviour for both MS2 and Qβ surrogates whereas it was particularly atypical for the GA surrogate. The infectious character of MS2 and Qβ bacteriophages was mostly removed after clarification followed by sand filtration processes (more than a 4.8-log reduction) while genomic copies were removed at more than a 4.0-log after the complete treatment process. On the contrary, GA bacteriophage was only slightly removed by clarification followed by sand filtration, with less than 1.7-log and 1.2-log reduction, respectively. After the complete treatment process achieved, GA bacteriophage was removed with less than 2.2-log and 1.6-log reduction, respectively. The effectiveness of the three UF membranes tested in terms of

Molecular dynamics simulation studies of the wild type and E92Q/N155H mutant of Elvitegravir-resistance HIV-1 integrase.

PubMed

Chen, Qi; Cheng, Xiaolin; Wei, Dongqing; Xu, Qin

2015-03-01

Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we herein use the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was used to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.

Membrane filtration immobilization technique-a simple and novel method for primary isolation and enrichment of bacteriophages.

PubMed

Ghugare, G S; Nair, A; Nimkande, V; Sarode, P; Rangari, P; Khairnar, K

2017-02-01

To develop a method for the isolation and enrichment of bacteriophages selectively against specific bacteria coupled with a membrane filtration technique. Rapid isolation and concentration of host-specific bacteriophages was achieved by exposure of the sample suspected to contain bacteriophages to a specific host immobilized on a 0·45 μm membrane in a membrane filtration unit. The principle behind this method is the exploitation of host-specific interaction of bacteriophages with their host and maximizing this interaction using a classic membrane filtration method. This provides a chance for each bacteriophage in the sample to interact with the specific host on the membrane filter fitted with a vacuum pump. Specific bacteriophages of the host are retained on the membrane along with its host cells due to the effect of adsorption and these adsorbed bacteriophages (along with their hosts) on the filter disc are then amplified and enriched in regular nutritive broth tryptose soya broth by incubation. With the help of the plaque assay method, host-specific phages of various bacterial species were isolated, segregated and enriched. The phage concentration method coupled with membrane filtration immobilization of host bacteria was able to isolate and enrich the host-specific bacteriophages by several fold using a lower quantity of an environmental water sample, or other phage suspensions. Enrichment of phages from single plaques was also achieved. The isolation and detection of host-specific bacteriophages from a low density bacteriophage water sample in a single step by the use of a simple and basic microbiological technique can be achieved. Enrichment of phages from low phage titre suspensions is also achieved very effectively. © 2016 The Society for Applied Microbiology.

Bacteriophage-Based Bacterial Wilt Biocontrol for an Environmentally Sustainable Agriculture

PubMed Central

Álvarez, Belén; Biosca, Elena G.

2017-01-01

Bacterial wilt diseases caused by Ralstonia solanacearum, R. pseudosolanacearum, and R. syzygii subsp. indonesiensis (former R. solanacearum species complex) are among the most important plant diseases worldwide, severely affecting a high number of crops and ornamentals. Difficulties of bacterial wilt control by non-biological methods are related to effectiveness, bacterial resistance and environmental impact. Alternatively, a great many biocontrol strategies have been carried out, with the advantage of being environmentally friendly. Advances in bacterial wilt biocontrol include an increasing interest in bacteriophage-based treatments as a promising re-emerging strategy. Bacteriophages against the bacterial wilt pathogens have been described with either lytic or lysogenic effect but, they were proved to be active against strains belonging to R. pseudosolanacearum and/or R. syzygii subsp. indonesiensis, not to the present R. solanacearum species, and only two of them demonstrated successful biocontrol potential in planta. Despite the publication of three patents on the topic, until now no bacteriophage-based product is commercially available. Therefore, there is still much to be done to incorporate valid bacteriophages in an integrated management program to effectively fight bacterial wilt in the field. PMID:28769942

Bacteriophage-Based Bacterial Wilt Biocontrol for an Environmentally Sustainable Agriculture.

PubMed

Álvarez, Belén; Biosca, Elena G

2017-01-01

Bacterial wilt diseases caused by Ralstonia solanacearum , R. pseudosolanacearum , and R. syzygii subsp. indonesiensis (former R. solanacearum species complex) are among the most important plant diseases worldwide, severely affecting a high number of crops and ornamentals. Difficulties of bacterial wilt control by non-biological methods are related to effectiveness, bacterial resistance and environmental impact. Alternatively, a great many biocontrol strategies have been carried out, with the advantage of being environmentally friendly. Advances in bacterial wilt biocontrol include an increasing interest in bacteriophage-based treatments as a promising re-emerging strategy. Bacteriophages against the bacterial wilt pathogens have been described with either lytic or lysogenic effect but, they were proved to be active against strains belonging to R. pseudosolanacearum and/or R. syzygii subsp. indonesiensis , not to the present R. solanacearum species, and only two of them demonstrated successful biocontrol potential in planta . Despite the publication of three patents on the topic, until now no bacteriophage-based product is commercially available. Therefore, there is still much to be done to incorporate valid bacteriophages in an integrated management program to effectively fight bacterial wilt in the field.

Self-Assembled Nanoporous Biofilms from Functionalized Nanofibrous M13 Bacteriophage.

PubMed

Devaraj, Vasanthan; Han, Jiye; Kim, Chuntae; Kang, Yong-Cheol; Oh, Jin-Woo

2018-06-12

Highly periodic and uniform nanostructures, based on a genetically engineered M13 bacteriophage, displayed unique properties at the nanoscale that have the potential for a variety of applications. In this work, we report a multilayer biofilm with self-assembled nanoporous surfaces involving a nanofiber-like genetically engineered 4E-type M13 bacteriophage, which was fabricated using a simple pulling method. The nanoporous surfaces were effectively formed by using the networking-like structural layers of the M13 bacteriophage during self-assembly. Therefore, an external template was not required. The actual M13 bacteriophage-based fabricated multilayered biofilm with porous nanostructures agreed well with experimental and simulation results. Pores formed in the final layer had a diameter of about 150⁻500 nm and a depth of about 15⁻30 nm. We outline a filter application for this multilayered biofilm that enables selected ions to be extracted from a sodium chloride solution. Here, we describe a simple, environmentally friendly, and inexpensive fabrication approach with large-scale production potential. The technique and the multi-layered biofilms produced may be applied to sensor, filter, plasmonics, and bio-mimetic fields.

Ultrastructure and Viral Metagenome of Bacteriophages from an Anaerobic Methane Oxidizing Methylomirabilis Bioreactor Enrichment Culture

PubMed Central

Gambelli, Lavinia; Cremers, Geert; Mesman, Rob; Guerrero, Simon; Dutilh, Bas E.; Jetten, Mike S. M.; Op den Camp, Huub J. M.; van Niftrik, Laura

2016-01-01

With its capacity for anaerobic methane oxidation and denitrification, the bacterium Methylomirabilis oxyfera plays an important role in natural ecosystems. Its unique physiology can be exploited for more sustainable wastewater treatment technologies. However, operational stability of full-scale bioreactors can experience setbacks due to, for example, bacteriophage blooms. By shaping microbial communities through mortality, horizontal gene transfer, and metabolic reprogramming, bacteriophages are important players in most ecosystems. Here, we analyzed an infected Methylomirabilis sp. bioreactor enrichment culture using (advanced) electron microscopy, viral metagenomics and bioinformatics. Electron micrographs revealed four different viral morphotypes, one of which was observed to infect Methylomirabilis cells. The infected cells contained densely packed ~55 nm icosahedral bacteriophage particles with a putative internal membrane. Various stages of virion assembly were observed. Moreover, during the bacteriophage replication, the host cytoplasmic membrane appeared extremely patchy, which suggests that the bacteriophages may use host bacterial lipids to build their own putative internal membrane. The viral metagenome contained 1.87 million base pairs of assembled viral sequences, from which five putative complete viral genomes were assembled and manually annotated. Using bioinformatics analyses, we could not identify which viral genome belonged to the Methylomirabilis- infecting bacteriophage, in part because the obtained viral genome sequences were novel and unique to this reactor system. Taken together these results show that new bacteriophages can be detected in anaerobic cultivation systems and that the effect of bacteriophages on the microbial community in these systems is a topic for further study. PMID:27877158

Isolation of bacteriophages from air using vacuum filtration technique: an improved and novel method.

PubMed

Magare, B; Nair, A; Khairnar, K

2017-10-01

Development of a simple and economical air sampler for isolation and enrichment of bacteriophages from air samples. A vacuum filtration unit with simple modifications was used for isolation of bacteriophages from air sampled in the lavatory. Air was sampled at the rate of 62 l min -1 by bubbling into Mcllvaine buffer for 30 min, which was used as bacteriophage solution for enrichment and plaque assessment against individual hosts. Alternatively, the aforementioned phage solution was enriched using a host consortium before plaque assessment. Phages were isolated in the range of 1-12 PFU per ml by the first method, whereas enrichment with host consortium gave phages around 10- to 1000-folds higher in number. Combining with established enrichment method, an improvement of about 10 times in phage isolation efficiency was attained. The method is very useful for studying the natural bacteriophages of air, requiring only a basic microbiological laboratory setup making it simple and economical. This study brings out a simple, economical air sampler for assessing air bacteriophages that can be employed by any microbial laboratory. Although various methods are available for studying bacteriophages in water and soil, very limited are available for air. To the best of our knowledge, the method developed in this study is unique in its design and concept for studying bacteriophages in air. The sampler is sterilizable by autoclaving and maintains a healthy rate of airflow provided by conventional vacuum pumps. The use of a nonspecific 'trapping solution' allows for the qualitative and quantitative study of air bacteriophages. © 2017 The Society for Applied Microbiology.

Isolation and characterization of bacteriophages specific to hydrogen-sulfide-producing bacteria.

PubMed

Gong, Chao; Heringa, Spencer; Singh, Randhir; Kim, Jinkyung; Jiang, Xiuping

2013-01-01

The objectives of this study were to isolate and characterize bacteriophages specific to hydrogen-sulfide-producing bacteria (SPB) from raw animal materials, and to develop a SPB-specific bacteriophage cocktail for rendering application. Meat, chicken offal, and feather samples collected from local supermarkets and rendering processing plants were used to isolate SPB (n = 142). Bacteriophages (n = 52) specific to SPB were isolated and purified from the above samples using 18 of those isolated SPB strains as hosts. The host ranges of bacteriophages against 5 selected SPB strains (Escherichia coli, Citrobacter freundii, and Hafnia alvei) were determined. Electron microscopy observation of 9 phages selected for the phage cocktail revealed that 6 phages belonged to the family of Siphoviridae and 3 belonged to the Myoviridae family. Restriction enzyme digestion analysis with endonuclease DraI detected 6 distinguished patterns among the 9 phages. Phage treatment prevented the growth of SPB for up to 10 h with multiplicity of infection ratios of 1, 10, 100, and 1000 in tryptic soy broth at 30 °C, and extended the lag phase of SPB growth for 2 h at 22 °C with multiplicities of infection of 10, 100, and 1000. These results suggest that the selected bacteriophage cocktail has a high potential for phage application to control SPB in raw animal materials destined for the rendering process.

Bacteriophage-based therapy in cystic fibrosis-associated Pseudomonas aeruginosa infections: rationale and current status

PubMed Central

Hraiech, Sami; Brégeon, Fabienne; Rolain, Jean-Marc

2015-01-01

Pulmonary infections involving Pseudomonas aeruginosa are among the leading causes of the deterioration of the respiratory status of cystic fibrosis (CF) patients. The emergence of multidrug-resistant strains in such populations, favored by iterative antibiotic cures, has led to the urgent need for new therapies. Among them, bacteriophage-based therapies deserve a focus. One century of empiric use in the ex-USSR countries suggests that bacteriophages may have beneficial effects against a large range of bacterial infections. Interest in bacteriophages has recently renewed in Western countries, and the in vitro data available suggest that bacteriophage-based therapy may be of significant interest for the treatment of pulmonary infections in CF patients. Although the clinical data concerning this specific population are relatively scarce, the beginning of the first large randomized study evaluating bacteriophage-based therapy in burn infections suggests that the time has come to assess the effectiveness of this new therapy in CF P. aeruginosa pneumonia. Consequently, the aim of this review is, after a brief history, to summarize the evidence concerning bacteriophage efficacy against P. aeruginosa and, more specifically, the in vitro studies, animal models, and clinical trials targeting CF. PMID:26213462

Amplifying genetic logic gates.

PubMed

Bonnet, Jerome; Yin, Peter; Ortiz, Monica E; Subsoontorn, Pakpoom; Endy, Drew

2013-05-03

Organisms must process information encoded via developmental and environmental signals to survive and reproduce. Researchers have also engineered synthetic genetic logic to realize simpler, independent control of biological processes. We developed a three-terminal device architecture, termed the transcriptor, that uses bacteriophage serine integrases to control the flow of RNA polymerase along DNA. Integrase-mediated inversion or deletion of DNA encoding transcription terminators or a promoter modulates transcription rates. We realized permanent amplifying AND, NAND, OR, XOR, NOR, and XNOR gates actuated across common control signal ranges and sequential logic supporting autonomous cell-cell communication of DNA encoding distinct logic-gate states. The single-layer digital logic architecture developed here enables engineering of amplifying logic gates to control transcription rates within and across diverse organisms.

Docking studies on a new human immunodeficiency virus integrase-Mg-DNA complex: phenyl ring exploration and synthesis of 1H-benzylindole derivatives through fluorine substitutions.

PubMed

Ferro, Stefania; De Luca, Laura; Barreca, Maria Letizia; Iraci, Nunzio; De Grazia, Sara; Christ, Frauke; Witvrouw, Myriam; Debyser, Zeger; Chimirri, Alba

2009-01-22

A new model of HIV-1 integrase-Mg-DNA complex that is useful for docking experiments has been built. It was used to study the binding mode of integrase strand transfer inhibitor 1 (CHI-1043) and other fluorine analogues. Molecular modeling results prompted us to synthesize the designed derivatives which showed potent enzymatic inhibition at nanomolar concentration, high antiviral activity, and low toxicity. Microwave assisted organic synthesis (MAOS) was employed in several steps of the synthetic pathway, thus reducing reaction times and improving yields.

GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data

PubMed Central

Vazquez-Vilar, Marta; Quijano-Rubio, Alfredo; Fernandez-del-Carmen, Asun; Sarrion-Perdigones, Alejandro; Ochoa-Fernandez, Rocio; Ziarsolo, Peio

2017-01-01

Abstract Modular DNA assembly simplifies multigene engineering in Plant Synthetic Biology. Furthermore, the recent adoption of a common syntax to facilitate the exchange of plant DNA parts (phytobricks) is a promising strategy to speed up genetic engineering. Following this lead, here, we present a platform for plant biodesign that incorporates functional descriptions of phytobricks obtained under pre-defined experimental conditions, and systematically registers the resulting information as metadata for documentation. To facilitate the handling of functional descriptions, we developed a new version (v3.0) of the GoldenBraid (GB) webtool that integrates the experimental data and displays it in the form of datasheets. We report the use of the Luciferase/Renilla (Luc/Ren) transient agroinfiltration assay in Nicotiana benthamiana as a standard to estimate relative transcriptional activities conferred by regulatory phytobricks, and show the consistency and reproducibility of this method in the characterization of a synthetic phytobrick based on the CaMV35S promoter. Furthermore, we illustrate the potential for combinatorial optimization and incremental innovation of the GB3.0 platform in two separate examples, (i) the development of a collection of orthogonal transcriptional regulators based on phiC31 integrase and (ii) the design of a small genetic circuit that connects a glucocorticoid switch to a MYB/bHLH transcriptional activation module. PMID:28053117

Bacteriophage Procurement for Therapeutic Purposes

PubMed Central

Weber-Dąbrowska, Beata; Jończyk-Matysiak, Ewa; Żaczek, Maciej; Łobocka, Małgorzata; Łusiak-Szelachowska, Marzanna; Górski, Andrzej

2016-01-01

Bacteriophages (phages), discovered 100 years ago, are able to infect and destroy only bacterial cells. In the current crisis of antibiotic efficacy, phage therapy is considered as a supplementary or even alternative therapeutic approach. Evolution of multidrug-resistant and pandrug-resistant bacterial strains poses a real threat, so it is extremely important to have the possibility to isolate new phages for therapeutic purposes. Our phage laboratory and therapy center has extensive experience with phage isolation, characterization, and therapeutic application. In this article we present current progress in bacteriophages isolation and use for therapeutic purposes, our experience in this field and its practical implications for phage therapy. We attempt to summarize the state of the art: properties of phages, the methods for their isolation, criteria of phage selection for therapeutic purposes and limitations of their use. Perspectives for the use of genetically engineered phages to specifically target bacterial virulence-associated genes are also briefly presented. PMID:27570518

Assessment of drinking water quality using indicator bacteria and bacteriophages.

PubMed

Méndez, Javier; Audicana, Ana; Cancer, Mercedes; Isern, Anna; Llaneza, Julian; Moreno, Belén; Navarro, Mercedes; Tarancón, M Lluisa; Valero, Fernando; Ribas, Ferran; Jofre, Juan; Lucena, Francisco

2004-09-01

Bacterial indicators and bacteriophages suggested as potential indicators of water quality were determined by public laboratories in water from springs, household water wells, and rural and metropolitan water supplies in north-eastern Spain. Indicator bacteria were detected more frequently than bacteriophages in springs, household water wells and rural water supplies. In contrast, positive bacteriophage detections were more numerous than those of bacteria in metropolitan water supplies. Most of the metropolitan water supply samples containing indicators had concentrations of chlorine below 0.1 mg l(-1), their indicator loads resembling more closely those of rural water supplies than any other samples taken from metropolitan water supplies. The number of samples from metropolitan water supplies containing more than 0.1 mg l(-1) of chlorine that contained phages clearly outnumbered those containing indicator bacteria. Some association was observed between rainfall and the presence of indicators. Sediments from service reservoirs and water from dead ends in the distribution network of one of the metropolitan water supplies were also tested. Bacterial indicators and phages were detected in a higher percentage than in samples of tap water from the same network. Additionally, indicator bacteria were detected more frequently than bacteriophages in sediments of service reservoirs and water from dead end samples. We conclude that naturally occurring indicator bacteria and bacteriophages respond differently to chlorination and behave differently in drinking water distribution networks. Moreover, this study has shown that testing for the three groups of phages in routine laboratories is easy to implement and feasible without the requirement for additional material resources for the laboratories.

Virulent Bacteriophages Can Target O104:H4 Enteroaggregative Escherichia coli in the Mouse Intestine

PubMed Central

Maura, Damien; Galtier, Matthieu; Le Bouguénec, Chantal

2012-01-01

In vivo bacteriophage targeting of enteroaggregative Escherichia coli (EAEC) was assessed using a mouse intestinal model of colonization with the O104:H4 55989Str strain and a cocktail of three virulent bacteriophages. The colonization model was shown to mimic asymptomatic intestinal carriage found in humans. The addition of the cocktail to drinking water for 24 h strongly decreased ileal and weakly decreased fecal 55989Str concentrations in a dose-dependent manner. These decreases in ileal and fecal bacterial concentrations were only transient, since 55989Str concentrations returned to their original levels 3 days later. These transient decreases were independent of the mouse microbiota, as similar results were obtained with axenic mice. We studied the infectivity of each bacteriophage in the ileal and fecal environments and found that 55989Str bacteria in the mouse ileum were permissive to all three bacteriophages, whereas those in the feces were permissive to only one bacteriophage. Our results provide the first demonstration that bacterial permissivity to infection with virulent bacteriophages is not uniform throughout the gut; this highlights the need for a detailed characterization of the interactions between bacteria and bacteriophages in vivo for the further development of phage therapy targeting intestinal pathogens found in the gut of asymptomatic human carriers. PMID:23006754

The Effectiveness of Bacteriophages against Methicillin-Resistant Staphylococcus aureus ST398 Nasal Colonization in Pigs.

PubMed

Verstappen, Koen M; Tulinski, Pawel; Duim, Birgitta; Fluit, Ad C; Carney, Jennifer; van Nes, Arie; Wagenaar, Jaap A

2016-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is an important colonizer in animals and an opportunistic pathogen in humans. In humans, MRSA can cause infections that might be difficult to treat because of antimicrobial resistance. The use of bacteriophages has been suggested as a potential approach for the control of MRSA colonization to minimize the-often occupational-exposure of humans. The aim of this study was to assess the efficacy of bacteriophage treatment on porcine nasal colonization with MRSA in vitro, in vivo, and ex vivo. The effectiveness of a bacteriophage combination of phage K*710 and P68 was assessed in vitro by incubating them with MRSA V0608892/1 (ST398) measuring the OD600 hourly. To study the in vivo effect, bacteriophages were administered in a gel developed for human application, which contain 109 plaque-forming units (pfu)/mL (K and P68 in a 19.25:1 ratio) for 5 days to piglets (N = 8) that were experimentally colonized with the MRSA strain. Eight piglets experimentally colonized were used as a negative control. The MRSA strain was also used to colonize porcine nasal mucosa explants and bacteriophages were applied to assess the ex vivo efficacy of treatment. Bacteriophages were effective in vitro. In vivo, sixteen piglets were colonized with MRSA but the number of CFU recovered after the application of the bacteriophages in 8 piglets was not reduced compared to the control animals (approx. 105 CFU/swab). In the ex vivo model, 108 CFU were used to establish colonization with MRSA; a reduction of colonization was not observed after application of bacteriophages. However, application of mupirocin both in vivo and ex vivo resulted in a near eradication of MRSA. i) The MRSA strain was killed in the presence of the bacteriophages phage K*710 and P68 in vitro. ii) Bacteriophages did not reduce porcine nasal colonization in vivo or ex vivo. Physiological in vivo and ex vivo conditions may explain these observations. Efficacy in the ex vivo

The Effectiveness of Bacteriophages against Methicillin-Resistant Staphylococcus aureus ST398 Nasal Colonization in Pigs

PubMed Central

Duim, Birgitta; Fluit, Ad C; Carney, Jennifer; van Nes, Arie; Wagenaar, Jaap A

2016-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is an important colonizer in animals and an opportunistic pathogen in humans. In humans, MRSA can cause infections that might be difficult to treat because of antimicrobial resistance. The use of bacteriophages has been suggested as a potential approach for the control of MRSA colonization to minimize the—often occupational—exposure of humans. The aim of this study was to assess the efficacy of bacteriophage treatment on porcine nasal colonization with MRSA in vitro, in vivo, and ex vivo. The effectiveness of a bacteriophage combination of phage K*710 and P68 was assessed in vitro by incubating them with MRSA V0608892/1 (ST398) measuring the OD600 hourly. To study the in vivo effect, bacteriophages were administered in a gel developed for human application, which contain 109 plaque-forming units (pfu)/mL (K and P68 in a 19.25:1 ratio) for 5 days to piglets (N = 8) that were experimentally colonized with the MRSA strain. Eight piglets experimentally colonized were used as a negative control. The MRSA strain was also used to colonize porcine nasal mucosa explants and bacteriophages were applied to assess the ex vivo efficacy of treatment. Bacteriophages were effective in vitro. In vivo, sixteen piglets were colonized with MRSA but the number of CFU recovered after the application of the bacteriophages in 8 piglets was not reduced compared to the control animals (approx. 105 CFU/swab). In the ex vivo model, 108 CFU were used to establish colonization with MRSA; a reduction of colonization was not observed after application of bacteriophages. However, application of mupirocin both in vivo and ex vivo resulted in a near eradication of MRSA. In conclusion: i) The MRSA strain was killed in the presence of the bacteriophages phage K*710 and P68 in vitro. ii) Bacteriophages did not reduce porcine nasal colonization in vivo or ex vivo. Physiological in vivo and ex vivo conditions may explain these observations. Efficacy

Treatment of Highly Virulent Extraintestinal Pathogenic Escherichia coli Pneumonia With Bacteriophages.

PubMed

Dufour, Nicolas; Debarbieux, Laurent; Fromentin, Mélanie; Ricard, Jean-Damien

2015-06-01

To study the effect of bacteriophage treatment on highly virulent extraintestinal Escherichia coli pneumonia in mice and compare it with conventional antimicrobial treatment. Animal investigation. University research laboratory. Pathogen-free 8-week-old Balb/cJRj male mice. Two bacteriophages (536_P1 and 536_P7) were isolated from sewage using strain 536, a highly virulent extraintestinal E. coli. Their in vitro and in vivo efficacy against strain 536 and a ventilator-associated pneumonia E. coli were tested. The first group of mice were infected by intranasal instillation of bioluminescent strain 536 and received 536_P1 intranasally, ceftriaxone, or control. The second group of mice was infected with the ventilator-associated pneumonia strain and received 536_P7. Adaptation of 536_P7 to this clinical isolate was also evaluated in vitro and in vivo. In vivo efficacy of bacteriophage and antibiotic treatment were assessed by recording bioluminescence for short-time periods and by recording body weight and survival of mice for longer periods. Both treatments improved survival compared with control (100% vs 0%), and in vivo bioluminescence recordings showed a similar rapid decrease of emitted light, suggesting prompt bacterial clearance. The majority of mice infected by the ventilator-associated pneumonia strain were not rescued by treatment with 536_P7; however, in vitro adaptation of this bacteriophage toward the ventilator-associated pneumonia strain led to isolate a variant which significantly improved in vivo treatment efficacy (animal survival increased from 20% to 75%). Bacteriophage treatment was as effective as antibiotherapy to provide 100% survival rate in a lethal model of highly virulent E. coli pneumonia. Adaptation of a bacteriophage is a rapid solution to improve its efficacy toward specific strains. These results suggest that phage therapy could be a promising therapeutic strategy for ventilator-associated pneumonia.

Identification and Characterization of T5-Like Bacteriophages Representing Two Novel Subgroups from Food Products

PubMed Central

Sváb, Domonkos; Falgenhauer, Linda; Rohde, Manfred; Szabó, Judit; Chakraborty, Trinad; Tóth, István

2018-01-01

During recent years, interest in the use of bacteriophages as biocontrol agents against foodborne pathogens has increased, particularly for members of the family Enterobacteriaceae, with pathogenic Escherichia coli, Shigella, and Salmonella strains among them. Here, we report the isolation and characterisation of 12 novel T5-like bacteriophages from confiscated food samples. All bacterophages effectively lysed E. coli K-12 strains and were able to infect pathogenic E. coli strains representing enterohaemorrhagic (EHEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), and enteroinvasive (EIEC) pathotypes, Shigella dysenteriae, S. sonnei strains, as well as multidrug-resistant (MDR) E. coli and multiple strains representing different Salmonella enterica serovars. All the bacteriophages exhibited Siphoviridae morphology. Whole genome sequencing of the novel T5-like bacteriophages showed that they represent two distinct groups, with the genome-based grouping correlating to the different host spectra. As these bacteriophages are of food origin, their stability and lack of any virulence genes, as well as their broad and mutually complementary host spectrum makes these new T5-like bacteriophages valuable candidates for use as biocontrol agents against foodborne pathogenic enterobacteria. PMID:29487585

[Immunodetection of bacteriophages by a piezoelectric resonator with lateral electric field].

PubMed

Gulii, O I; Zaitsev, B D; Shikhabudinov, A M; Teplykh, A A; Borodina, I A; Pavlii, S A; Larionova, O S; Fomin, A S; Staroverov, S A; Dykman, L A; Ignatov, O V

2016-01-01

It has been demonstrated that electroacoustic analysis with polyclonal antibodies can be used for bacteriophage detection. The frequency dependences of the real and imaginary parts of electrical impedance of a resonator with a viral suspension with antibodies were shown to be essentially different from the dependences of a resonator with control viral suspension without antibodies. It was shown that ΦAl-Sp59b bacteriophages were detected with the use of antibodies in the presence of foreign virus particles. The ΦAl-Sp59b bacteriophage content in the analyzed suspension was ~1010–106 phages/mL; the time of analysis was no more than 5 min. The optimally informative parameter for obtaining reliable information was the change in the real or imaginary part of electrical impedance at a fixed frequency near the resonance upon the addition of specific antibodies to the analyzed suspension. It was demonstrated that the interaction between bacteriophages and antibodies can be recorded, offering good prospects for the development of a biological sensor for liquid-phase identification and virus detection.

MetaPhinder-Identifying Bacteriophage Sequences in Metagenomic Data Sets.

PubMed

Jurtz, Vanessa Isabell; Villarroel, Julia; Lund, Ole; Voldby Larsen, Mette; Nielsen, Morten

Bacteriophages are the most abundant biological entity on the planet, but at the same time do not account for much of the genetic material isolated from most environments due to their small genome sizes. They also show great genetic diversity and mosaic genomes making it challenging to analyze and understand them. Here we present MetaPhinder, a method to identify assembled genomic fragments (i.e.contigs) of phage origin in metagenomic data sets. The method is based on a comparison to a database of whole genome bacteriophage sequences, integrating hits to multiple genomes to accomodate for the mosaic genome structure of many bacteriophages. The method is demonstrated to out-perform both BLAST methods based on single hits and methods based on k-mer comparisons. MetaPhinder is available as a web service at the Center for Genomic Epidemiology https://cge.cbs.dtu.dk/services/MetaPhinder/, while the source code can be downloaded from https://bitbucket.org/genomicepidemiology/metaphinder or https://github.com/vanessajurtz/MetaPhinder.

Effects of bacteriophage on the quality and shelf life of Paralichthys olivaceus during chilled storage.

PubMed

Li, Meng; Lin, Hong; Khan, Muhammad Naseem; Wang, Jingxue; Kong, Linghong

2014-06-01

The microbiological spoilage of fishery foods is mainly due to specific spoilage organisms (SSOs), with Shewanella putrefaciens being the SSO of most chilled marine fish. Bacteriophages have shown excellent capability to control micro-organisms. The aim of this study was to determine a specific bacteriophage to prevent spoilage by reducing SSO (S. putrefaciens) levels in the marine fish Paralichthys olivaceus (olive flounder) under chilled storage. Chilled flounder fillets were inoculated with S. putrefaciens and treated with different concentrations of bacteriophage Spp001 ranging from 10(4) to 10(8) plaque-forming units (pfu) mL(-1) . Bacterial growth (including total viable count and SSO) of the bacteriophage-treated groups was significantly inhibited compared with that of the negative control group (P < 0.05). Sensory evaluation and biochemical parameters revealed that the bacteriophage could extend the shelf life of chilled flounder fillets (from <4 to 14 days) with good esthetic quality, even at low temperature (4 °C). Furthermore, bacteriophage concentrations of 10(6) and 10(8) pfu mL(-1) were more effective than the chemical preservative potassium sorbate (5 g L(-1) ). The bacteriophage Spp001 offered effective biocontrol of S. putrefaciens under chilled conditions, retaining the quality characteristics of spiked fish fillets, and thus could be a potential candidate for use in chilled fish fillet biopreservation. © 2013 Society of Chemical Industry.

Pulmonary Bacteriophage Therapy on Pseudomonas aeruginosa Cystic Fibrosis Strains: First Steps Towards Treatment and Prevention

PubMed Central

Morello, Eric; Saussereau, Emilie; Maura, Damien; Huerre, Michel; Touqui, Lhousseine; Debarbieux, Laurent

2011-01-01

Multidrug-resistant bacteria are the cause of an increasing number of deadly pulmonary infections. Because there is currently a paucity of novel antibiotics, phage therapy—the use of specific viruses that infect bacteria—is now more frequently being considered as a potential treatment for bacterial infections. Using a mouse lung-infection model caused by a multidrug resistant Pseudomonas aeruginosa mucoid strain isolated from a cystic fibrosis patient, we evaluated bacteriophage treatments. New bacteriophages were isolated from environmental samples and characterized. Bacteria and bacteriophages were applied intranasally to the immunocompetent mice. Survival was monitored and bronchoalveolar fluids were analysed. Quantification of bacteria, bacteriophages, pro-inflammatory and cytotoxicity markers, as well as histology and immunohistochemistry analyses were performed. A curative treatment (one single dose) administrated 2 h after the onset of the infection allowed over 95% survival. A four-day preventive treatment (one single dose) resulted in a 100% survival. All of the parameters measured correlated with the efficacy of both curative and preventive bacteriophage treatments. We also showed that in vitro optimization of a bacteriophage towards a clinical strain improved both its efficacy on in vivo treatments and its host range on a panel of 20 P. aeruginosa cystic fibrosis strains. This work provides an incentive to develop clinical studies on pulmonary bacteriophage therapy to combat multidrug-resistant lung infections. PMID:21347240

Genomic and proteomic characterization of SuMu, a Mu-like bacteriophage infecting Haemophilus parasuis

PubMed Central

2012-01-01

Background Haemophilus parasuis, the causative agent of Glässer’s disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer’s disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate. Results Characterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome�

Genomic and proteomic characterization of SuMu, a Mu-like bacteriophage infecting Haemophilus parasuis.

PubMed

Zehr, Emilie S; Tabatabai, Louisa B; Bayles, Darrell O

2012-07-23

Haemophilus parasuis, the causative agent of Glässer's disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer's disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate. Characterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome's G + C content was

Occurrence of bacteriophages infecting Aeromonas, Enterobacter, and Klebsiella in water and association with contamination sources in Thailand.

PubMed

Wangkahad, Bencharong; Bosup, Suchada; Mongkolsuk, Skorn; Sirikanchana, Kwanrawee

2015-06-01

The co-residence of bacteriophages and their bacterial hosts in humans, animals, and environmental sources directed the use of bacteriophages to track the origins of the pathogenic bacteria that can be found in contaminated water. The objective of this study was to enumerate bacteriophages of Aeromonas caviae (AecaKS148), Enterobacter sp. (EnspKS513), and Klebsiella pneumoniae (KlpnKS648) in water and evaluate their association with contamination sources (human vs. animals). Bacterial host strains were isolated from untreated wastewater in Bangkok, Thailand. A double-layer agar technique was used to detect bacteriophages. All three bacteriophages were detected in polluted canal samples, with likely contamination from human wastewater, whereas none was found in non-polluted river samples. AecaKS148 was found to be associated with human fecal sources, while EnspKS513 and KlpnKS648 seemed to be equally prevalent in both human and animal fecal sources. Both bacteriophages were also present in polluted canals that could receive contamination from other fecal sources or the environment. In conclusion, all three bacteriophages were successfully monitored in Bangkok, Thailand. This study provided an example of bacteriophages for potential use as source identifiers of pathogen contamination. The results from this study will assist in controlling sources of pathogen contamination, especially in developing countries.

An Ensemble Method to Distinguish Bacteriophage Virion from Non-Virion Proteins Based on Protein Sequence Characteristics.

PubMed

Zhang, Lina; Zhang, Chengjin; Gao, Rui; Yang, Runtao

2015-09-09

Bacteriophage virion proteins and non-virion proteins have distinct functions in biological processes, such as specificity determination for host bacteria, bacteriophage replication and transcription. Accurate identification of bacteriophage virion proteins from bacteriophage protein sequences is significant to understand the complex virulence mechanism in host bacteria and the influence of bacteriophages on the development of antibacterial drugs. In this study, an ensemble method for bacteriophage virion protein prediction from bacteriophage protein sequences is put forward with hybrid feature spaces incorporating CTD (composition, transition and distribution), bi-profile Bayes, PseAAC (pseudo-amino acid composition) and PSSM (position-specific scoring matrix). When performing on the training dataset 10-fold cross-validation, the presented method achieves a satisfactory prediction result with a sensitivity of 0.870, a specificity of 0.830, an accuracy of 0.850 and Matthew's correlation coefficient (MCC) of 0.701, respectively. To evaluate the prediction performance objectively, an independent testing dataset is used to evaluate the proposed method. Encouragingly, our proposed method performs better than previous studies with a sensitivity of 0.853, a specificity of 0.815, an accuracy of 0.831 and MCC of 0.662 on the independent testing dataset. These results suggest that the proposed method can be a potential candidate for bacteriophage virion protein prediction, which may provide a useful tool to find novel antibacterial drugs and to understand the relationship between bacteriophage and host bacteria. For the convenience of the vast majority of experimental Int. J. Mol. Sci. 2015, 16,21735 scientists, a user-friendly and publicly-accessible web-server for the proposed ensemble method is established.

Interference with propagation of typing bacteriophages by extrachromosomal elements in Salmonella typhimurium: bacteriophage type 505.

PubMed Central

van Embden, J D; van Leeuwen, W J; Guinée, P A

1976-01-01

Samonella typhimurium bacteriophage type 505 is the most frequently encountered phage type in the Netherlands and its neighboring countries. Phage type 505 was analyzed with regard o the interference with propagation of the typing phages by the prophages and plasmids, present in the type strain S. typhimurium 505... Images PMID:783145

76 FR 66187 - Bacteriophage of Clavibacter Michiganensis Subspecies Michiganensis; Exemption From the...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-26

... bacteria, which means they attach to, infect, and reproduce in bacteria, and are host-specific for bacteria... bacteria. In addition, there is no evidence for bacteriophage infecting any other life form, including humans, except bacteria (Refs. 7, 12, and 13). Humans and other animals commonly consume bacteriophage as...

Molecular Biology and Biotechnology of Bacteriophage

NASA Astrophysics Data System (ADS)

Onodera, Kazukiyo

The development of the molecular biology of bacteriophage such as T4, lambda and filamentous phages was described and the process that the fundamental knowledge obtained in this field has subsequently led us to the technology of phage display was introduced.

Comparative persistence of human sewage-specific enterococcal bacteriophages in freshwater and seawater.

PubMed

Booncharoen, Namfon; Mongkolsuk, Skorn; Sirikanchana, Kwanrawee

2018-07-01

Enterococcus faecalis bacteria have been recently reported for their ability to host bacteriophages that are specifically from human sewage, suggesting their application to track human fecal contamination in water resources. However, little is known about the survivability of sewage-specific enterococcal bacteriophages in various water matrices under ambient and storage conditions. In this study, bacteriophages that were derived from the Thailand-isolated E. faecalis strains AIM06 and SR14 exhibited morphologies consistent with the Siphoviridae, Podoviridae, and Myoviridae families. Four representative bacteriophages were separately spiked into environmental water samples (n = 7) comprising freshwater and seawater with low- and high-pollution (LF, HF, LS, and HS, respectively) levels, defined according to Thailand Water Quality Standards. All bacteriophages decayed fastest in HS or HF samples at 30 °C, reaching a 5-log 10 reduction in 2.2 to 9.8 days, and slowest in LS samples, requiring 8.8 to 23.5 days. The decay rates were 5 to 53 times lower at a storage temperature of 5 °C. HF samples could be stored for as little as 2.5 days to prevent the decay of 50% of the phages. Myoviridae phages decayed faster than Siphoviridae phages and Podoviridae phages in most water matrices at 30 °C. Moreover, the decay rates were 1.8 to 92 times slower in filtered samples, emphasizing a strong role for water constituents, i.e., suspended solids and natural microorganisms, in phage persistence. This study emphasized that differential enterococcal bacteriophage persistence should be considered when planning the monitoring and interpreting of fecal sources by microbial source tracking.

Use of encapsulated bacteriophages to enhance farm to fork food safety.

PubMed

Hussain, Malik A; Liu, Huan; Wang, Qi; Zhong, Fang; Guo, Qian; Balamurugan, Sampathkumar

2017-09-02

Bacteriophages have been successfully applied to control the growth of pathogens in foods and to reduce the colonization and shedding of pathogens by food animals. They are set to play a dominant role in food safety in the future. However, many food-processing operations and the microenvironments in food animals' guts inactivate phages and reduce their infectivity. Encapsulation technologies have been used successfully to protect phages against extreme environments, and have been shown to preserve their activity and enable their release in targeted environments. A number of encapsulation technologies have shown potential for use with bacteriophages. This review discusses the current state of knowledge about the use of encapsulation technologies with bacteriophages to control pathogens in foods and food animals.

Occurrence and numbers of bacteriophages and bacterial indicators in faeces of yellow-legged seagull (Larus cachinnans).

PubMed

Muniesa, M; Jofre, J; Lucena, F

1999-12-01

Faeces from feral populations of yellow-legged seagulls from the northern coastal area of Catalonia (North-eastern Spain) contained variable amounts of faecal coliforms, faecal streptococci, somatic coliphages, F-specific bacteriophages and Bacteroides fragilis bacteriophages. Occurrence and numbers of bacterial indicators and bacteriophages in the faeces of yellow-legged seagulls are in the ranges described in the faeces of different animals. The ratios between numbers of bacterial indicators and numbers of bacteriophages are much higher in faeces of seagulls than in treated or raw sewage contributed by out-falls of the same area.

Small Molecule Inhibitors of Drug Resistant Forms of HIV-1 Integrase | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute discovered small-molecule compounds containing 1-hydroxy-2-oxo-1,8-naphthyridine moieties whose activity against HIV-1 integrase mutants confer resistance to currently approved INSTIs. Preliminary rodent efficacy, metabolic, and pharmacokinetic studies have been completed by the NCI researchers. The National Cancer Institute seeks partners to commercialize this class of compounds through licensing or co-development.

Methods for generation of reporter phages and immobilization of active bacteriophages on a polymer surface

NASA Technical Reports Server (NTRS)

Morgan, Mark Thomas (Inventor); Kothapalli, Aparna (Inventor); Applegate, Bruce Michael (Inventor); Perry, Lynda Louise (Inventor)

2012-01-01

Novel reporter bacteriophages are provided. Provided are compositions and methods that allow bacteriophages that are used for specific detection or killing of E. coli 0157:H7 to be propagated in nonpathogenic E. coli, thereby eliminating the safety and security risks of propagation in E. coli 0157:H7. Provided are compositions and methods for attaching active bacteriophages to the surface of a polymer in order to kill target bacteria with which the phage comes into contact. Provided are modified bacteriophages immobilized to a surface, which capture E. coli 0157:H7 and cause the captured cells to emit light or fluorescence, allowing detection of the bacteria in a sample.

Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry.

PubMed

Borrenberghs, Doortje; Dirix, Lieve; De Wit, Flore; Rocha, Susana; Blokken, Jolien; De Houwer, Stéphanie; Gijsbers, Rik; Christ, Frauke; Hofkens, Johan; Hendrix, Jelle; Debyser, Zeger

2016-11-10

Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies.

Bacteriophages as vehicles for gene delivery into mammalian cells: prospects and problems.

PubMed

Bakhshinejad, Babak; Sadeghizadeh, Majid

2014-10-01

The identification of more efficient gene delivery vehicles (GDVs) is essential to fulfill the expectations of clinical gene therapy. Bacteriophages, due to their excellent safety profile, extreme stability under a variety of harsh environmental conditions and the capability for being genetically manipulated, have drawn a flurry of interest to be applied as a newly arisen category of gene delivery platforms. The incessant evolutionary interaction of bacteriophages with human cells has turned them into a part of our body's natural ecosystem. However, these carriers represent several barriers to gene transduction of mammalian cells. The lack of evolvement of specialized machinery for targeted cellular internalization, endosomal, lysosomal and proteasomal escape, cytoplasmic entry, nuclear localization and intranuclear transcription poses major challenges to the expression of the phage-carried gene. In this review, we describe pros and cons of bacteriophages as GDVs, provide an insight into numerous barriers that bacteriophages face for entry into and subsequent trafficking inside mammalian cells and elaborate on the strategies used to bypass these barriers. Tremendous genetic flexibility of bacteriophages to undergo numerous surface modifications through phage display technology has proven to be a turning point in the uncompromising efforts to surmount the limitations of phage-mediated gene expression. The revelatory outcomes of the studies undertaken within the recent years have been promising for phage-mediated gene delivery to move from concept to reality.

QSAR study of curcumine derivatives as HIV-1 integrase inhibitors.

PubMed

Gupta, Pawan; Sharma, Anju; Garg, Prabha; Roy, Nilanjan

2013-03-01

A QSAR study was performed on curcumine derivatives as HIV-1 integrase inhibitors using multiple linear regression. The statistically significant model was developed with squared correlation coefficients (r(2)) 0.891 and cross validated r(2) (r(2) cv) 0.825. The developed model revealed that electronic, shape, size, geometry, substitution's information and hydrophilicity were important atomic properties for determining the inhibitory activity of these molecules. The model was also tested successfully for external validation (r(2) pred = 0.849) as well as Tropsha's test for model predictability. Furthermore, the domain analysis was carried out to evaluate the prediction reliability of external set molecules. The model was statistically robust and had good predictive power which can be successfully utilized for screening of new molecules.

ADSORPTION OF BACTERIOPHAGES ON CLAY MINERALS

EPA Science Inventory

Theability to predict the fate of microorganisms in soil is dependent on an understanding of the process of their sorption on soil and subsurface materials. Presently, we have focused on studying the thermodynamics of sorption of bacteriophages (T-2, MS-2, and

Soil-based systemic delivery and phyllosphere in vivo propagation of bacteriophages: Two possible strategies for improving bacteriophage persistence for plant disease control.

PubMed

Iriarte, Fanny B; Obradović, Aleksa; Wernsing, Mine H; Jackson, Lee E; Balogh, Botond; Hong, Jason A; Momol, M Timur; Jones, Jeffrey B; Vallad, Gary E

2012-10-01

Soil-based root applications and attenuated bacterial strains were evaluated as means to enhance bacteriophage persistence on plants for bacterial disease control. In addition, the systemic nature of phage applied to tomato roots was also evaluated. Several experiments were conducted applying either single phages or phage mixtures specific for Ralstonia solanacearum , Xanthomonas perforans or X. euvesicatoria to soil surrounding tomato plants and measuring the persistence and translocation of the phages over time. In general, all phages persisted in the roots of treated plants and were detected in stems and leaves; although phage level varied and persistence in stems and leaves was at a much lower level compared with persistence in roots. Bacterial wilt control was typically best if the phage or phage mixtures were applied to the soil surrounding tomatoes at the time of inoculation, less effective if applied 3 days before inoculation, and ineffective if applied 3 days after inoculation. The use of an attenuated X. perforans strain was also evaluated to improve the persistence of phage populations on tomato leaf surfaces. In greenhouse and field experiments, foliar applications of an attenuated mutant X. perforans 91-118:∆ OPGH strain prior to phage applications significantly improved phage persistence on tomato foliage compared with untreated tomato foliage. Both the soil-based bacteriophage delivery and the use of attenuated bacterial strains improved bacteriophage persistence on respective root and foliar tissues, with evidence of translocation with soil-based bacteriophage applications. Both strategies could lead to improved control of bacterial pathogens on plants.

Distinct Stabilities of the Structurally Homologous Heptameric Co-Chaperonins GroES and gp31

NASA Astrophysics Data System (ADS)

Dyachenko, Andrey; Tamara, Sem; Heck, Albert J. R.

2018-05-01

The GroES heptamer is the molecular co-chaperonin that partners with the tetradecamer chaperonin GroEL, which assists in the folding of various nonnative polypeptide chains in Escherichia coli. Gp31 is a structural and functional analogue of GroES encoded by the bacteriophage T4, becoming highly expressed in T4-infected E. coli, taking over the role of GroES, favoring the folding of bacteriophage proteins. Despite being slightly larger, gp31 is quite homologous to GroES in terms of its tertiary and quaternary structure, as well as in its function and mode of interaction with the chaperonin GroEL. Here, we performed a side-by-side comparison of GroES and gp31 heptamer complexes by (ion mobility) tandem mass spectrometry. Surprisingly, we observed quite distinct fragmentation mechanisms for the GroES and gp31 heptamers, whereby GroES displays a unique and unusual bimodal charge distribution in its released monomers. Not only the gas-phase dissociation but also the gas-phase unfolding of GroES and gp31 were found to be very distinct. We rationalize these observations with the similar discrepancies we observed in the thermal unfolding characteristics and surface contacts within GroES and gp31 in the solution. From our data, we propose a model that explains the observed simultaneous dissociation pathways of GroES and the differences between GroES and gp31 gas-phase dissociation and unfolding. We conclude that, although GroES and gp31 exhibit high homology in tertiary and quaternary structure, they are quite distinct in their solution and gas-phase (un)folding characteristics and stability. [Figure not available: see fulltext.

Use of a bacteriophage cocktail to control Salmonella in food and the food industry.

PubMed

Spricigo, Denis Augusto; Bardina, Carlota; Cortés, Pilar; Llagostera, Montserrat

2013-07-15

The use of lytic bacteriophages for the biocontrol of food-borne pathogens in food and in the food industry is gaining increasing acceptance. In this study, the effectiveness of a bacteriophage cocktail composed of three different lytic bacteriophages (UAB_Phi 20, UAB_Phi78, and UAB_Phi87) was determined in four different food matrices (pig skin, chicken breasts, fresh eggs, and packaged lettuce) experimentally contaminated with Salmonella enterica serovar Typhimurium and S. enterica serovar Enteritidis. A significant bacterial reduction (>4 and 2 log/cm(2) for S. Typhimurium and S. Enteritidis, respectively; p≤0.005) was obtained in pig skin sprayed with the bacteriophage cocktail and then incubated at 33 °C for 6h. Significant decreases in the concentration of S. Typhimurium and S. Enteritidis were also measured in chicken breasts dipped for 5 min in a solution containing the bacteriophage cocktail and then refrigerated at 4 °C for 7 days (2.2 and 0.9 log10 cfu/g, respectively; p≤0.0001) as well as in lettuce similarly treated for 60 min at room temperature (3.9 and 2.2 log10 cfu/g, respectively; p≤0.005). However, only a minor reduction of the bacterial concentration (0.9 log10 cfu/cm(2) of S. Enteritidis and S. Typhimurium; p≤0.005) was achieved in fresh eggs sprayed with the bacteriophage cocktail and then incubated at 25 °C for 2 h. These results show the potential effectiveness of this bacteriophage cocktail as a biocontrol agent of Salmonella in several food matrices under conditions similar to those used in their production. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization and formulation into solid dosage forms of a novel bacteriophage lytic against Klebsiella oxytoca.

PubMed

Brown, Teagan L; Petrovski, Steve; Hoyle, Dannielle; Chan, Hiu Tat; Lock, Peter; Tucci, Joseph

2017-01-01

To isolate and characterize bacteriophage lytic for the opportunistic pathogen Klebsiella oxytoca and their formulation into a range of solid dosage forms for in-vitro testing. We report the isolation, genomic and functional characterization of a novel bacteriophage lytic for Klebsiella oxytoca, which does not infect the closely related Klebsiella pneumoniae. This bacteriophage was formulated into suppositories and troches and shown to be released and lyse underlying Klebsiella oxytoca bacteria in an in-vitro model. These bacteriophage formulations were stable for at least 49 days at 4°C. The successful in-vitro assay of these formulations here suggests that they could potentially be tested in-vivo to determine whether such a therapeutic approach could modulate the gut microbiome, and control Klebsiella oxytoca overgrowth, during antibiotic therapy regimes. This study reports a novel bacteriophage specific for Klebsiella oxytoca which can be formulated into solid dosage forms appropriate for potential delivery in testing as a therapy to modulate gut microbiome during antibiotic therapies.

Survival studies of a temperate and lytic bacteriophage in bovine faeces and slurry.

PubMed

Nyambe, S; Burgess, C; Whyte, P; Bolton, D

2016-10-01

Cattle are the main reservoir of verocytotoxigenic Escherichia coli (VTEC), food-borne pathogens that express verocytotoxins (vtx) encoded by temperate bacteriophage. Bovine faeces and unturned manure heaps can support the survival of VTEC and may propagate and transmit VTEC. This study investigated the survival of a vtx2 bacteriophage, φ24B ::Kan, in bovine faeces and slurry. The survival of an anti-Escherichia coli O157:H7 lytic bacteriophage, e11/2, was examined in the same matrices, as a possible bio-control option for VTEC. Samples were inoculated with φ24B ::Kan and/or e11/2 bacteriophage at a concentration of 7-8 log10  PFU g(-1)  (faeces) or ml(-1) (slurry), stored at 4 and 14°C and examined every 2 days for 36 days. The ability of φ24B ::Kan to transduce E. coli cells was examined. Moreover, E. coli concentrations in the faeces and slurry were monitored throughout the experiment as were the pH and aw (faeces only). Both bacteriophages survived well in faeces and slurry. In addition, φ24B ::Kan was able to form lysogens. φ24B ::Kan and e11/2 phage can survive and remain infective in bovine faeces and slurry for at least 30 days under representative Irish temperatures. Bovine faeces and slurry may act as a reservoir for vtx bacteriophages. The survival of the anti-O157 phage suggests it may be a suitable bio-control option in these matrices. © 2016 The Society for Applied Microbiology.

MetaPhinder—Identifying Bacteriophage Sequences in Metagenomic Data Sets

PubMed Central

Villarroel, Julia; Lund, Ole; Voldby Larsen, Mette; Nielsen, Morten

2016-01-01

Bacteriophages are the most abundant biological entity on the planet, but at the same time do not account for much of the genetic material isolated from most environments due to their small genome sizes. They also show great genetic diversity and mosaic genomes making it challenging to analyze and understand them. Here we present MetaPhinder, a method to identify assembled genomic fragments (i.e.contigs) of phage origin in metagenomic data sets. The method is based on a comparison to a database of whole genome bacteriophage sequences, integrating hits to multiple genomes to accomodate for the mosaic genome structure of many bacteriophages. The method is demonstrated to out-perform both BLAST methods based on single hits and methods based on k-mer comparisons. MetaPhinder is available as a web service at the Center for Genomic Epidemiology https://cge.cbs.dtu.dk/services/MetaPhinder/, while the source code can be downloaded from https://bitbucket.org/genomicepidemiology/metaphinder or https://github.com/vanessajurtz/MetaPhinder. PMID:27684958

FRNA Bacteriophages as Viral Indicators of Faecal Contamination in Mexican Tropical Aquatic Systems.

PubMed

Arredondo-Hernandez, Luis Jose Rene; Diaz-Avalos, Carlos; Lopez-Vidal, Yolanda; Castillo-Rojas, Gonzalo; Mazari-Hiriart, Marisa

2017-01-01

A particular challenge to water safety in populous intertropical regions is the lack of reliable faecal indicators to detect microbiological contamination of water, while the numerical relationships of specific viral indicators remain largely unexplored. The aim of this study was to investigate the numerical relationships of FRNA-bacteriophage genotypes, adenovirus 41, and human adenoviruses (HADV) in Mexican surface water systems to assess sewage contamination. We studied the presence of HADV, HADV41 and FRNA bacteriophage genotypes in water samples and quantified by qPCR and RT-qPCR. Virus and water quality indicator variances, as analyzed by principal component analysis and partial least squared regression, followed along the major percentiles of water faecal enterococci. FRNA bacteriophages adequately deciphered viral and point source water contamination. The strongest correlation for HADV was with FRNA bacteriophage type II, in water samples higher than the 50th percentiles of faecal enterococci, thus indicating urban pollution. FRNA bacteriophage genotypes I and III virus indicator performances were assisted by their associations with electrical conductivity and faecal enterococci. In combination, our methods are useful for inferring water quality degradation caused by sewage contamination. The methods used have potential for determining source contamination in water and, specifically, the presence of enteric viruses where clean and contaminated water have mixed.

FRNA Bacteriophages as Viral Indicators of Faecal Contamination in Mexican Tropical Aquatic Systems

PubMed Central

Diaz-Avalos, Carlos; Lopez-Vidal, Yolanda; Castillo-Rojas, Gonzalo; Mazari-Hiriart, Marisa

2017-01-01

A particular challenge to water safety in populous intertropical regions is the lack of reliable faecal indicators to detect microbiological contamination of water, while the numerical relationships of specific viral indicators remain largely unexplored. The aim of this study was to investigate the numerical relationships of FRNA-bacteriophage genotypes, adenovirus 41, and human adenoviruses (HADV) in Mexican surface water systems to assess sewage contamination. We studied the presence of HADV, HADV41 and FRNA bacteriophage genotypes in water samples and quantified by qPCR and RT-qPCR. Virus and water quality indicator variances, as analyzed by principal component analysis and partial least squared regression, followed along the major percentiles of water faecal enterococci. FRNA bacteriophages adequately deciphered viral and point source water contamination. The strongest correlation for HADV was with FRNA bacteriophage type II, in water samples higher than the 50th percentiles of faecal enterococci, thus indicating urban pollution. FRNA bacteriophage genotypes I and III virus indicator performances were assisted by their associations with electrical conductivity and faecal enterococci. In combination, our methods are useful for inferring water quality degradation caused by sewage contamination. The methods used have potential for determining source contamination in water and, specifically, the presence of enteric viruses where clean and contaminated water have mixed. PMID:28114378

Transformation of Clostridium acetobutylicum Protoplasts with Bacteriophage DNA

PubMed Central

Reid, Sharon J.; Allcock, Errol R.; Jones, David T.; Woods, David R.

1983-01-01

Techniques for the transformation of Clostridium acetobutylicum protoplasts with bacteriophage DNA are described. Transformation required regeneration of protoplasts and a 2-h eclipse period. PMID:16346174

Isolation and Characterization of the Lytic Cold-Active Bacteriophage MYSP06 from the Mingyong Glacier in China.

PubMed

Li, Mingyuan; Wang, Jilian; Zhang, Qi; Lin, Lianbing; Kuang, Anxin; Materon, Luis Alberto; Ji, Xiuling; Wei, Yunlin

2016-02-01

As unique ecological systems, glaciers are characterized by low temperatures and low nutrient levels, which allow them to be considered as “living fossils” for the purpose of researching the evolution of life and the environmental evolution of the earth. Glaciers are also natural microbial “reservoirs”. In this work, a lytic cold-active bacteriophage designated MYSP06 was isolated from Janthinobacterium sp. MYB06 from the Mingyong Glacier in China, and its major characteristics were determined. Electron microscopy revealed that bacteriophage MYSP06 had an isometric head (74 nm) and a long tail (10 nm in width, 210 nm in length). It was classified as a Siphoviridae with an approximate genome size of 65–70 kb. A one-step growth curve revealed that the latent and burst periods were 95 and 65 min, respectively, with an average burst size of 16 bacteriophage particles per infected cell. The bacteriophage particles (100 %) adsorbed to the host cells within 10 min after infection. Moreover, the pH value and thermal stability of bacteriophage MYSP06 were also investigated. The maximum stability of the bacteriophage was observed at the optimal pH 7.0, and the bacteriophage became completely unstable at the extremely alkaline pH 11.0; however, it was comparatively stable at the acidic alkaline pH 6.0. As MYSP06 is a cold-active bacteriophage with a lower production temperature, its characterization and its relationship with its host Janthinobacterium sp. MYB06 deserve further study.

Engineered enzymatically active bacteriophages and methods of uses thereof

DOEpatents

Collins, James J [Newton, MA; Kobayashi, Hideki [Yokohama, JP; Kearn, Mads [Ottawa, CA; Araki, Michihiro [Minatoku, JP; Friedland, Ari [Boston, MA; Lu, Timothy Kuan-Ta [Palo Alto, CA

2012-05-22

The present invention provides engineered bacteriophages that express at least one biofilm degrading enzyme on their surface and uses thereof for degrading bacterial biofilms. The invention also provides genetically engineered bacteriophages expressing the biofilm degrading enzymes and proteins necessary for the phage to replicate in different naturally occurring biofilm producing bacteria. The phages of the invention allow a method of biofilm degradation by the use of one or only a few administration of the phage because the system using these phages is self perpetuating, and capable of degrading biofilm even when the concentration of bacteria within the biofilm is low.

Access to bacteriophage therapy: discouraging experiences from the human cell and tissue legal framework.

PubMed

Verbeken, G; Huys, I; De Vos, D; De Coninck, A; Roseeuw, D; Kets, E; Vanderkelen, A; Draye, J P; Rose, T; Jennes, S; Ceulemans, C; Pirnay, J P

2016-02-01

Cultures of human epithelial cells (keratinocytes) are used as an additional surgical tool to treat critically burnt patients. Initially, the production environment of keratinocyte grafts was regulated exclusively by national regulations. In 2004, the European Tissues and Cells Directive 2004/23/EC (transposed into Belgian Law) imposed requirements that resulted in increased production costs and no significant increase in quality and/or safety. In 2007, Europe published Regulation (EC) No. 1394/2007 on Advanced Therapy Medicinal Products. Overnight, cultured keratinocytes became (arguably) 'Advanced' Therapy Medicinal Products to be produced as human medicinal products. The practical impact of these amendments was (and still is) considerable. A similar development appears imminent in bacteriophage therapy. Bacteriophages are bacterial viruses that can be used for tackling the problem of bacterial resistance development to antibiotics. Therapeutic natural bacteriophages have been in clinical use for almost 100 years. Regulators today are framing the (re-)introduction of (natural) bacteriophage therapy into 'modern western' medicine as biological medicinal products, also subject to stringent regulatory medicinal products requirements. In this paper, we look back on a century of bacteriophage therapy to make the case that therapeutic natural bacteriophages should not be classified under the medicinal product regulatory frames as they exist today. It is our call to authorities to not repeat the mistake of the past. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A filamentous bacteriophage targeted to carcinoembryonic antigen induces tumor regression in mouse models of colorectal cancer.

PubMed

Murgas, Paola; Bustamante, Nicolás; Araya, Nicole; Cruz-Gómez, Sebastián; Durán, Eduardo; Gaete, Diana; Oyarce, César; López, Ernesto; Herrada, Andrés Alonso; Ferreira, Nicolás; Pieringer, Hans; Lladser, Alvaro

2018-02-01

Colorectal cancer is a deadly disease, which is frequently diagnosed at advanced stages, where conventional treatments are no longer effective. Cancer immunotherapy has emerged as a new form to treat different malignancies by turning-on the immune system against tumors. However, tumors are able to evade antitumor immune responses by promoting an immunosuppressive microenvironment. Single-stranded DNA containing M13 bacteriophages are highly immunogenic and can be specifically targeted to the surface of tumor cells to trigger inflammation and infiltration of activated innate immune cells, overcoming tumor-associated immunosuppression and promoting antitumor immunity. Carcinoembryonic antigen (CEA) is highly expressed in colorectal cancers and has been shown to promote several malignant features of colorectal cancer cells. In this work, we targeted M13 bacteriophage to CEA, a tumor-associated antigen over-expressed in a high proportion of colorectal cancers but largely absent in normal cells. The CEA-targeted M13 bacteriophage was shown to specifically bind to purified CEA and CEA-expressing tumor cells in vitro. Both intratumoral and systemic administration of CEA-specific bacteriophages significantly reduced tumor growth of mouse models of colorectal cancer, as compared to PBS and control bacteriophage administration. CEA-specific bacteriophages promoted tumor infiltration of neutrophils and macrophages, as well as maturation dendritic cells in tumor-draining lymph nodes, suggesting that antitumor T-cell responses were elicited. Finally, we demonstrated that tumor protection provided by CEA-specific bacteriophage particles is mediated by CD8 + T cells, as depletion of circulating CD8 + T cells completely abrogated antitumor protection. In summary, we demonstrated that CEA-specific M13 bacteriophages represent a potential immunotherapy against colorectal cancer.

Enhancement of the antimicrobial properties of bacteriophage-K via stabilization using oil-in-water nano-emulsions.

PubMed

Esteban, Patricia Perez; Alves, Diana R; Enright, Mark C; Bean, Jessica E; Gaudion, Alison; Jenkins, A T A; Young, Amber E R; Arnot, Tom C

2014-01-01

Bacteriophage therapy is a promising new treatment that may help overcome the threat posed by antibiotic-resistant pathogenic bacteria, which are increasingly identified in hospitalized patients. The development of biocompatible and sustainable vehicles for incorporation of viable bacterial viruses into a wound dressing is a promising alternative. This article evaluates the antimicrobial efficacy of Bacteriophage K against Staphylococcus aureus over time, when stabilized and delivered via an oil-in-water nano-emulsion. Nano-emulsions were formulated via thermal phase inversion emulsification, and then bacterial growth was challenged with either native emulsion, or emulsion combined with Bacteriophage K. Bacteriophage infectivity, and the influence of storage time of the preparation, were assessed by turbidity measurements of bacterial samples. Newly prepared Bacteriophage K/nano-emulsion formulations have greater antimicrobial activity than freely suspended bacteriophage. The phage-loaded emulsions caused rapid and complete bacterial death of three different strains of S. aureus. The same effect was observed for preparations that were either stored at room temperature (18-20°C), or chilled at 4°C, for up to 10 days of storage. A response surface design of experiments was used to gain insight on the relative effects of the emulsion formulation on bacterial growth and phage lytic activity. More diluted emulsions had a less significant effect on bacterial growth, and diluted bacteriophage-emulsion preparations yielded greater antibacterial activity. The enhancement of bacteriophage activity when delivered via nano-emulsions is yet to be reported. This prompts further investigation into the use of these formulations for the development of novel anti-microbial wound management strategies. © 2014 American Institute of Chemical Engineers.

Bacteriophages as indicators of human and animal faecal contamination in raw and treated wastewaters from Tunisia.

PubMed

Yahya, M; Hmaied, F; Jebri, S; Jofre, J; Hamdi, M

2015-05-01

We aimed at quantifying bacteriophages in raw and treated wastewaters of human and animal origin in Tunisia to assess their usefulness for tracking the origin of faecal pollution and in the follow-up of effectiveness of water treatments process. The concentrations of bacteriophages in wastewater samples were determined by double layer agar technique. Somatic coliphages and F-specific RNA bacteriophages were present in all types of samples in high concentrations. The values of Escherichia coli were variable depending on geographical location. On the other hand, bacteriophages infecting strain GA17 were detected preferably when human faecal contamination was occurred. Bacteriophages appear as a feasible and widely applicable manner to detect faecal contamination in Tunisia. On the other hand, phages infecting GA17 could be good markers for tracking the origin of faecal pollution in the area studied. The reuse of treated wastewaters can be a solution to meet the needs of water in the geographical area of study. Bacteriophages seem to predict differently the presence of faecal contamination in water than bacterial indicators. Consequently, they can be a valuable additional tool to improve water resources management for minimizing health risks. © 2015 The Society for Applied Microbiology.

Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials

PubMed Central

Merabishvili, Maya; Pirnay, Jean-Paul; Verbeken, Gilbert; Chanishvili, Nina; Tediashvili, Marina; Lashkhi, Nino; Glonti, Thea; Krylov, Victor; Mast, Jan; Van Parys, Luc; Lavigne, Rob; Volckaert, Guido; Mattheus, Wesley; Verween, Gunther; De Corte, Peter; Rose, Thomas; Jennes, Serge; Zizi, Martin; De Vos, Daniel; Vaneechoutte, Mario

2009-01-01

We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on succesive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, φKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee. PMID:19300511

Mutation of M13 Bacteriophage Major Coat Protein for Increased Conjugation to Exogenous Compounds.

PubMed

Tridgett, Matthew; Lloyd, James R; Kennefick, Jack; Moore-Kelly, Charles; Dafforn, Timothy R

2018-06-20

Over the past ten years there has been increasing interest in the conjugation of exogenous compounds to the surface of the M13 bacteriophage. M13 offers a convenient scaffold for the development of nanoassemblies with useful functions, such as highly specific drug delivery and pathogen detection. However, the progress of these technologies has been hindered by the limited efficiency of conjugation to the bacteriophage. Here we generate a mutant version of M13 with an additional lysine residue expressed on the outer surface of the M13 major coat protein, pVIII. We show that this mutation is accommodated by the bacteriophage and that up to an additional 520 exogenous groups can be attached to the bacteriophage surface via amine-directed conjugation. These results could aid the development of high payload drug delivery nanoassemblies and pathogen detection systems with increased sensitivity.

Anticancer activity of bacteriophage T4 and its mutant HAP1 in mouse experimental tumour models.

PubMed

Dabrowska, Krystyna; Opolski, Adam; Wietrzyk, Joanna; Switala-Jelen, Kinga; Godlewska, Joanna; Boratynski, Janusz; Syper, Danuta; Weber-Dabrowska, Beata; Gorski, Andrzej

2004-01-01

Previously, we have shown the ability of the bacteriophage T4 and its substrain HAP1 (selected for a higher affinity to melanoma cells) to reveal antimetastatic activity in a mouse melanoma model. Here, we investigated the potential phage anticancer activity in primary tumour models. Mice were inoculated subcutaneously with B16 or LLC cells (collected from in vitro culture). Bacteriophages T4 and HAP1 were injected intraperitoneally daily (8 x 10(8)pfu/mouse, except the experiment concerning the dose-dependence). Treatment with purified preparations of bacteriophage T4 resulted in significant reduction of tumour size, the effect being dose-dependent. HAP1 was more effective than T4 and its activity was also dose-dependent. Parallel experiments with non-purified bacteriophage lysates resulted in significant stimulation of tumour growth. These data suggest that purified bacteriophages may inhibit tumour growth, a phenomenon with potentially important clinical implications in oncology.

Efficacies of Cabotegravir and Bictegravir against drug-resistant HIV-1 integrase mutants.

PubMed

Smith, Steven J; Zhao, Xue Zhi; Burke, Terrence R; Hughes, Stephen H

2018-05-16

Integrase strand transfer inhibitors (INSTIs) are the class of antiretroviral (ARV) drugs most recently approved by the FDA for the treatment of HIV-1 infections. INSTIs block the strand transfer reaction catalyzed by HIV-1 integrase (IN) and have been shown to potently inhibit infection by wild-type HIV-1. Of the three current FDA-approved INSTIs, Dolutegravir (DTG), has been the most effective, in part because treatment does not readily select for resistant mutants. However, recent studies showed that when INSTI-experienced patients are put on a DTG-salvage therapy, they have reduced response rates. Two new INSTIs, Cabotegravir (CAB) and Bictegravir (BIC), are currently in late-stage clinical trials. Both CAB and BIC had much broader antiviral profiles than RAL and EVG against the INSTI-resistant single, double, and triple HIV-1 mutants used in this study. BIC was more effective than DTG against several INSTI-resistant mutants. Overall, in terms of their ability to inhibit a broad range of INSTI-resistant IN mutants, BIC was superior to DTG, and DTG was superior to CAB. Modeling the binding of CAB, BIC, and DTG within the active site of IN suggested that the "left side" of the INSTI pharmacophore (the side away from the viral DNA) was important in determining the ability of the compound to inhibit the IN mutants we tested. Of the two INSTIs in late stage clinical trials, BIC appears to be better able to inhibit the replication of a broad range of IN mutants. BIC retained potency against several of the INSTI-resistant mutants that caused a decrease in susceptibility to DTG.

Characterization and formulation into solid dosage forms of a novel bacteriophage lytic against Klebsiella oxytoca

PubMed Central

Petrovski, Steve; Hoyle, Dannielle; Chan, Hiu Tat; Lock, Peter; Tucci, Joseph

2017-01-01

Aim To isolate and characterize bacteriophage lytic for the opportunistic pathogen Klebsiella oxytoca and their formulation into a range of solid dosage forms for in-vitro testing. Methods and results We report the isolation, genomic and functional characterization of a novel bacteriophage lytic for Klebsiella oxytoca, which does not infect the closely related Klebsiella pneumoniae. This bacteriophage was formulated into suppositories and troches and shown to be released and lyse underlying Klebsiella oxytoca bacteria in an in-vitro model. These bacteriophage formulations were stable for at least 49 days at 4°C. Conclusions The successful in-vitro assay of these formulations here suggests that they could potentially be tested in-vivo to determine whether such a therapeutic approach could modulate the gut microbiome, and control Klebsiella oxytoca overgrowth, during antibiotic therapy regimes. Significance and impact of the study This study reports a novel bacteriophage specific for Klebsiella oxytoca which can be formulated into solid dosage forms appropriate for potential delivery in testing as a therapy to modulate gut microbiome during antibiotic therapies. PMID:28817689

Effectiveness of cooking to reduce norovirus and infectious F-specific RNA bacteriophage concentrations in Mytilus edulis.

PubMed

Flannery, J; Rajko-Nenow, P; Winterbourn, J B; Malham, S K; Jones, D L

2014-08-01

The aim of this study was to determine if domestic cooking practices can reduce concentrations of norovirus (NoV) and F-specific RNA (FRNA) bacteriophage in experimentally contaminated mussels. Mussels (n = 600) contaminated with NoV and FRNA bacteriophage underwent four different cooking experiments performed in triplicate at ~70°C and >90°C. Concentrations of infectious FRNA bacteriophage (using a plaque assay) were compared with concentrations of FRNA bacteriophage and NoV determined using a standardised RT-qPCR. Initial concentrations of infectious FRNA bacteriophage (7·05 log10  PFU g(-1) ) in mussels were not significantly reduced in simmering water (~70°C); however, cooking at higher temperatures (>90°C) reduced infectious FRNA bacteriophage to undetected levels within 3 min. Further investigation determined the time required for a 1-log reduction of infectious FRNA bacteriophage at 90°C to be 42 s therefore a >3-log reduction in infectious virus can be obtained by heating mussel digestive tissue to 90°C for 126 s. Domestic cooking practices based on shell opening alone do not inactivate infectious virus in mussels, however, cooking mussels at high temperatures is effective to reduce infectious virus concentrations and the risk of illness in consumers. The data will contribute towards evidence-based cooking recommendations for shellfish to provide a safe product for human consumption. © 2014 The Society for Applied Microbiology.

M13 Bacteriophage Based Protein Sensors

NASA Astrophysics Data System (ADS)

Lee, Ju Hun

Despite significant progress in biotechnology and biosensing, early detection and disease diagnosis remains a critical issue for improving patient survival rates and well-being. Many of the typical detection schemes currently used possess issues such as low sensitivity and accuracy and are also time consuming to run and expensive. In addition, multiplexed detection remains difficult to achieve. Therefore, developing advanced approaches for reliable, simple, quantitative analysis of multiple markers in solution that also are highly sensitive are still in demand. In recent years, much of the research has primarily focused on improving two key components of biosensors: the bio-recognition agent (bio-receptor) and the transducer. Particular bio-receptors that have been used include antibodies, aptamers, molecular imprinted polymers, and small affinity peptides. In terms of transducing agents, nanomaterials have been considered as attractive candidates due to their inherent nanoscale size, durability and unique chemical and physical properties. The key focus of this thesis is the design of a protein detection and identification system that is based on chemically engineered M13 bacteriophage coupled with nanomaterials. The first chapter provides an introduction of biosensors and M13 bacteriophage in general, where the advantages of each are provided. In chapter 2, an efficient and enzyme-free sensor is demonstrated from modified M13 bacteriophage to generate highly sensitive colorimetric signals from gold nanocrystals. In chapter 3, DNA conjugated M13 were used to enable facile and rapid detection of antigens in solution that also provides modalities for identification. Lastly, high DNA loadings per phage was achieved via hydrozone chemistry and these were applied in conjunction with Raman active DNA-gold/silver core/shell nanoparticles toward highly sensitive SERS sensing.

In vitro Effectiveness of Commercial Bacteriophage Cocktails on Diverse Extended-Spectrum Beta-Lactamase Producing Escherichia coli Strains.

PubMed

Gundogdu, Aycan; Bolkvadze, Darajen; Kilic, Huseyin

2016-01-01

The objective of this study is to determine the in vitro susceptibility of Georgian bacteriophage cocktails on multidrug resistant (MDR) extended-spectrum beta-lactamase producing Escherichia coli (ESBL-EC) isolated from patients' blood and urine cultures. A total of 615 E. coli isolates were included in this study. Phene Plate (PhP)-typing and phylogenetic grouping were used for the typing. Antimicrobial resistance profiles and ESBL production of all isolates were confirmed according to Clinical and Laboratory Standards Institute (CLSI) criteria. The activities of four bacteriophage cocktails (Enko-phage, SES-bacteriophage, Pyo-bacteriophage, and Intesti-bacteriophage) were determined against 142 ESBL-EC using in vitro spot tests. According to this, Enko-phage were active against 87.3% of the tested strains while that ratio was 81.7% for Intesti-bacteriophage, 81.7% for Pyo-bacteriophage, and 59.2% for SES-bacteriophage cocktails. Based on the contingency tests, the phage cocktails were observed to be statistically significantly ( p < 0.001) more effective on ESBL-EC strains belonging to phylogenetic groups D and B2. The employed phage cocktails were found to be affective against all tested resistant types. These results are promising especially for the infections that are caused by MDR pathogens that are difficult to treat. As this is a preliminary step to the potential clinical trials to be designed for the country, in vitro confirmation of their success on a MDR ESBL-EC collection should be accepted as an initial action, which is encouraging to consider clinical trials of phage therapy especially in countries which are not introduce phage therapy.

In vitro Effectiveness of Commercial Bacteriophage Cocktails on Diverse Extended-Spectrum Beta-Lactamase Producing Escherichia coli Strains

PubMed Central

Gundogdu, Aycan; Bolkvadze, Darajen; Kilic, Huseyin

2016-01-01

The objective of this study is to determine the in vitro susceptibility of Georgian bacteriophage cocktails on multidrug resistant (MDR) extended-spectrum beta-lactamase producing Escherichia coli (ESBL-EC) isolated from patients’ blood and urine cultures. A total of 615 E. coli isolates were included in this study. Phene Plate (PhP)-typing and phylogenetic grouping were used for the typing. Antimicrobial resistance profiles and ESBL production of all isolates were confirmed according to Clinical and Laboratory Standards Institute (CLSI) criteria. The activities of four bacteriophage cocktails (Enko-phage, SES-bacteriophage, Pyo-bacteriophage, and Intesti-bacteriophage) were determined against 142 ESBL-EC using in vitro spot tests. According to this, Enko-phage were active against 87.3% of the tested strains while that ratio was 81.7% for Intesti-bacteriophage, 81.7% for Pyo-bacteriophage, and 59.2% for SES-bacteriophage cocktails. Based on the contingency tests, the phage cocktails were observed to be statistically significantly (p < 0.001) more effective on ESBL-EC strains belonging to phylogenetic groups D and B2. The employed phage cocktails were found to be affective against all tested resistant types. These results are promising especially for the infections that are caused by MDR pathogens that are difficult to treat. As this is a preliminary step to the potential clinical trials to be designed for the country, in vitro confirmation of their success on a MDR ESBL-EC collection should be accepted as an initial action, which is encouraging to consider clinical trials of phage therapy especially in countries which are not introduce phage therapy. PMID:27857711

Guidelines for Bacteriophage Product Certification.

PubMed

Fauconnier, Alan

2018-01-01

Following decades in the wilderness, bacteriophage therapy is now appearing as a credible antimicrobial strategy. However, this reemerging therapy does not rekindle without raising sensitive regulatory concerns. Indeed, whereas the European regulatory framework has been basically implemented to tackle ready-to-use pharmaceuticals produced on a large scale, bacteriophage therapy relies on a dynamic approach requiring a regulation on personalized medicine, nonexistent at present. Because of this, no guideline are currently available for addressing the scientific and regulatory issues specifically related to phage therapy medicinal products (PTMP).Pending to the implementation of an appropriate regulatory framework and to the development of ensuing guidelines, several avenues which might lead to PTMP regulatory compliance are explored here. Insights might come from the multi-strain dossier approach set up for particular animal vaccines, from the homologous group concept developed for the allergen products or from the licensing process for veterinary autogenous vaccines. Depending on national legislations, customized preparations prescribed as magistral formulas or to be used on a named-patient basis are possible regulatory approaches to be considered. However, these schemes are not optimal and should thus be regarded as transitional.

Multiplex PCR for the detection and identification of dairy bacteriophages in milk.

PubMed

del Rio, B; Binetti, A G; Martín, M C; Fernández, M; Magadán, A H; Alvarez, M A

2007-02-01

Bacteriophage infections of starter lactic acid bacteria are a serious risk in the dairy industry. Phage infection can lead to slow lactic acid production or even the total failure of fermentation. The associated economic losses can be substantial. Rapid and sensitive methods are therefore required to detect and identify phages at all stages of the manufacture of fermented dairy products. This study describes a simple and rapid multiplex PCR method that, in a single reaction, detects the presence of bacteriophages infecting Streptococcus thermophilus and Lactobacillus delbrueckii, plus three genetically distinct 'species' of Lactococcus lactis phages commonly found in dairy plants (P335, 936 and c2). Available bacteriophage genome sequences were examined and the conserved regions used to design five pairs of primers, one for each of the above bacteriophage species. These primers were designed to generate specific fragments of different size depending on the species. Since this method can detect the above phages in untreated milk and can be easily incorporated into dairy industry routines, it might be readily used to earmark contaminated milk for use in processes that do not involve susceptible starter organisms or for use in those that involve phage-deactivating conditions.

Bacteriophages for detection and control of bacterial pathogens in food and food-processing environment.

PubMed

Brovko, Lubov Y; Anany, Hany; Griffiths, Mansel W

2012-01-01

This chapter presents recent advances in bacteriophage research and their application in the area of food safety. Section 1 describes general facts on phage biology that are relevant to their application for control and detection of bacterial pathogens in food and environmental samples. Section 2 summarizes the recently acquired data on application of bacteriophages to control growth of bacterial pathogens and spoilage organisms in food and food-processing environment. Section 3 deals with application of bacteriophages for detection and identification of bacterial pathogens. Advantages of bacteriophage-based methods are presented and their shortcomings are discussed. The chapter is intended for food scientist and food product developers, and people in food inspection and health agencies with the ultimate goal to attract their attention to the new developing technology that has a tremendous potential in providing means for producing wholesome and safe food. Copyright © 2012 Elsevier Inc. All rights reserved.

Co-option of bacteriophage lysozyme genes by bivalve genomes.

PubMed

Ren, Qian; Wang, Chunyang; Jin, Min; Lan, Jiangfeng; Ye, Ting; Hui, Kaimin; Tan, Jingmin; Wang, Zheng; Wyckoff, Gerald J; Wang, Wen; Han, Guan-Zhu

2017-01-01

Eukaryotes have occasionally acquired genetic material through horizontal gene transfer (HGT). However, little is known about the evolutionary and functional significance of such acquisitions. Lysozymes are ubiquitous enzymes that degrade bacterial cell walls. Here, we provide evidence that two subclasses of bivalves (Heterodonta and Palaeoheterodonta) acquired a lysozyme gene via HGT, building on earlier findings. Phylogenetic analyses place the bivalve lysozyme genes within the clade of bacteriophage lysozyme genes, indicating that the bivalves acquired the phage-type lysozyme genes from bacteriophages, either directly or through intermediate hosts. These bivalve lysozyme genes underwent dramatic structural changes after their co-option, including intron gain and fusion with other genes. Moreover, evidence suggests that recurrent gene duplication occurred in the bivalve lysozyme genes. Finally, we show the co-opted lysozymes exhibit a capacity for antibacterial action, potentially augmenting the immune function of related bivalves. This represents an intriguing evolutionary strategy in the eukaryote-microbe arms race, in which the genetic materials of bacteriophages are co-opted by eukaryotes, and then used by eukaryotes to combat bacteria, using a shared weapon against a common enemy. © 2017 The Authors.

Two-Stage Dynamics of In Vivo Bacteriophage Genome Ejection

NASA Astrophysics Data System (ADS)

Chen, Yi-Ju; Wu, David; Gelbart, William; Knobler, Charles M.; Phillips, Rob; Kegel, Willem K.

2018-04-01

Biopolymer translocation is a key step in viral infection processes. The transfer of information-encoding genomes allows viruses to reprogram the cell fate of their hosts. Constituting 96% of all known bacterial viruses [A. Fokine and M. G. Rossmann, Molecular architecture of tailed double-stranded DNA phages, Bacteriophage 4, e28281 (2014)], the tailed bacteriophages deliver their DNA into host cells via an "ejection" process, leaving their protein shells outside of the bacteria; a similar scenario occurs for mammalian viruses like herpes, where the DNA genome is ejected into the nucleus of host cells, while the viral capsid remains bound outside to a nuclear-pore complex. In light of previous experimental measurements of in vivo bacteriophage λ ejection, we analyze here the physical processes that give rise to the observed dynamics. We propose that, after an initial phase driven by self-repulsion of DNA in the capsid, the ejection is driven by anomalous diffusion of phage DNA in the crowded bacterial cytoplasm. We expect that this two-step mechanism is general for phages that operate by pressure-driven ejection, and we discuss predictions of our theory to be tested in future experiments.

Control of Listeria monocytogenes growth in soft cheeses by bacteriophage P100.

PubMed

Silva, Elaine Nóbrega Gibson; Figueiredo, Ana Cláudia Leite; Miranda, Fernanda Araújo; de Castro Almeida, Rogeria Comastri

2014-01-01

The purpose of this study was to determine the effect of bacteriophage P100 on strains of Listeria monocytogenes in artificially inoculated soft cheeses. A mix of L. monocytogenes 1/2a and Scott A was inoculated in Minas Frescal and Coalho cheeses (approximately 10(5) cfu/g) with the bacteriophage added thereafter (8.3 × 10(7) PFU/g). Samples were analyzed immediately, and then stored at 10 °C for seven days. At time zero, 30 min post-infection, the bacteriophage P100 reduced L. monocytogenes counts by 2.3 log units in Minas Frescal cheese and by 2.1 log units in Coalho cheese, compared to controls without bacteriophage. However, in samples stored under refrigeration for seven days, the bacteriophage P100 was only weakly antilisterial, with the lowest decimal reduction (DR) for the cheeses: 1.0 log unit for Minas Frescal and 0.8 log units for Coalho cheese. The treatment produced a statistically significant decrease in the counts of viable cells (p < 0.05) and in all assays performed, we observed an increase of approximately one log cycle in the number of viable cells of L. monocytogenes in the samples under refrigeration for seven days. Moreover, a smaller effect of phages was observed. These results, along with other published data, indicate that the effectiveness of the phage treatment depends on the initial concentration of L. monocytogenes, and that a high concentration of phages per unit area is required to ensure sustained inactivation of target pathogens on food surfaces.

Antimicrobial bacteriophage-derived proteins and therapeutic applications

USDA-ARS?s Scientific Manuscript database

Antibiotics have the remarkable power to control bacterial infections. Unfortunately, widespread use, whether regarded as prudent or not, has favored the emergence and persistence of antibiotic resistant strains of human pathogenic bacteria, resulting in a global health threat. Bacteriophages (pha...

Hexagonally packed DNA within bacteriophage T7 stabilized by curvature stress.

PubMed Central

Odijk, T

1998-01-01

A continuum computation is proposed for the bending stress stabilizing DNA that is hexagonally packed within bacteriophage T7. Because the inner radius of the DNA spool is rather small, the stress of the curved DNA genome is strong enough to balance its electrostatic self-repulsion so as to form a stable hexagonal phase. The theory is in accord with the microscopically determined structure of bacteriophage T7 filled with DNA within the experimental margin of error. PMID:9726924

Evaluation of consumers’ perception and willingness to pay for bacteriophage treated fresh produce

PubMed Central

Naanwaab, Cephas; Yeboah, Osei-Agyeman; Ofori Kyei, Foster; Sulakvelidze, Alexander; Goktepe, Ipek

2014-01-01

Food-borne illnesses caused by bacteria such as enterohemorrhagic E. coli and Salmonella spp. take a significant toll on American consumers’ health; they also cost the United States an estimated $77.7 billion annually in health care and other losses.1 One novel modality for improving the safety of foods is application of lytic bacteriophages directly onto foods, in order to reduce or eliminate their contamination with specific foodborne bacterial pathogens. The main objective of this study was to assess consumers’ perception about foods treated with bacteriophages and examine their willingness to pay (WTP) an additional amount (10–30 cents/lb) for bacteriophage-treated fresh produce. The study utilized a survey questionnaire administered by telephone to consumers in 4 different states: Alabama, Georgia, North Carolina, and South Carolina. The results show that consumers are in general willing to pay extra for bacteriophage-treated fresh produce if it improves their food safety. However, income, race, and the state where a consumer lives are significant determinants in their WTP. PMID:26713224

Methods for Initial Characterization of Campylobacter jejuni Bacteriophages.

PubMed

Sørensen, Martine Camilla Holst; Gencay, Yilmaz Emre; Brøndsted, Lone

2017-01-01

Here we describe an initial characterization of Campylobacter jejuni bacteriophages by host range analysis, genome size determination by pulsed-field gel electrophoresis, and receptor-type identification by screening mutants for phage sensitivity.

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

PubMed

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

2017-01-01

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

Evaluation of the broad-spectrum lytic capability of bacteriophage cocktails against various Salmonella serovars and their effects on weaned pigs infected with Salmonella Typhimurium.

PubMed

Seo, Byoung-Joo; Song, Eu-Tteum; Lee, Kichan; Kim, Jong-Won; Jeong, Chang-Gi; Moon, Sung-Hyun; Son, Jee Soo; Kang, Sang Hyeon; Cho, Ho-Seong; Jung, Byeong Yeal; Kim, Won-Il

2018-06-06

The broad-spectrum lytic capability of Salmonella bacteriophages against various Salmonella species was evaluated to determine their potential as an alternative for antibiotics, and the safety and preventive effects of the bacteriophages were assessed on mice and pigs. Four bacteriophage cocktails were prepared using 13 bacteriophages, and the lytic capability of the four bacteriophage cocktails was tested using Salmonella reference strains and field isolates. Bacteriophage cocktail C (SEP-1, SGP-1, STP-1, SS3eP-1, STP-2, SChP-1, SAP-1, SAP-2; ≥10 9 pfu/ml) showed the best lytic activity against the Salmonella reference strains (100% of 34) and field isolates (92.5% of 107). Fifty mice were then orally inoculated with bacteriophage cocktail C to determine the distribution of bacteriophages in various organs, blood and feces. The effects of bacteriophages on Salmonella infection in weaned pigs (n=15) were also evaluated through an experimental challenge with Salmonella Typhimurium after treatment with bacteriophage cocktail C. All mice exhibited distribution of the bacteriophages in all organs, blood and feces until 15 days post infection (dpi). After 35 dpi, bacteriophages were not detected in any of these specimens. As demonstrated in a pig challenge study, treatment with bacteriophage cocktail C reduced the level of Salmonella shedding in feces. The metagenomic analyses of these pig feces also revealed that bacteriophage treatment decreased the number of species of the Enterobacteriaceae family without significant disturbance to the normal fecal flora. This study showed that bacteriophages effectively controlled Salmonella in a pig challenge model and could be a good alternative for antibiotics to control Salmonella infection.

Modeling removal of bacteriophages MS2 and PRD1 by dune recharge at Castricum, Netherlands

NASA Astrophysics Data System (ADS)

Schijven, Jack F.; Hoogenboezem, Wim; Hassanizadeh, S. Majid; Peters, Jos H.

1999-04-01

Removal of model viruses by dune recharge was studied at a field site in the dune area of Castricum, Netherlands. Recharge water was dosed with bacteriophages MS2 and PRD1 for 11 days at a constant concentration in a 10- by 15-m compartment that was isolated in a recharge basin. Breakthrough was monitored for 120 days at six wells with their screens along a flow line. Concentrations of both phages were reduced about 3 log10 within the first 2.4 m and another 5 log10 in a linear fashion within the following 27 m. A model accounting for one-site kinetic attachment as well as first-order inactivation was employed to simulate the bacteriophage breakthrough curves. The major removal process was found to be attachment of the bacteriophages. Detachment was very slow. After passage of the pulse of dosed bacteriophages, there was a long tail whose slope corresponds to the inactivation rate coefficient of 0.07-0.09 day-1 for attached bacteriophages. The end of the rising and the start of the declining limbs of the breakthrough curves could not be simulated completely, probably because of an as yet unknown process.

40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

Code of Federal Regulations, 2011 CFR

2011-07-01

... bacteriophages; temporary exemption from the requirement of a tolerance. 180.1301 Section 180.1301 Protection of... bacteriophages; temporary exemption from the requirement of a tolerance. A temporary exemption from the requirement of a tolerance is established for residues of lytic bacteriophages that are specific to...

40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

Code of Federal Regulations, 2014 CFR

2014-07-01

... bacteriophages; temporary exemption from the requirement of a tolerance. 180.1301 Section 180.1301 Protection of... bacteriophages; temporary exemption from the requirement of a tolerance. A temporary exemption from the requirement of a tolerance is established for residues of lytic bacteriophages that are specific to...

40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

Code of Federal Regulations, 2013 CFR

2013-07-01

... bacteriophages; temporary exemption from the requirement of a tolerance. 180.1301 Section 180.1301 Protection of... bacteriophages; temporary exemption from the requirement of a tolerance. A temporary exemption from the requirement of a tolerance is established for residues of lytic bacteriophages that are specific to...

40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

Code of Federal Regulations, 2012 CFR

2012-07-01

... bacteriophages; temporary exemption from the requirement of a tolerance. 180.1301 Section 180.1301 Protection of... bacteriophages; temporary exemption from the requirement of a tolerance. A temporary exemption from the requirement of a tolerance is established for residues of lytic bacteriophages that are specific to...

Contribution of silent mutations to thermal adaptation of RNA bacteriophage Qβ.

PubMed

Kashiwagi, Akiko; Sugawara, Ryu; Sano Tsushima, Fumie; Kumagai, Tomofumi; Yomo, Tetsuya

2014-10-01

Changes in protein function and other biological properties, such as RNA structure, are crucial for adaptation of organisms to novel or inhibitory environments. To investigate how mutations that do not alter amino acid sequence may be positively selected, we performed a thermal adaptation experiment using the single-stranded RNA bacteriophage Qβ in which the culture temperature was increased from 37.2°C to 41.2°C and finally to an inhibitory temperature of 43.6°C in a stepwise manner in three independent lines. Whole-genome analysis revealed 31 mutations, including 14 mutations that did not result in amino acid sequence alterations, in this thermal adaptation. Eight of the 31 mutations were observed in all three lines. Reconstruction and fitness analyses of Qβ strains containing only mutations observed in all three lines indicated that five mutations that did not result in amino acid sequence changes but increased the amplification ratio appeared in the course of adaptation to growth at 41.2°C. Moreover, these mutations provided a suitable genetic background for subsequent mutations, altering the fitness contribution from deleterious to beneficial. These results clearly showed that mutations that do not alter the amino acid sequence play important roles in adaptation of this single-stranded RNA virus to elevated temperature. Recent studies using whole-genome analysis technology suggested the importance of mutations that do not alter the amino acid sequence for adaptation of organisms to novel environmental conditions. It is necessary to investigate how these mutations may be positively selected and to determine to what degree such mutations that do not alter amino acid sequences contribute to adaptive evolution. Here, we report the roles of these silent mutations in thermal adaptation of RNA bacteriophage Qβ based on experimental evolution during which Qβ showed adaptation to growth at an inhibitory temperature. Intriguingly, four synonymous mutations and

Interrogating HIV integrase for compounds that bind- a SAMPL challenge

NASA Astrophysics Data System (ADS)

Peat, Thomas S.; Dolezal, Olan; Newman, Janet; Mobley, David; Deadman, John J.

2014-04-01

Tremendous gains and novel methods are often developed when people are challenged to do something new or difficult. This process is enhanced when people compete against each other-this can be seen in sport as well as in science and technology (e.g. the space race). The SAMPL challenges, like the CASP challenges, aim to challenge modellers and software developers to develop new ways of looking at molecular interactions so the community as a whole can progress in the accurate prediction of these interactions. In order for this challenge to occur, data must be supplied so the prospective test can be done. We have supplied unpublished data related to a drug discovery program run several years ago on HIV integrase for the SAMPL4 challenge. This paper describes the methods used to obtain these data and the chemistry involved.

Liposome-Encapsulated Bacteriophages for Enhanced Oral Phage Therapy against Salmonella spp.

PubMed Central

Colom, Joan; Cano-Sarabia, Mary; Otero, Jennifer; Cortés, Pilar

2015-01-01

Bacteriophages UAB_Phi20, UAB_Phi78, and UAB_Phi87 were encapsulated in liposomes, and their efficacy in reducing Salmonella in poultry was then studied. The encapsulated phages had a mean diameter of 309 to 326 nm and a positive charge between +31.6 and +35.1 mV (pH 6.1). In simulated gastric fluid (pH 2.8), the titer of nonencapsulated phages decreased by 5.7 to 7.8 log units, whereas encapsulated phages were significantly more stable, with losses of 3.7 to 5.4 log units. The liposome coating also improved the retention of bacteriophages in the chicken intestinal tract. When cocktails of the encapsulated and nonencapsulated phages were administered to broilers, after 72 h the encapsulated phages were detected in 38.1% of the animals, whereas the nonencapsulated phages were present in only 9.5%. The difference was significant. In addition, in an in vitro experiment, the cecal contents of broilers promoted the release of the phages from the liposomes. In broilers experimentally infected with Salmonella, the daily administration of the two cocktails for 6 days postinfection conferred similar levels of protection against Salmonella colonization. However, once treatment was stopped, protection by the nonencapsulated phages disappeared, whereas that provided by the encapsulated phages persisted for at least 1 week, showing the enhanced efficacy of the encapsulated phages in protecting poultry against Salmonella over time. The methodology described here allows the liposome encapsulation of phages of different morphologies. The preparations can be stored for at least 3 months at 4°C and could be added to the drinking water and feed of animals. PMID:25956778

A review of current methods using bacteriophages in live animals, food and animal products intended for human consumption.

PubMed

Cooper, Ian R

2016-11-01

Bacteriophages are utilised in the food industry as biocontrol agents to reduce the load of bacteria, and thus reduce potential for human infection. This review focuses on current methods using bacteriophages within the food chain. Limitations of research will be discussed, and the potential for future food-based bacteriophage research. Copyright © 2016 Elsevier B.V. All rights reserved.

Encapsulation Strategies of Bacteriophage (Felix O1) for Oral Therapeutic Application.

PubMed

Islam, Golam S; Wang, Qi; Sabour, Parviz M

2018-01-01

Due to emerging antibiotic-resistant strains among the pathogens, a variety of strategies, including therapeutic application of bacteriophages, have been suggested as a possible alternative to antibiotics in food animal production. As pathogen-specific biocontrol agents, bacteriophages are being studied intensively. Primarily their applications in the food industry and animal production have been recognized in the USA and Europe, for pathogens including Salmonella, Campylobacter, Escherichia coli, and Listeria. However, the viability of orally administered phage may rapidly reduce under the harsh acidic conditions of the stomach, presence of enzymes and bile. It is evident that bacteriophages, intended for phage therapy by oral administration, require efficient protection from the acidic environment of the stomach and should remain active in the animal's gastrointestinal tract where pathogen colonizes. Encapsulation of phages by spray drying or extrusion methods can protect phages from the simulated hostile gut conditions and help controlled release of phages to the digestive system when appropriate formulation strategy is implemented.

Removal of adenovirus, calicivirus, and bacteriophages by conventional drinking water treatment.

PubMed

Abbaszadegan, Morteza; Monteiro, Patricia; Nwachuku, Nena; Alum, Absar; Ryu, Hodon

2008-02-01

This study was conducted to evaluate the removal of adenovirus, feline calicivirus (FCV), and bacteriophages MS-2, fr, PRD-1, and Phi X-174 during conventional drinking water treatment using ferric chloride as a coagulant. Adenovirus and FCV were removed to a greater extent than PRD-1 and Phi X-174, indicating that these bacteriophages may be appropriate surrogates for both adenovirus and FCV. Of the four bacteriophages studied in the pilot plant, MS-2 was removed to the greatest extent (5.1 log), followed by fr (4.9 log), PRD-1 (3.5 log), and Phi X-174 (1.3 log). The virus removal trend in the pilot-scale testing was similar to the bench-scale testing; however, the bench-scale testing seemed to provide a conservative estimate of the pilot plant performance. In the pilot-scale testing, MS-2 and fr were removed with the greatest efficiency during filtration, whereas PRD-1 and Phi X-174 showed the greatest removal during sedimentation.

21 CFR 866.2050 - Staphylococcal typing bacteriophage.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Staphylococcal typing bacteriophage. 866.2050 Section 866.2050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2050...

21 CFR 866.2050 - Staphylococcal typing bacteriophage.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Staphylococcal typing bacteriophage. 866.2050 Section 866.2050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2050...

21 CFR 866.2050 - Staphylococcal typing bacteriophage.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Staphylococcal typing bacteriophage. 866.2050 Section 866.2050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2050...

A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

PubMed

Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

2014-07-01

The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Bacteriophage use to control Salmonella biofilm on surfaces present in chicken slaughterhouses.

PubMed

Garcia, Keila Carolina de Ornellas Dutka; Corrêa, Isadora Mainieri de Oliveira; Pereira, Larissa Quinto; Silva, Tarcísio Macedo; Mioni, Mateus de Souza Ribeiro; Izidoro, Ana Carolina de Moraes; Bastos, Igor Henrique Vellano; Gonçalves, Guilherme Augusto Marietto; Okamoto, Adriano Sakai; Andreatti Filho, Raphael Lucio

2017-09-01

Foodborne diseases represent a major risk to public health worldwide. Pathogenic bacteria can live in the form of biofilm within the food industry, providing a permanent source of contamination. The aim of this study was to evaluate the influence of the types of adhesion surfaces on Salmonella biofilm formation at eight different times, and analyze the action time of a bacteriophage pool on established biofilms. Most of the samples used were classified as weak biofilm producers, with serovars Enteritidis and Heidelberg showing the highest frequency of biofilm formation. Glass and stainless steel surfaces significantly favored biofilm formation at 60 and 36 h of incubation respectively, but the polyvinyl chloride surface did not favor biofilm production, suggesting that the type of material may interfere with production. The bacteriophage pool action period focused on 3 h, but treatment of 9 h on glass surface biofilms was superior to other treatments because it affected the largest number of samples. These results suggests that some surface types and Salmonella serotypes may promote biofilm formation and indicate bacteriophages as an alternative to control biofilms. But further studies are required to prove the effectiveness and safety of bacteriophage therapy as an alternative in the antimicrobial control in the processing plants. © 2017 Poultry Science Association Inc.

Susceptibility of Escherichia coli isolated from uteri of postpartum dairy cows to antibiotic and environmental bacteriophages. Part II: In vitro antimicrobial activity evaluation of a bacteriophage cocktail and several antibiotics.

PubMed

Santos, T M A; Gilbert, R O; Caixeta, L S; Machado, V S; Teixeira, L M; Bicalho, R C

2010-01-01

The use of pathogenic-specific antimicrobials, as proposed by bacteriophage therapy, is expected to reduce the incidence of resistance development. Eighty Escherichia coli isolated from uteri of Holstein dairy cows were phenotypically characterized for antimicrobial resistance to ampicillin, ceftiofur, chloramphenicol, florfenicol, spectinomycin, streptomycin, and tetracycline by broth microdilution method. The lytic activity of a bacteriophage cocktail against all isolates was performed by a similar method. Additionally, the effect of different concentrations of antimicrobials and multiplicities of infections (MOI) of the bacteriophage cocktail on E. coli growth curve was measured. Isolates exhibited resistance to ampicillin (33.7%), ceftiofur (1.2%), chloramphenicol (100%), and florfenicol (100%). All strains were resistant to at least 2 of the antimicrobial agents tested; multidrug resistance (>or=3 of 7 antimicrobials tested) was observed in 35% of E. coli isolates. The major multidrug resistance profile was found for ampicillin-chloramphenicol-florfenicol, which was observed in more than 96.4% of the multidrug-resistant isolates. The bacteriophage cocktail preparation showed strong antimicrobial activity against multidrug-resistant E. coli. Multiplicity of infection as low as 10(-4) affected the growth of the E. coli isolates. The ratio of 10 bacteriophage particles per bacterial cell (MOI=10(1)) was efficient in inhibiting at least 50% of all isolates. Higher MOI should be tested in future in vitro studies to establish ratios that completely inhibit bacterial growth during longer periods. All isolates resistant to florfenicol were resistant to chloramphenicol and, because florfenicol was recently introduced into veterinary clinics, this finding suggests that the selection pressure of chloramphenicol, as well as other antimicrobials, may still play a relevant role in the emergence and dissemination of florfenicol resistance in E. coli. The bacteriophage

In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections.

PubMed

Mendes, João J; Leandro, Clara; Mottola, Carla; Barbosa, Raquel; Silva, Filipa A; Oliveira, Manuela; Vilela, Cristina L; Melo-Cristino, José; Górski, Andrzej; Pimentel, Madalena; São-José, Carlos; Cavaco-Silva, Patrícia; Garcia, Miguel

2014-08-01

In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy. © 2014 The Authors.

Bacteriophages and medical oncology: targeted gene therapy of cancer.

PubMed

Bakhshinejad, Babak; Karimi, Marzieh; Sadeghizadeh, Majid

2014-08-01

Targeted gene therapy of cancer is of paramount importance in medical oncology. Bacteriophages, viruses that specifically infect bacterial cells, offer a variety of potential applications in biomedicine. Their genetic flexibility to go under a variety of surface modifications serves as a basis for phage display methodology. These surface manipulations allow bacteriophages to be exploited for targeted delivery of therapeutic genes. Moreover, the excellent safety profile of these viruses paves the way for their potential use as cancer gene therapy platforms. The merge of phage display and combinatorial technology has led to the emergence of phage libraries turning phage display into a high throughput technology. Random peptide libraries, as one of the most frequently used phage libraries, provide a rich source of clinically useful peptide ligands. Peptides are known as a promising category of pharmaceutical agents in medical oncology that present advantages such as inexpensive synthesis, efficient tissue penetration and the lack of immunogenicity. Phage peptide libraries can be screened, through biopanning, against various targets including cancer cells and tissues that results in obtaining cancer-homing ligands. Cancer-specific peptides isolated from phage libraries show huge promise to be utilized for targeting of various gene therapy vectors towards malignant cells. Beyond doubt, bacteriophages will play a more impressive role in the future of medical oncology.

Use of bacteriophage to prevent Pseudomonas aeruginosa contamination and fouling in Jet A aviation fuel.

PubMed

Bojanowski, Caitlin L; Crookes-Goodson, Wendy J; Robinson, Jayne B

2016-11-01

In the present study, the use of bacteriophages to prevent growth and/or biofouling by Pseudomonas aeruginosa PAO1 was investigated in microcosms containing Jet A aviation fuel as the carbon source. Bacteriophages were found to be effective at preventing biofilm formation but did not always prevent planktonic growth in the microcosms. This result was at odds with experiments conducted in nutrient-rich medium, demonstrating the necessity to test antimicrobial and antifouling strategies under conditions as near as possible to the 'real world'. The success of the bacteriophages at preventing biofilm formation makes them potential candidates as antifouling agents for fuel systems.

Complete Nucleotide Sequence Analysis of a Novel Bacillus subtilis-Infecting Bacteriophage BSP10 and Its Effect on Poly-Gamma-Glutamic Acid Degradation

PubMed Central

Ghosh, Kuntal; Senevirathne, Amal; Kang, Hai Seong; Hyun, Woo Bin; Kim, Ji Eun; Kim, Kwang-Pyo

2018-01-01

While the harmful effects of lactic acid bacterial bacteriophages in the dairy industry are well-established, the importance of Bacillus subtilis-infecting bacteriophages on soybean fermentation is poorly-studied. In this study, we isolated a B. subtilis-infecting bacteriophage BSP10 from Meju (a brick of dried fermented soybean) and further characterized it. This Myoviridae family bacteriophage exhibited a narrow host range against B. subtilis strains (17/52, 32.7%). The genome of bacteriophage BSP10 is 153,767 bp long with 236 open reading frames and 5 tRNAs. Comparative genomics (using dot plot, progressiveMauve alignment, heat-plot, and BLASTN) and phylogenetic analysis strongly suggest its incorporation as a new species in the Nit1virus genus. Furthermore, bacteriophage BSP10 was efficient in the growth inhibition of B. subtilis ATCC 15245 in liquid culture and in Cheonggukjang (a soybean fermented food) fermentation. Artificial contamination of as low as 102 PFU/g of bacteriophage BSP10 during Cheonggukjang fermentation significantly reduced bacterial numbers by up to 112 fold in comparison to the control (no bacteriophage). Moreover, for the first time, we experimentally proved that B. subtilis-infecting bacteriophage greatly enhanced poly-γ-glutamic acid degradation during soybean fermentation, which is likely to negatively affect the functionalities of Cheonggukjang. PMID:29734701

Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors.

PubMed

Latham, Catherine F; La, Jennifer; Tinetti, Ricky N; Chalmers, David K; Tachedjian, Gilda

2016-01-01

Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (<300 Da) screened using primarily biophysical assays. FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment-based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs.

Engineering of M13 Bacteriophage for Development of Tissue Engineering Materials.

PubMed

Jin, Hyo-Eon; Lee, Seung-Wuk

2018-01-01

M13 bacteriophages have several qualities that make them attractive candidates as building blocks for tissue regenerating scaffold materials. Through genetic engineering, a high density of functional peptides and proteins can be simultaneously displayed on the M13 bacteriophage's outer coat proteins. The resulting phage can self-assemble into nanofibrous network structures and can guide the tissue morphogenesis through proliferation, differentiation and apoptosis. In this manuscript, we will describe methods to develop major coat-engineered M13 phages as a basic building block and aligned tissue-like matrices to develop regenerative nanomaterials.

Attenuation and colloidal mobilization of bacteriophages in natural sediments under anoxic as compared to oxic conditions.

PubMed

Klitzke, Sondra; Schroeder, Jendrik; Selinka, Hans-Christoph; Szewzyk, Regine; Chorus, Ingrid

2015-06-15

Redox conditions are known to affect the fate of viruses in porous media. Several studies report the relevance of colloid-facilitated virus transport in the subsurface, but detailed studies on the effect of anoxic conditions on virus retention in natural sediments are still missing. Therefore, we investigated the fate of viruses in natural flood plain sediments with different sesquioxide contents under anoxic conditions by considering sorption to the solid phase, sorption to mobilized colloids, and inactivation in the aqueous phase. Batch experiments were conducted under oxic and anoxic conditions at pH values between 5.1 and 7.6, using bacteriophages MS2 and PhiX174 as model viruses. In addition to free and colloid-associated bacteriophages, dissolved and colloidal concentrations of Fe, Al and organic C as well as dissolved Ca were determined. Results showed that regardless of redox conditions, bacteriophages did not adsorb to mobilized colloids, even under favourable charge conditions. Under anoxic conditions, attenuation of bacteriophages was dominated by sorption over inactivation, with MS2 showing a higher degree of sorption than PhiX174. Inactivation in water was low under anoxic conditions for both bacteriophages with about one log10 decrease in concentration during 16 h. Increased Fe/Al concentrations and a low organic carbon content of the sediment led to enhanced bacteriophage removal under anoxic conditions. However, even in the presence of sufficient Fe/A-(hydr)oxides on the solid phase, bacteriophage sorption was low. We presume that organic matter may limit the potential retention of sesquioxides in anoxic sediments and should thus be considered for the risk assessment of virus breakthrough in the subsurface. Copyright © 2015 Elsevier B.V. All rights reserved.

Bacteriophages infecting Bacteroides as a marker for microbial source tracking.

PubMed

Jofre, Joan; Blanch, Anicet R; Lucena, Francisco; Muniesa, Maite

2014-05-15

Bacteriophages infecting certain strains of Bacteroides are amid the numerous procedures proposed for tracking the source of faecal pollution. These bacteriophages fulfil reasonably well most of the requirements identified as appropriate for a suitable marker of faecal sources. Thus, different host strains are available that detect bacteriophages preferably in water contaminated with faecal wastes corresponding to different animal species. For phages found preferably in human faecal wastes, which are the ones that have been more extensively studied, the amounts of phages found in waters contaminated with human fecal samples is reasonably high; these amounts are invariable through the time; their resistance to natural and anthropogenic stressors is comparable to that of other relatively resistant indicator of faecal pollution such us coliphages; the abundance ratios of somatic coliphages and bacteriophages infecting Bacteroides thetaiotaomicron GA17 are unvarying in recent and aged contamination; and standardised detection methods exist. These methods are easy, cost effective and provide data susceptible of numerical analysis. In contrast, there are some uncertainties regarding their geographical stability, and consequently suitable hosts need to be isolated for different geographical areas. However, a feasible method has been described to isolate suitable hosts in a given geographical area. In summary, phages infecting Bacteroides are a marker of faecal sources that in our opinion merits being included in the "toolbox" for microbial source tracking. However, further research is still needed in order to make clear some uncertainties regarding some of their characteristics and behaviour, to compare their suitability to the one of emerging methods such us targeting Bacteroidetes by qPCR assays; or settling molecular methods for their determination. Copyright © 2014 Elsevier Ltd. All rights reserved.

Bacteriophage application on red meats and poultry: Effects on Salmonella population in final ground products.

PubMed

Yeh, Y; Purushothaman, P; Gupta, N; Ragnone, M; Verma, S C; de Mello, A S

2017-05-01

This research was conducted to study the effects of bacteriophage application during tumbling on Salmonella populations in ground meat and poultry. Red meat trim and poultry were inoculated with a Salmonella cocktail to result in a contamination level of 7logCFU/g in ground products. A commercial preparation containing bacteriophages S16 and Felix-O1a (FO1a) was applied during tumbling at 10 7 and 10 8 PFU/ml. Samples were held at 4°C for 6h and 18h (red meat) and 30min and 6h (poultry). Overall, bacteriophage application on trim reduced 1 and 0.8logCFU/g of Salmonella in ground beef and ground pork, respectively. For ground chicken and ground turkey, Salmonella was reduced by 1.1 and 0.9logCFU/g, respectively. This study shows that bacteriophage application during tumbling of red meat trim and poultry can provide additional Salmonella control in ground products. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay.

PubMed

Diamond, Tracy L; Bushman, Frederic D

2006-01-01

Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn2+ binds tightly to the sulfur atoms of the cysteine residues, but Mg2+ does not. We found that Mn2+, but not Mg2+, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5' hydroxyl groups as well as 3' hydroxyls as nucleophiles. Lastly, we show that Mn2+ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold.

Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay

PubMed Central

Diamond, Tracy L.; Bushman, Frederic D.

2006-01-01

Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn2+ binds tightly to the sulfur atoms of the cysteine residues, but Mg2+ does not. We found that Mn2+, but not Mg2+, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5′ hydroxyl groups as well as 3′ hydroxyls as nucleophiles. Lastly, we show that Mn2+ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold. PMID:17085478

Molecular and chemical engineering of bacteriophages for potential medical applications.

PubMed

Hodyra, Katarzyna; Dąbrowska, Krystyna

2015-04-01

Recent progress in molecular engineering has contributed to the great progress of medicine. However, there are still difficult problems constituting a challenge for molecular biology and biotechnology, e.g. new generation of anticancer agents, alternative biosensors or vaccines. As a biotechnological tool, bacteriophages (phages) offer a promising alternative to traditional approaches. They can be applied as anticancer agents, novel platforms in vaccine design, or as target carriers in drug discovery. Phages also offer solutions for modern cell imaging, biosensor construction or food pathogen detection. Here we present a review of bacteriophage research as a dynamically developing field with promising prospects for further development of medicine and biotechnology.

The integrase of the long terminal repeat-retrotransposon tf1 has a chromodomain that modulates integrase activities.

PubMed

Hizi, Amnon; Levin, Henry L

2005-11-25

Chromodomains in a variety of proteins mediate the formation of heterochromatin by interacting directly with histone H3, DNA, or RNA. A diverse family of long terminal repeat (LTR)-retrotransposons possesses chromodomains in their integrases (IN), suggesting that the chromodomains may control integration. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is highly active and possesses a chromodomain in the COOH terminus of its IN. To test this chromodomain for a role in integration, recombinant INs with and without the chromodomain were assayed for activity in in vitro reactions. The full-length IN had integration activity with oligonucleotide substrates that modeled both the insertion reaction and a reverse reaction known as disintegration. The INs of retroviruses possess an additional activity termed 3' processing that must remove 2-3 nucleotides from the 3' ends of the viral cDNA before insertion can occur. These additional nucleotides are added during reverse transcription because of the position of the minus strand primer downstream of the LTR. The position of the primer for Tf1 suggests no nucleotides are added 3' of the LTR. It was therefore surprising that Tf1 IN was capable of 3' cleavage. The most unexpected result reported here was that the IN lacking the chromodomain had significantly higher activity and substantially reduced substrate specificity. These results reveal that both the activity and specificity of enzymes can be modulated by their chromodomains.

[Isolation and characterization of a lytic bacteriophage from Mingyong glacier melt water].

PubMed

Li, Mingyuan; Ji, Xiuling; Wang, Baoqiang; Zhang, Qi; Lin, Lianbing; Zhang, Bing; Wei, Yunlin

2012-02-04

Glacier is a unique ecological system. This study focused on the isolation and characterization of a cold-active bateriophage from Mingyong glacier area in northwest Yunnan. Bacterial strains isolated from glacial melt water were used as host cells to isolate and purify bacteriophages by double-layer plate method. The morphology of the isolated phages and their host strains were observed by electron microscope. Restriction fragment length polymorphism (RFLP) analysis of genomic DNA, constituent proteins and physiological analysis of the bacteriophages were further carried out to characterize the phages. A lytic cold-active bacteriophage, designated as MYSP03, was isolated from Mingyong glacier. Its host strain MYB03 was identified as a member of genus Flavobacterium, based on the 16S rRNA sequence analysis. The bacteriophage MYSP03 has a isometric head (about 72 nm in diameter) and a long tail (about 240 nm in length and 10 nm in width), but no envelope was detected. Physiological analysis results showed that MYSP03 had infection activity at 4 degrees C, and clear and transparent plaques were formed on double-layer plates between 4 and 20 degrees C. Its optimum infection temperature was 10 degrees C and optimal pH 9.4, respectively. It is insensitive to chloroform. Furthermore, the genome of MYSP03 consists of double-stranded DNA and is approximately 66 kb.

Characterization of a novel lytic bacteriophage from an industrial Escherichia coli fermentation process and elimination of virulence using a heterologous CRISPR-Cas9 system.

PubMed

Halter, Mathew C; Zahn, James A

2018-03-01

Bacterial-bacteriophage interactions are a well-studied and ecologically-important aspect of microbiology. Many commercial fermentation processes are susceptible to bacteriophage infections due to the use of high-density, clonal cell populations. Lytic infections of bacterial cells in these fermentations are especially problematic due to their negative impacts on product quality, asset utilization, and fouling of downstream equipment. Here, we report the isolation and characterization of a novel lytic bacteriophage, referred to as bacteriophage DTL that is capable of rapid lytic infections of an Escherichia coli K12 strain used for commercial production of 1,3-propanediol (PDO). The bacteriophage genome was sequenced and annotated, which identified 67 potential open-reading frames (ORF). The tail fiber ORF, the largest in the genome, was most closely related to bacteriophage RTP, a T1-like bacteriophage reported from a commercial E. coli fermentation process in Germany. To eliminate virulence, both a fully functional Streptococcus thermophilus CRISPR3 plasmid and a customized S. thermophilus CRISPR3 plasmid with disabled spacer acquisition elements and seven spacers targeting the bacteriophage DTL genome were constructed. Both plasmids were separately integrated into a PDO production strain, which was subsequently infected with bacteriophage DTL. The native S. thermophilus CRISPR3 operon was shown to decrease phage susceptibility by approximately 96%, while the customized CRISPR3 operon provided complete resistance to bacteriophage DTL. The results indicate that the heterologous bacteriophage-resistance system described herein is useful in eliminating lytic infections of bacteriophage DTL, which was prevalent in environment surrounding the manufacturing facility.

Screening of Pro-Asp Sequences Exposed on Bacteriophage M13 as an Ideal Anchor for Gold Nanocubes.

PubMed

Lee, Hwa Kyoung; Lee, Yujean; Kim, Hyori; Lee, Hye-Eun; Chang, Hyejin; Nam, Ki Tae; Jeong, Dae Hong; Chung, Junho

2017-09-15

Bacteriophages are thought to be ideal vehicles for linking antibodies to nanoparticles. Here, we define the sequence of peptides exposed as a fusion protein on M13 bacteriophages to yield optimal binding of gold nanocubes and efficient bacteriophage amplification. We generated five helper bacteriophage libraries using AE(X) 2 DP, AE(X) 3 DP, AE(X) 4 DP, AE(X) 5 DP, and AE(X) 6 DP as the exposed portion of pVIII, in which X was a randomized amino acid residue encoded by the nucleotide sequence NNK. Efficient phage amplification was achievable only in the AE(X) 2 DP, AE(X) 3 DP, and AE(X) 4 DP libraries. Through biopanning with gold nanocubes, we enriched the phage clones and selected the clone with the highest fold change after enrichment. This clone displayed Pro-Asp on the surface of the bacteriophage and had amplification yields similar to those of the wild-type helper bacteriophage (VCSM13). The clone displayed even binding of gold nanocubes along its length and minimal aggregation after binding. We conclude that, for efficient amplification, the exposed pVIII amino acid length should be limited to six residues and Ala-Glu-Pro-Asp-Asp-Pro (AEPDDP) is the ideal fusion protein sequence for guaranteeing the optimal formation of a complex with gold nanocubes.

STUDIES ON THE BACTERIOPHAGE OF D'HÉRELLE

PubMed Central

Hetler, D. M.; Bronfenbrenner, J.

1928-01-01

1. During the process of lysis by bacteriophage, there is an appreciable increase in the amount of free amino acid present in the culture. 2. The increase of free amino acid is due to hydrolysis of bacterial protein. PMID:19869482

Bacteriophage Transduction in Staphylococcus aureus.

PubMed

Olson, Michael E

2016-01-01

The genetic manipulation of Staphylococcus aureus for molecular experimentation is a valuable tool for assessing gene function and virulence. Genetic variability between strains coupled with difficult laboratory techniques for strain construction is a frequent roadblock in S. aureus research. Bacteriophage transduction greatly increases the speed and ease of S. aureus studies by allowing movement of chromosomal markers and plasmids between strains. This technique enables the S. aureus research community to focus investigations on clinically relevant isolates.

Bacteriophages: the possible solution to treat infections caused by pathogenic bacteria.

PubMed

El-Shibiny, Ayman; El-Sahhar, Salma

2017-11-01

Since their discovery in 1915, bacteriophages have been used to treat bacterial infections in animals and humans because of their unique ability to infect their specific bacterial hosts without affecting other bacterial populations. The research carried out in this field throughout the 20th century, largely in Georgia, part of USSR and Poland, led to the establishment of phage therapy protocols. However, the discovery of penicillin and sulfonamide antibiotics in the Western World during the 1930s was a setback in the advancement of phage therapy. The misuse of antibiotics has reduced their efficacy in controlling pathogens and has led to an increase in the number of antibiotic-resistant bacteria. As an alternative to antibiotics, bacteriophages have become a topic of interest with the emergence of multidrug-resistant bacteria, which are a threat to public health. Recent studies have indicated that bacteriophages can be used indirectly to detect pathogenic bacteria or directly as biocontrol agents. Moreover, they can be used to develop new molecules for clinical applications, vaccine production, drug design, and in the nanomedicine field via phage display.

Targeted binding of the M13 bacteriophage to thiamethoxam organic crystals.

PubMed

Cho, Whirang; Fowler, Jeffrey D; Furst, Eric M

2012-04-10

Phage display screening with a combinatorial library was used to identify M13-type bacteriophages that express peptides with selective binding to organic crystals of thiamethoxam. The six most strongly binding phages exhibit at least 1000 times the binding affinity of wild-type M13 and express heptapeptide sequences that are rich in hydrophobic, hydrogen-bonding amino acids and proline. Among the peptide sequences identified, M13 displaying the pIII domain heptapeptide ASTLPKA exhibits the strongest binding to thiamethoxam in competitive binding assays. Electron and confocal microscopy confirm the specific binding affinity of ASTLPKA to thiamethoxam. Using atomic force microscope (AFM) probes functionalized with ASTLPKA expressing phage, we found that the average adhesion force between the bacteriophage and a thiamethoxam surface is 1.47 ± 0.80 nN whereas the adhesion force of wild-type M13KE phage is 0.18 ± 0.07 nN. Such a strongly binding bacteriophage could be used to modify the surface chemistry of thiamethoxam crystals and other organic solids with a high degree of specificity. © 2012 American Chemical Society

Comparative genomic analysis of novel bacteriophages infecting Vibrio parahaemolyticus isolated from western and southern coastal areas of Korea.

PubMed

Yu, Junhyeok; Lim, Jeong-A; Kwak, Su-Jin; Park, Jong-Hyun; Chang, Hyun-Joo

2018-05-01

Vibrio parahaemolyticus, a foodborne pathogen, has become resistant to antibiotics. Therefore, alternative bio-control agents such bacteriophage are urgently needed for its control. Six novel bacteriophages specific to V. parahaemolyticus (vB_VpaP_KF1~2, vB_VpaS_KF3~6) were characterized at the molecular level in this study. Genomic similarity analysis revealed that these six bacteriophages could be divided into two groups with different genomic features, phylogenetic grouping, and morphologies. Two groups of bacteriophages had their own genes with different mechanisms for infection, assembly, and metabolism. Our results could be used as a future reference to study phage genomics or apply phages in future bio-control studies.

Isolation and characterization of two bacteriophages with strong in vitro antimicrobial activity against Pseudomonas aeruginosa isolated from dogs with ocular infections.

PubMed

Santos, Thiago M A; Ledbetter, Eric C; Caixeta, Luciano S; Bicalho, Marcela L S; Bicalho, Rodrigo C

2011-08-01

To isolate and characterize bacteriophages with strong in vitro lytic activity against various pathogenic Pseudomonas aeruginosa strains isolated from dogs with ocular infections. 26 genetically distinct P aeruginosa isolates. P aeruginosa strains were derived from dogs with naturally acquired ulcerative keratitis. From a large-scale screening for bacteriophages with potential therapeutic benefit against canine ocular infections, 2 bacteriophages (P2S2 and P5U5) were selected; host ranges were determined, and phage nucleic acid type and genetic profile were identified via enzymatic digestion. Electron microscopy was used to characterize bacteriophage ultrastructure. Bacteriophage temperature and pH stabilities were assessed by use of double-layer agar overlay titration. A cocultivation assay was used to evaluate the effect of the bacteriophages on bacterial host growth. P5U5 was active against all P aeruginosa isolates, whereas P2S2 formed lytic plaques on plates of 21 (80.8%) isolates. For each bacteriophage, the genomic nucleic acid was DNA; each was genetically distinct. Ultrastructurally, P2S2 and P5U5 appeared likely to belong to the Podoviridae and Siphoviridae families, respectively. The bacteriophages were stable within a pH range of 4 to 12; however, titers of both bacteriophages decreased following heating for 10 to 50 minutes at 45° or 60°C. Growth of each P aeruginosa isolate was significantly inhibited in coculture with P2S2 or P5U5; the dose response was related to the plaque-forming unit-to-CFU ratios. Bacteriophages P2S2 and P5U5 appear to be good candidates for phage treatment of infection caused by pathogenic P aeruginosa in dogs.

Characterization of tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurochkina, Lidia P., E-mail: lpk@ibch.r; Aksyuk, Anastasia A.; Sachkova, Maria Yu.

2009-12-20

The tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa encoded by gene 29 was identified and its expression system was developed. Localization of the protein on the virion was confirmed by immunoelectron microscopy. Properties of gene product (gp) 29 were studied by electron microscopy, immunoblotting and limited trypsinolysis. Recombinant gp29 assembles into the regular tubular structures (polysheaths) of variable length. Trypsin digestion of gp29 within polysheaths or extended sheath of virion results in specific cleavage of the peptide bond between Arg135 and Asp136. However, this cleavage does not affect polymeric structure of polysheaths, sheaths and viral infectivity. Digestion bymore » trypsin of the C-truncated gp29 mutant, lacking the ability to self-assemble, results in formation of a stable protease-resistant fragment. Although there is no sequence homology of phiKZ proteins to proteins of other bacteriophages, some characteristic biochemical properties of gp29 revealed similarities to the tail sheath protein of bacteriophage T4.« less

Structure of the bacteriophage T4 long tail fiber receptor-binding tip

PubMed Central

Bartual, Sergio G.; Otero, José M.; Garcia-Doval, Carmela; Llamas-Saiz, Antonio L.; Kahn, Richard; Fox, Gavin C.; van Raaij, Mark J.

2010-01-01

Bacteriophages are the most numerous organisms in the biosphere. In spite of their biological significance and the spectrum of potential applications, little high-resolution structural detail is available on their receptor-binding fibers. Here we present the crystal structure of the receptor-binding tip of the bacteriophage T4 long tail fiber, which is highly homologous to the tip of the bacteriophage lambda side tail fibers. This structure reveals an unusual elongated six-stranded antiparallel beta-strand needle domain containing seven iron ions coordinated by histidine residues arranged colinearly along the core of the biological unit. At the end of the tip, the three chains intertwine forming a broader head domain, which contains the putative receptor interaction site. The structure reveals a previously unknown beta-structured fibrous fold, provides insights into the remarkable stability of the fiber, and suggests a framework for mutations to expand or modulate receptor-binding specificity. PMID:21041684

Liposome-Encapsulated Bacteriophages for Enhanced Oral Phage Therapy against Salmonella spp.

PubMed

Colom, Joan; Cano-Sarabia, Mary; Otero, Jennifer; Cortés, Pilar; Maspoch, Daniel; Llagostera, Montserrat

2015-07-01

Bacteriophages UAB_Phi20, UAB_Phi78, and UAB_Phi87 were encapsulated in liposomes, and their efficacy in reducing Salmonella in poultry was then studied. The encapsulated phages had a mean diameter of 309 to 326 nm and a positive charge between +31.6 and +35.1 mV (pH 6.1). In simulated gastric fluid (pH 2.8), the titer of nonencapsulated phages decreased by 5.7 to 7.8 log units, whereas encapsulated phages were significantly more stable, with losses of 3.7 to 5.4 log units. The liposome coating also improved the retention of bacteriophages in the chicken intestinal tract. When cocktails of the encapsulated and nonencapsulated phages were administered to broilers, after 72 h the encapsulated phages were detected in 38.1% of the animals, whereas the nonencapsulated phages were present in only 9.5%. The difference was significant. In addition, in an in vitro experiment, the cecal contents of broilers promoted the release of the phages from the liposomes. In broilers experimentally infected with Salmonella, the daily administration of the two cocktails for 6 days postinfection conferred similar levels of protection against Salmonella colonization. However, once treatment was stopped, protection by the nonencapsulated phages disappeared, whereas that provided by the encapsulated phages persisted for at least 1 week, showing the enhanced efficacy of the encapsulated phages in protecting poultry against Salmonella over time. The methodology described here allows the liposome encapsulation of phages of different morphologies. The preparations can be stored for at least 3 months at 4°C and could be added to the drinking water and feed of animals. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Bacteriophages of methanotrophic bacteria.

PubMed Central

Tyutikov, F M; Bespalova, I A; Rebentish, B A; Aleksandrushkina, N N; Krivisky, A S

1980-01-01

Bacteriophages of methanotrophic bacteria have been found in 16 out of 88 studied samples (underground waters, pond water, soil, gas and oil installation waters, fermentor cultural fluids, bacterial paste, and rumen of cattle) taken in different geographic zones of the Soviet Union. Altogether, 23 phage strains were isolated: 10 strains that specifically lysed only Methylosinus sporium strains, 2 strains that each lysed 1 of 5 Methylosinus trichosporium strains studied, and 11 strains that lysed Flavobacterium gasotypicum and, at the same time, 1 M. sporium strain. By fine structure, the phages were divided into two types (with very short or long noncontractile tails); by host range and serological properties, they fell into three types. One-step growth characteristics of the phages differed only slightly; the latent period varied from 6 to 8 h, the rise period varied from 4 to 6 h, and the average burst size was 100. All phages had guanine- and cytosine-rich double-stranded deoxyribonucleic acid consisting of common nitrogen bases. The molecular mass of the deoxyribonucleic acid as determined by restriction endonuclease analysis was 29.4 X 10(6) for M. sporium phages and 44 X 10(6) for F. gasotypicum phages. By all of the above-mentioned properties, all phages within each of the groups were completely identical to one another, but differed from phages of other groups. Bacteriophages lysing M. sporium and M. trichosporium GB2 were identical to phages M1 and M4, respectively, which were isolated earlier in the German Democratic Republic on the same methanotrophic species. Images PMID:6774962

BACTERIOPHAGE TRANSPORT IN SANDY SOIL AND FRACTURED TUFF

EPA Science Inventory

Bacteriophage transport was investigated in laboratory column experiments using sandy soil, a controlled field study in a sandy wash, and laboratory experiments using fractured rock. In the soil columns, the phage MS-2 exhibited significant dispersion and was excluded from 35 to ...

Polymorphism of DNA conformation inside the bacteriophage capsid.

PubMed

Leforestier, Amélie

2013-03-01

Double-stranded DNA bacteriophage genomes are packaged into their icosahedral capsids at the highest densities known so far (about 50 % w:v). How the molecule is folded at such density and how its conformation changes upon ejection or packaging are fascinating questions still largely open. We review cryo-TEM analyses of DNA conformation inside partially filled capsids as a function of the physico-chemical environment (ions, osmotic pressure, temperature). We show that there exists a wide variety of DNA conformations. Strikingly, the different observed structures can be described by some of the different models proposed over the years for DNA organisation inside bacteriophage capsids: either spool-like structures with axial or concentric symmetries, or liquid crystalline structures characterised by a DNA homogeneous density. The relevance of these conformations for the understanding of DNA folding and unfolding upon ejection and packaging in vivo is discussed.

Bacteriophages and their applications in the diagnosis and treatment of hepatitis B virus infection.

PubMed

Bakhshinejad, Babak; Sadeghizadeh, Majid

2014-09-07

Hepatitis B virus (HBV) infection is a major global health challenge leading to serious disorders such as cirrhosis and hepatocellular carcinoma. Currently, there exist various diagnostic and therapeutic approaches for HBV infection. However, prevalence and hazardous effects of chronic viral infection heighten the need to develop novel methodologies for the detection and treatment of this infection. Bacteriophages, viruses that specifically infect bacterial cells, with a long-established tradition in molecular biology and biotechnology have recently been introduced as novel tools for the prevention, diagnosis and treatment of HBV infection. Bacteriophages, due to tremendous genetic flexibility, represent potential to undergo a huge variety of surface modifications. This property has been the rationale behind introduction of phage display concept. This powerful approach, together with combinatorial chemistry, has shaped the concept of phage display libraries with diverse applications for the detection and therapy of HBV infection. This review aims to offer an insightful overview of the potential of bacteriophages in the development of helpful prophylactic (vaccine design), diagnostic and therapeutic strategies for HBV infection thereby providing new perspectives to the growing field of bacteriophage researches directing towards HBV infection.

Bacteriophages and their applications in the diagnosis and treatment of hepatitis B virus infection

PubMed Central

Bakhshinejad, Babak; Sadeghizadeh, Majid

2014-01-01

Hepatitis B virus (HBV) infection is a major global health challenge leading to serious disorders such as cirrhosis and hepatocellular carcinoma. Currently, there exist various diagnostic and therapeutic approaches for HBV infection. However, prevalence and hazardous effects of chronic viral infection heighten the need to develop novel methodologies for the detection and treatment of this infection. Bacteriophages, viruses that specifically infect bacterial cells, with a long-established tradition in molecular biology and biotechnology have recently been introduced as novel tools for the prevention, diagnosis and treatment of HBV infection. Bacteriophages, due to tremendous genetic flexibility, represent potential to undergo a huge variety of surface modifications. This property has been the rationale behind introduction of phage display concept. This powerful approach, together with combinatorial chemistry, has shaped the concept of phage display libraries with diverse applications for the detection and therapy of HBV infection. This review aims to offer an insightful overview of the potential of bacteriophages in the development of helpful prophylactic (vaccine design), diagnostic and therapeutic strategies for HBV infection thereby providing new perspectives to the growing field of bacteriophage researches directing towards HBV infection. PMID:25206272

Quick bacteriophage-mediated bioluminescence assay for detecting Staphylococcus spp. in sonicate fluid of orthopaedic artificial joints.

PubMed

Šuster, Katja; Podgornik, Aleš; Cör, Andrej

2017-07-01

Staphylococcus spp. accounts for up to two thirds of all microorganisms causing prosthetic joint infections, with Staphylococcus aureus and Staphylococcus epidermidis being the major cause. The present study describes a diagnostic model to detect staphylococci using a specific bacteriophage and bioluminescence detection, exploring the possibility of its use on sonicate fluid of orthopaedic artificial joints. Intracellular adenosine-5'-triphosphate release by bacteriophage mediated lysis of staphylococci was assessed to determine optimal parameters for detection. With the optimized method, a limit of detection of around 103 CFU/mL was obtained after incubation with bacteriophage for 2 h. Importantly, sonicate fluid did not prevent the ability of bacteriophage to infect bacteria and all simulated infected sonicate fluid as well as 6 clinical samples with microbiologically proven staphylococcal infection were detected as positive. The total assay took approximately 4 h. Collectively, the results indicate that the developed method promises a rapid, inexpensive and specific diagnostic detection of staphylococci in sonicate fluid of infected prosthetic joints. In addition, the unlimited pool of different existing bacteriophages, with different specificity for all kind of bacteria gives the opportunity for further investigations, improvements of the current model and implementation in other medical fields for the purpose of the establishment of a rapid diagnosis.

Bacteriophages to combat foodborne infections caused by food contamination by bacteria of the Campylobacter genus.

PubMed

Myga-Nowak, Magdalena; Godela, Agnieszka; Głąb, Tomasz; Lewańska, Monika; Boratyński, Janusz

2016-09-26

It is estimated that each year more than 2 million people suffer from diarrheal diseases, resulting from the consumption of contaminated meat. Foodborne infections are most frequently caused by small Gram-negative rods Campylobacter. The hosts of these bacteria are mainly birds wherein they are part of the normal intestinal flora. During the commercial slaughter, there is a likelihood of contamination of carcasses by the bacteria found in the intestinal content. In Europe, up to 90% of poultry flocks can be a reservoir of the pathogen. According to the European Food Safety Authority report from 2015, the number of reported and confirmed cases of human campylobacteriosis exceeds 200 thousands per year, and such trend remains at constant level for several years. The occurrence of growing antibiotic resistance in bacteria forces the limitation of antibiotic use in the animal production. Therefore, the European Union allows only using stringent preventive and hygienic treatment on farms. Achieving Campylobacter free chickens using these methods is possible, but difficult to implement and expensive. Utilization of bacterial viruses - bacteriophages, can be a path to provide the hygienic conditions of poultry production and food processing. Formulations applied in the food protection should contain strictly lytic bacteriophages, be non-pyrogenic and retain long lasting biological activity. Currently, on the market there are available commercial bacteriophage preparations for agricultural use, but neither includes phages against Campylobacter. However, papers on the application of bacteriophages against Campylobacter in chickens and poultry products were published in the last few years. In accordance with the estimates, 2-logarithm reduction of Campylobacter in poultry carcases will contribute to the 30-fold reduction in the incidence of campylobacteriosis in humans. Research on bacteriophages against Campylobacter have cognitive and economic importance. The paper

Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

PubMed Central

Filippov, Andrey A.; Sergueev, Kirill V.; He, Yunxiu; Huang, Xiao-Zhe; Gnade, Bryan T.; Mueller, Allen J.; Fernandez-Prada, Carmen M.; Nikolich, Mikeljon P.

2011-01-01

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. PMID:21980477

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

PubMed

Filippov, Andrey A; Sergueev, Kirill V; He, Yunxiu; Huang, Xiao-Zhe; Gnade, Bryan T; Mueller, Allen J; Fernandez-Prada, Carmen M; Nikolich, Mikeljon P

2011-01-01

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. 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. 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.

RGD peptide-displaying M13 bacteriophage/PLGA nanofibers as cell-adhesive matrices for smooth muscle cells

NASA Astrophysics Data System (ADS)

Shin, Yong Cheol; Lee, Jong Ho; Jin, Oh Seong; Lee, Eun Ji; Jin, Lin Hua; Kim, Chang-Seok; Hong, Suck Won; Han, Dong-Wook; Kim, Chuntae; Oh, Jin-Woo

2015-01-01

Extracellular matrices (ECMs) are network structures that play an essential role in regulating cellular growth and differentiation. In this study, novel nanofibrous matrices were fabricated by electrospinning M13 bacteriophage and poly(lactic- co-glycolic acid) (PLGA) and were shown to be structurally and functionally similar to natural ECMs. A genetically-engineered M13 bacteriophage was constructed to display Arg-Gly-Asp (RGD) peptides on its surface. The physicochemical properties of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage)/PLGA nanofibers were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. We used immunofluorescence staining to confirm that M13 bacteriophages were homogenously distributed in RGD-M13 phage/PLGA matrices. Furthermore, RGD-M13 phage/PLGA nanofibrous matrices, having excellent biocompatibility, can enhance the behaviors of vascular smooth muscle cells. This result suggests that RGD-M13 phage/PLGA nanofibrous matrices have potentials to serve as tissue engineering scaffolds.

6-(1-Benzyl-1H-pyrrol-2-yl)-2,4-dioxo-5-hexenoic acids as dual inhibitors of recombinant HIV-1 integrase and ribonuclease H, synthesized by a parallel synthesis approach.

PubMed

Costi, Roberta; Métifiot, Mathieu; Esposito, Francesca; Cuzzucoli Crucitti, Giuliana; Pescatori, Luca; Messore, Antonella; Scipione, Luigi; Tortorella, Silvano; Zinzula, Luca; Novellino, Ettore; Pommier, Yves; Tramontano, Enzo; Marchand, Christophe; Di Santo, Roberto

2013-11-14

The increasing efficiency of HAART has helped to transform HIV/AIDS into a chronic disease. Still, resistance and drug-drug interactions warrant the development of new anti-HIV agents. We previously discovered hit 6, active against HIV-1 replication and targeting RNase H in vitro. Because of its diketo-acid moiety, we speculated that this chemotype could serve to develop dual inhibitors of both RNase H and integrase. Here, we describe a new series of 1-benzyl-pyrrolyl diketohexenoic derivatives, 7a-y and 8a-y, synthesized following a parallel solution-phase approach. Those 50 analogues have been tested on recombinant enzymes (RNase H and integrase) and in cell-based assays. Approximately half (22) exibited inhibition of HIV replication. Compounds 7b, 7u, and 8g were the most active against the RNase H activity of reverse-transcriptase, with IC50 values of 3, 3, and 2.5 μM, respectively. Compound 8g was also the most potent integrase inhibitor with an IC50 value of 26 nM.

Inactivation of F-specific bacteriophages during flocculation with polyaluminum chloride - a mechanistic study.

PubMed

Kreißel, Katja; Bösl, Monika; Hügler, Michael; Lipp, Pia; Franzreb, Matthias; Hambsch, Beate

2014-03-15

Bacteriophages are often used as surrogates for enteric viruses in spiking experiments to determine the efficiencies of virus removal of certain water treatment measures, like e.g. flocculation or filtration steps. Such spiking experiments with bacteriophages are indispensable if the natural virus concentrations in the raw water of water treatment plants are too low to allow the determination of elimination levels over several orders of magnitude. In order to obtain reliable results from such spiking tests, it is essential that bacteriophages behave comparable to viruses and remain stable during the experiments. To test this, the influence of flocculation parameters on the bacteriophages MS2, Qβ and phiX174 was examined. Notably, the F-specific phages MS2 and Qβ were found to be inactivated in flocculation processes with polyaluminum chloride (PACl). In contrast, other aluminum coagulants like AlCl3 or Al2(SO4)3 did not show a comparable effect on MS2 in this study. In experiments testing the influence of different PACl species on MS2 and Qβ inactivation during flocculation, it could be shown that cationic dissolved PACl species (Al13) interacted with the MS2 surface and hereby reduced the surviving phage fraction to c/c0 values below 1*10(-4) even at very low PACl concentrations of 7 μmol Al/L. Other inactivation mechanisms like the irreversible adsorption of phages to the floc structure or the damage of phage surfaces due to entrapment into the floc during coagulation and floc formation do not seem to contribute to the low surviving fraction found for both F-specific bacteriophages. Furthermore, no influence of phage agglomeration or pH drops during the flocculation process on phage inactivation could be observed. The somatic coliphage phiX174 in contrast did not show sensitivity to chemical stress and in accordance only slight interaction between Al13 and the phage surface was observed. Consequently, F-specific phages like MS2 should not be used as

In vitro evaluation of a novel bacteriophage cocktail as a preventative for bovine coliform mastitis.

PubMed

Porter, J; Anderson, J; Carter, L; Donjacour, E; Paros, M

2016-03-01

The objective of this study was to investigate the potential use of bacteriophage in preventing Escherichia coli mastitis on dairies. A cocktail consisting of 4 distinct bacteriophages was generated by screening against 36 E. coli isolates from dairy cows in Washington State with clinical mastitis. The bacteriophage significantly inhibited growth of 58% of the Washington State isolates and 54% of E. coli mastitis isolates from New York State, suggesting that the cocktail of phages had a relatively broad spectrum of action against relevant strains from 2 distinct geographies. The ability to suppress bacterial growth of these isolates in a liquid growth medium was not affected by the ratio of bacteriophage particles to bacterial cells (multiplicity of infection, MOI). For those E. coli that were completely inhibited by the phage cocktail, an MOI as low as 10 had the same effect as 10 µg/mL of ceftiofur on the growth rate of E. coli over a 12-h period using optical density measurements. A 3.3- to 5.6-log reduction of growth was achieved when E. coli was co-incubated with our phage cocktail in raw milk over a 12-h period at physiologic temperature. A modified gentamicin protection assay using bovine mammary epithelial cells provided a model to test whether bacteriophage could prevent cell attachment and invasion by chronic coliform mastitis strains. Pretreatment of cell cultures with the phage cocktail significantly reduced adhesion and intracellular survival of E. coli compared with controls. When combined with a bismuth-based teat sealant, the phage cocktail was able to inhibit bacterial growth when challenged with 1.6 × 10(3) cfu/mL of a clinical mastitis E. coli strain. In vitro results show bactericidal activity by our phage in raw milk and mammary tissue culture systems. Before a bacteriophage-based dry-cow treatment becomes a potential option for dairies, in vivo studies must be able to demonstrate that a specific dose of bacteriophage can protect cows from

Identification of novel bacteriophage peptides using a combination of gene sequence LC-MS-MS analysis and BLASTP

USDA-ARS?s Scientific Manuscript database

Introduction: In an effort to characterize novel bacteriophage with lytic activity against pathogenic E.coli associated with foodborne illness, gene sequencing and mass spectrometry have been used to identify expressed peptides which differentiate isolated bacteriophage from other known phage. Here,...

Comparative analysis of the biological and physical properties of Enterococcus faecalis bacteriophage vB_EfaS_GEC-EfS_3 and Streptococcus mitis bacteriophage vB_SmM_GEC-SmitisM_2.

PubMed

Rigvava, Sophio; Tchgkonia, Irina; Jgenti, Darejan; Dvalidze, Teona; Carpino, James; Goderdzishvili, Marina

2013-01-01

Enterococcus faecalis and Streptococcus mitis are common commensal inhabitants of the human gastrointestinal and genitourinary tracts. However, both species can be opportunistic pathogens and cause disease in nosocomial settings. These infections can be difficult to treat because of the frequency of antibiotic resistance among these strains. Bacteriophages are often suggested as an alternative therapeutic agent against these infections. In this study, E. faecalis and S. mitis strains were isolated from female patients with urinary tract infections. Bacteriophages active against these strains were isolated from sewage water from the Mtkvari River. Two phages, designated vB_EfaS_GEC-EfS_3 (Syphoviridae) and vB_SmM_GEC-SmitisM_2 (Myoviridae), were specific for E. faecalis and S. mitis, respectively. Each phage's growth patterns and adsorption rates were quantified. Sensitivity to ultraviolet light and temperature was determined, as was host range and serology. The S. mitis bacteriophage was found to be more resistant to ultraviolet light and exposure to high temperatures than the E. faecalis bacteriophage, despite having a much greater rate of replication. While each phage was able to infect a broad range of strains of the same species as the host species from which they were isolated, they were unable to infect other host species tested.

Top-down effects of a lytic bacteriophage and protozoa on bacteria in aqueous and biofilm phases.

PubMed

Zhang, Ji; Ormälä-Odegrip, Anni-Maria; Mappes, Johanna; Laakso, Jouni

2014-12-01

Lytic bacteriophages and protozoan predators are the major causes of bacterial mortality in natural microbial communities, which also makes them potential candidates for biological control of bacterial pathogens. However, little is known about the relative impact of bacteriophages and protozoa on the dynamics of bacterial biomass in aqueous and biofilm phases. Here, we studied the temporal and spatial dynamics of bacterial biomass in a microcosm experiment where opportunistic pathogenic bacteria Serratia marcescens was exposed to particle-feeding ciliates, surface-feeding amoebas, and lytic bacteriophages for 8 weeks, ca. 1300 generations. We found that ciliates were the most efficient enemy type in reducing bacterial biomass in the open water, but least efficient in reducing the biofilm biomass. Biofilm was rather resistant against bacterivores, but amoebae had a significant long-term negative effect on bacterial biomass both in the open-water phase and biofilm. Bacteriophages had only a minor long-term effect on bacterial biomass in open-water and biofilm phases. However, separate short-term experiments with the ancestral bacteriophages and bacteria revealed that bacteriophages crash the bacterial biomass dramatically in the open-water phase within the first 24 h. Thereafter, the bacteria evolve phage-resistance that largely prevents top-down effects. The combination of all three enemy types was most effective in reducing biofilm biomass, whereas in the open-water phase the ciliates dominated the trophic effects. Our results highlight the importance of enemy feeding mode on determining the spatial distribution and abundance of bacterial biomass. Moreover, the enemy type can be crucially important predictor of whether the rapid defense evolution can significantly affect top-down regulation of bacteria.

Characterization of a novel bacteriophage, Phda1, infecting the histamine-producing Photobacterium damselae subsp. damselae.

PubMed

Yamaki, S; Kawai, Y; Yamazaki, K

2015-06-01

Photobacterium damselae subsp. damselae is a potent histamine-producing micro-organism. The aim of this study was to isolate and characterize a bacteriophage Phda1 that infected P. damselae subsp. damselae to inhibit its growth and histamine accumulation. Phda1 was isolated from a raw oyster, and the host range, morphology and the bacteriophage genome size were analysed. Phda1 formed a clear plaque only against P. damselae subsp. damselae JCM8969 among five Gram-positive and 32 Gram-negative bacterial strains tested. Phda1 belongs to the family Myoviridae, and its genome size was estimated as 35·2-39·5 kb. According to the one-step growth curve analysis, the latent period, rise period and burst size of Phda1 were 60 min, 50 min and 19 plaque-forming units per infected cell, respectively. Divalent cations, especially Ca(2+) and Mg(2+) , strongly improved Phda1 adsorption to the host cells and its propagation. Phda1 treatment delayed the growth and histamine production of P. damselae subsp. damselae in an in vitro challenge test. The bacteriophage Phda1 might serve as a potential antimicrobial agent to inhibit the histamine poisoning caused by P. damselae subsp. damselae. This is the first description of a bacteriophage specifically infecting P. damselae subsp. damselae and its potential applications. Bacteriophage therapy could prove useful in the prevention of histamine poisoning. © 2015 The Society for Applied Microbiology.

21 CFR 172.785 - Listeria-specific bacteriophage preparation.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Listeria-specific bacteriophage preparation. 172.785 Section 172.785 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO...

Abundance of Antibiotic Resistance Genes in Bacteriophage following Soil Fertilization with Dairy Manure or Municipal Biosolids, and Evidence for Potential Transduction

PubMed Central

Ross, Joseph

2015-01-01

Animal manures and municipal biosolids recycled onto crop production land carry antibiotic-resistant bacteria that can influence the antibiotic resistome of agricultural soils, but little is known about the contribution of bacteriophage to the dissemination of antibiotic resistance genes (ARGs) in this context. In this work, we quantified a set of ARGs in the bacterial and bacteriophage fractions of agricultural soil by quantitative PCR. All tested ARGs were present in both the bacterial and phage fractions. We demonstrate that fertilization of soil with dairy manure or human biosolids increases ARG abundance in the bacterial fraction but not the bacteriophage fraction and further show that pretreatment of dairy manure can impact ARG abundance in the bacterial fraction. Finally, we show that purified bacteriophage can confer increased antibiotic resistance to soil bacteria when combined with selective pressure. The results indicate that soilborne bacteriophage represents a substantial reservoir of antibiotic resistance and that bacteriophage could play a significant role in the horizontal transfer of resistance genes in the context of an agricultural soil microbiome. Overall, our work reinforces the advisability of composting or digesting fecal material prior to field application and suggests that application of some antibiotics at subclinical concentrations can promote bacteriophage-mediated horizontal transfer of ARGs in agricultural soil microbiomes. PMID:26341211

Insights into Bacteriophage Application in Controlling Vibrio Species

PubMed Central

Letchumanan, Vengadesh; Chan, Kok-Gan; Pusparajah, Priyia; Saokaew, Surasak; Duangjai, Acharaporn; Goh, Bey-Hing; Ab Mutalib, Nurul-Syakima; Lee, Learn-Han

2016-01-01

Bacterial infections from various organisms including Vibrio sp. pose a serious hazard to humans in many forms from clinical infection to affecting the yield of agriculture and aquaculture via infection of livestock. Vibrio sp. is one of the main foodborne pathogens causing human infection and is also a common cause of losses in the aquaculture industry. Prophylactic and therapeutic usage of antibiotics has become the mainstay of managing this problem, however, this in turn led to the emergence of multidrug resistant strains of bacteria in the environment; which has raised awareness of the critical need for alternative non-antibiotic based methods of preventing and treating bacterial infections. Bacteriophages – viruses that infect and result in the death of bacteria – are currently of great interest as a highly viable alternative to antibiotics. This article provides an insight into bacteriophage application in controlling Vibrio species as well underlining the advantages and drawbacks of phage therapy. PMID:27486446

Bacteriophages and phage-inspired nanocarriers for targeted delivery of therapeutic cargos.

PubMed

Karimi, Mahdi; Mirshekari, Hamed; Moosavi Basri, Seyed Masoud; Bahrami, Sajad; Moghoofei, Mohsen; Hamblin, Michael R

2016-11-15

The main goal of drug delivery systems is to target therapeutic cargoes to desired cells and to ensure their efficient uptake. Recently a number of studies have focused on designing bio-inspired nanocarriers, such as bacteriophages, and synthetic carriers based on the bacteriophage structure. Bacteriophages are viruses that specifically recognize their bacterial hosts. They can replicate only inside their host cell and can act as natural gene carriers. Each type of phage has a particular shape, a different capacity for loading cargo, a specific production time, and their own mechanisms of supramolecular assembly, that have enabled them to act as tunable carriers. New phage-based technologies have led to the construction of different peptide libraries, and recognition abilities provided by novel targeting ligands. Phage hybridization with non-organic compounds introduces new properties to phages and could be a suitable strategy for construction of bio-inorganic carriers. In this review we try to cover the major phage species that have been used in drug and gene delivery systems, and the biological application of phages as novel targeting ligands and targeted therapeutics. Copyright © 2016 Elsevier B.V. All rights reserved.

Evidence of translation efficiency adaptation of the coding regions of the bacteriophage lambda.

PubMed

Goz, Eli; Mioduser, Oriah; Diament, Alon; Tuller, Tamir

2017-08-01

Deciphering the way gene expression regulatory aspects are encoded in viral genomes is a challenging mission with ramifications related to all biomedical disciplines. Here, we aimed to understand how the evolution shapes the bacteriophage lambda genes by performing a high resolution analysis of ribosomal profiling data and gene expression related synonymous/silent information encoded in bacteriophage coding regions.We demonstrated evidence of selection for distinct compositions of synonymous codons in early and late viral genes related to the adaptation of translation efficiency to different bacteriophage developmental stages. Specifically, we showed that evolution of viral coding regions is driven, among others, by selection for codons with higher decoding rates; during the initial/progressive stages of infection the decoding rates in early/late genes were found to be superior to those in late/early genes, respectively. Moreover, we argued that selection for translation efficiency could be partially explained by adaptation to Escherichia coli tRNA pool and the fact that it can change during the bacteriophage life cycle.An analysis of additional aspects related to the expression of viral genes, such as mRNA folding and more complex/longer regulatory signals in the coding regions, is also reported. The reported conclusions are likely to be relevant also to additional viruses. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Application of bacteriophages specific to hydrogen sulfide-producing bacteria in raw poultry by-products.

PubMed

Gong, Chao; Liu, Xiaohua; Jiang, Xiuping

2014-03-01

Hydrogen sulfide-producing bacteria (SPB) can spoil raw animal materials and release harmful hydrogen sulfide (H2S) gas. The objective of this study was to apply a SPB-specific bacteriophage cocktail to control H2S production by SPB in different raw poultry by-products in the laboratory (20, 30, and 37°C) and greenhouse (average temperature 29 to 31°C, humidity 34.8 to 59.8%, and light intensity 604.8 Wm(2)) by simulating transportation and a rendering facility. The amount of H2S production was determined using either test strips impregnated with lead acetate or a H2S monitor. In the laboratory, phage treatment applied to fresh chicken meat inoculated with SPB, spoiled chicken meat, chicken guts, and chicken feathers reduced H2S production by approximately 25 to 69% at temperatures from 20 to 37°C. In the greenhouse, phage treatment achieved approximately a 30 to 85% reduction of H2S yield in chicken offal and feathers. Among all phage treatments, multiplicity of infection (MOI) of 100 exhibited the highest inhibitory activities against SPB on H2S production. Several factors such as initial SPB level, temperature, and MOI affect lytic activities of bacteriophages. Our study demonstrated that the phage cocktail is effective to reduce the production of H2S by SPB significantly in raw animal materials. This biological control method can control SPB in raw poultry by-products at ambient temperatures, leading to a safer working environment and high quality product with less nutrient degradation for the rendering industry.

M13 bacteriophage-activated superparamagnetic beads for affinity separation.

PubMed

Muzard, Julien; Platt, Mark; Lee, Gil U

2012-08-06

The growth of the biopharmaceutical industry has created a demand for new technologies for the purification of genetically engineered proteins.The efficiency of large-scale, high-gradient magnetic fishing could be improved if magnetic particles offering higher binding capacity and magnetization were available. This article describes several strategies for synthesizing microbeads that are composed of a M13 bacteriophage layer assembled on a superparamagnetic core. Chemical cross-linking of the pVIII proteins to a carboxyl-functionalized bead produces highly responsive superparamagnetic particles (SPM) with a side-on oriented, adherent virus monolayer. Also, the genetic manipulation of the pIII proteins with a His(6) peptide sequence allows reversible assembly of the bacteriophage on a nitrilotriacetic-acid-functionalized core in an end-on configuration. These phage-magnetic particles are successfully used to separate antibodies from high-protein concentration solutions in a single step with a >90% purity. The dense magnetic core of these particles makes them five times more responsive to magnetic fields than commercial materials composed of polymer-(iron oxide) composites and a monolayer of phage could produce a 1000 fold higher antibody binding capacity. These new bionanomaterials appear to be well-suited to large-scale high-gradient magnetic fishing separation and promise to be cost effective as a result of the self-assembling and self-replicating properties of genetically engineered M13 bacteriophage. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bacteriophages in the Dairy Environment: From Enemies to Allies

PubMed Central

Fernández, Lucía; Escobedo, Susana; Gutiérrez, Diana; Portilla, Silvia; García, Pilar; Rodríguez, Ana

2017-01-01

The history of dairy farming goes back thousands of years, evolving from a traditional small-scale production to the industrialized manufacturing of fermented dairy products. Commercialization of milk and its derived products has been very important not only as a source of nourishment but also as an economic resource. However, the dairy industry has encountered several problems that have to be overcome to ensure the quality and safety of the final products, as well as to avoid economic losses. Within this context, it is interesting to highlight the role played by bacteriophages, or phages, viruses that infect bacteria. Indeed, bacteriophages were originally regarded as a nuisance, being responsible for fermentation failure and economic losses when infecting lactic acid bacteria, but are now considered promising antimicrobials to fight milk-borne pathogens without contributing to the increase in antibiotic resistance. PMID:29117107

UPTAKE OF BACTERIOPHAGE F2 THROUGH PLANT ROOTS

EPA Science Inventory

A model system was designed to measure viral uptake through the roots of plants and translocation to distal plant parts. For this study, uptake of bacteriophage f2 was measured in corn and bean plants growing in hydroponic solutions. Few phage were detected in plants with uncut r...

Bacteriophages encode factors required for protection in a symbiotic mutualism.

PubMed

Oliver, Kerry M; Degnan, Patrick H; Hunter, Martha S; Moran, Nancy A

2009-08-21

Bacteriophages are known to carry key virulence factors for pathogenic bacteria, but their roles in symbiotic bacteria are less well understood. The heritable symbiont Hamiltonella defensa protects the aphid Acyrthosiphon pisum from attack by the parasitoid Aphidius ervi by killing developing wasp larvae. In a controlled genetic background, we show that a toxin-encoding bacteriophage is required to produce the protective phenotype. Phage loss occurs repeatedly in laboratory-held H. defensa-infected aphid clonal lines, resulting in increased susceptibility to parasitism in each instance. Our results show that these mobile genetic elements can endow a bacterial symbiont with benefits that extend to the animal host. Thus, phages vector ecologically important traits, such as defense against parasitoids, within and among symbiont and animal host lineages.

Double-stranded DNA organization in bacteriophage heads: an alternative toroid-based model.

PubMed Central

Hud, N V

1995-01-01

Studies of the organization of double-stranded DNA within bacteriophage heads during the past four decades have produced a wealth of data. However, despite the presentation of numerous models, the true organization of DNA within phage heads remains unresolved. The observations of toroidal DNA structures in electron micrographs of phage lysates have long been cited as support for the organization of DNA in a spool-like fashion. This particular model, like all other models, has not been found to be consistent will all available data. Recently we proposed that DNA within toroidal condensates produced in vitro is organized in a manner significantly different from that suggested by the spool model. This new toroid model has allowed the development of an alternative model for DNA organization within bacteriophage heads that is consistent with a wide range of biophysical data. Here we propose that bacteriophage DNA is packaged in a toroid that is folded into a highly compact structure. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:8534805

Biomimetic self-templating optical structures fabricated by genetically engineered M13 bacteriophage.

PubMed

Kim, Won-Geun; Song, Hyerin; Kim, Chuntae; Moon, Jong-Sik; Kim, Kyujung; Lee, Seung-Wuk; Oh, Jin-Woo

2016-11-15

Here, we describe a highly sensitive and selective surface plasmon resonance sensor system by utilizing self-assembly of genetically engineered M13 bacteriophage. About 2700 copies of genetically expressed peptide copies give superior selectivity and sensitivity to M13 phage-based SPR sensor. Furthermore, the sensitivity of the M13 phage-based SPR sensor was enhanced due to the aligning of receptor matrix in specific direction. Incorporation of specific binding peptide (His Pro Gln: HPQ) gives M13 bacteriophage high selectivity for the streptavidin. Our M13 phage-based SPR sensor takes advantage of simplicity of self-assembly compared with relatively complex photolithography techniques or chemical conjugations. Additionally, designed structure which is composed of functionalized M13 bacteriophage can simultaneously improve the sensitivity and selectivity of SPR sensor evidently. By taking advantages of the genetic engineering and self-assembly, we propose the simple method for fabricating novel M13 phage-based SPR sensor system which has a high sensitivity and high selectivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Novel bacteriophage therapy for controlling metallo-beta-lactamase producing Pseudomonas aeruginosa infection in Catfish

PubMed Central

2013-01-01

Background The bacteriophage therapy is an effective antimicrobial approach with potentially important applications in medicine and biotechnology which can be seen as an additional string in the bow. Emerging drug resistant bacteria in aquaculture industry due to unrestricted use of antibiotics warrants more sustainable and environmental friendly strategies for controlling fish infections. The isolated bacteria from fish lesions was characterised based on isolation on selective and differential medium like Pseudomonas agar, gram staining, biochemical tests and 16SrRNA sequencing. The metallo-beta-lactamase (MBL) producing bacterial isolate was evaluated using Imipenem - Ethylenediaminetetraacetic acid (EDTA) disk method. The specific bacteriophage was isolated and concentrated using coal bed developed in our lab at CSIR-NEERI. The isolated and enriched bacteriophage was characterised by nucleotide sequencing and electron microscopy. The phage therapy was applied for treating ulcerative lesion in fish. Results The pathogenic bacterium responsible for causing ulcerative lesions in catfish species (Clarias gariepinus) was identified as Pseudomonas aeruginosa. One out of twenty P. aeruginosa isolate showing multi drug resistance (MDR) was incidentally found to be MBL producing as determined by Imipenem-EDTA disk method. The phage therapy effectively cured the ulcerative lesions of the infected fish in 8–10 days of treatment, with a sevenfold reduction of the lesion with untreated infection control. Conclusion Bacteriophage therapy can have potential applications soon as an alternative or as a complement to antibiotic treatment in the aquaculture. We present bacteriophage therapy as a treatment method for controlling MDR P. aeruginosa infection in C. gariepinus. To the best of our knowledge this is a first report of application of phage therapy against MBL producing P. aeruginosa isolated from aquatic ecosystem. PMID:24369750

Novel bacteriophage therapy for controlling metallo-beta-lactamase producing Pseudomonas aeruginosa infection in catfish.

PubMed

Khairnar, Krishna; Raut, Mahendra P; Chandekar, Rajshree H; Sanmukh, Swapnil G; Paunikar, Waman N

2013-12-26

The bacteriophage therapy is an effective antimicrobial approach with potentially important applications in medicine and biotechnology which can be seen as an additional string in the bow. Emerging drug resistant bacteria in aquaculture industry due to unrestricted use of antibiotics warrants more sustainable and environmental friendly strategies for controlling fish infections.The isolated bacteria from fish lesions was characterised based on isolation on selective and differential medium like Pseudomonas agar, gram staining, biochemical tests and 16SrRNA sequencing. The metallo-beta-lactamase (MBL) producing bacterial isolate was evaluated using Imipenem - Ethylenediaminetetraacetic acid (EDTA) disk method. The specific bacteriophage was isolated and concentrated using coal bed developed in our lab at CSIR-NEERI. The isolated and enriched bacteriophage was characterised by nucleotide sequencing and electron microscopy. The phage therapy was applied for treating ulcerative lesion in fish. The pathogenic bacterium responsible for causing ulcerative lesions in catfish species (Clarias gariepinus) was identified as Pseudomonas aeruginosa. One out of twenty P. aeruginosa isolate showing multi drug resistance (MDR) was incidentally found to be MBL producing as determined by Imipenem-EDTA disk method. The phage therapy effectively cured the ulcerative lesions of the infected fish in 8-10 days of treatment, with a sevenfold reduction of the lesion with untreated infection control. Bacteriophage therapy can have potential applications soon as an alternative or as a complement to antibiotic treatment in the aquaculture. We present bacteriophage therapy as a treatment method for controlling MDR P. aeruginosa infection in C. gariepinus. To the best of our knowledge this is a first report of application of phage therapy against MBL producing P. aeruginosa isolated from aquatic ecosystem.

From Bits and Pieces to Whole Phage to Nanomachines: Pathogen Detection Using Bacteriophages.

PubMed

Anany, H; Chou, Y; Cucic, S; Derda, R; Evoy, S; Griffiths, M W

2017-02-28

The innate specificity of bacteriophages toward their hosts makes them excellent candidates for the development of detection assays. They can be used in many ways to detect pathogens, and each has its own advantages and disadvantages. Whole bacteriophages can carry reporter genes to alter the phenotype of the target. Bacteriophages can act as staining agents or the progeny of the infection process can be detected, which further increases the sensitivity of the detection assay. Compared with whole-phage particles, use of phage components as probes offers other advantages: for example, smaller probe size to enhance binding activity, phage structures that can be engineered for better affinity, as well as specificity, binding properties, and robustness. When no natural binding with the target exists, phages can be used as vehicles to identify new protein-ligand interactions necessary for diagnostics. This review comprehensively summarizes many uses of phages as detection tools and points the way toward how phage-based technologies may be improved.

Bacteriophage cocktail and multi-strain probiotics in the feed for weanling pigs: effects on intestine morphology and targeted intestinal coliforms and Clostridium.

PubMed

Kim, J S; Hosseindoust, A; Lee, S H; Choi, Y H; Kim, M J; Lee, J H; Kwon, I K; Chae, B J

2017-01-01

Two experiments were conducted to investigate the effects of dietary supplementation of bacteriophage cocktail, probiotics and a combination of these two supplements on performance and gut health of weanling pigs. In Experiment 1, 150 weaned piglets were randomly allotted to three treatments on the basis of BW. The dietary treatments included a basal diet supplemented with 0 (control), 1.0 and 1.5 g/kg bacteriophage cocktail. Pigs fed 1.0 and 1.5 g/kg bacteriophage product had greater (P<0.05) average daily gain (ADG), apparent total tract digestibility of dry matter from day 22 to 35, ileal Lactobacillus spp., villus height (duodenum and jejunum), and fewer coliforms (ileum) and Clostridium spp. (ileum). In Experiment 2, 200 weaned piglets were randomly allotted to four treatments. Dietary treatments included basal diet, basal diet supplemented with 3.0 g/kg fermented probiotic product (P), 1.0 g/kg bacteriophage cocktail (B) and combination of 1.0 g/kg bacteriophage cocktail and 3.0 g/kg fermented probiotic product. Pigs fed bacteriophage cocktail diets had greater (P<0.05) overall ADG, gain to feed ratio (G : F), fecal score from day 8 to day 21, and pigs fed bacteriophage cocktail diets had fewer coliforms (ileum) Clostridium spp. (ileum and cecum). Probiotics significantly increased G : F, colonization of Lactobacillus spp. in ileum. At day 35, bacteriophage treatment group showed greater (P<0.05) villus height of the duodenum, but a deeper crypt in duodenum. The present results indicate that the bacteriophage cocktail had a potential to enhance the performance and gut health of weanling pigs, however their combination with probiotics did not show an interaction.

Elucidating the pH-Dependent Structural Transition of T7 Bacteriophage Endolysin.

PubMed

Sharma, Meenakshi; Kumar, Dinesh; Poluri, Krishna Mohan

2016-08-23

Bacteriophages are the most abundant and diverse biological entities on earth. Bacteriophage endolysins are unique peptidoglycan hydrolases and have huge potential as effective enzybiotics in various infectious models. T7 bacteriophage endolysin (T7L), also known as N-acetylmuramoyl-l-alanine amidase or T7 lysozyme, is a 17 kDa protein that lyses a range of Gram-negative bacteria by hydrolyzing the amide bond between N-acetylmuramoyl residues and the l-alanine of the peptidoglycan layer. Although the activity profiles of several of the T7 family members have been known for many years, the molecular basis for their pH-dependent differential activity is not clear. In this study, we explored the pH-induced structural, stability, and activity characteristics of T7L by applying a variety of biophysical techniques and protein nuclear magnetic resonance (NMR) spectroscopy. Our studies established a reversible structural transition of T7L below pH 6 and the formation of a partially denatured conformation at pH 3. This low-pH conformation is thermally stable and exposed its hydrophobic pockets. Further, NMR relaxation measurements and structural analysis unraveled that T7L is highly dynamic in its native state and a network of His residues are responsible for the observed pH-dependent conformational dynamics and transitions. As bacteriophage chimeric and engineered endolysins are being developed as novel therapeutics against multiple drug resistance pathogens, we believe that our results are of great help in designing these entities as broadband antimicrobial and/or antibacterial agents.

The roles of bacteriophages in membrane-based water and wastewater treatment processes: A review.

PubMed

Wu, Bing; Wang, Rong; Fane, Anthony G

2017-03-01

Membrane filtration processes have been widely applied in water and wastewater treatment for many decades. Concerns related to membrane treatment effectiveness, membrane lifespan, and membrane fouling control have been paid great attention. To achieve sustainable membrane operation with regards to low energy and maintenance cost, monitoring membrane performance and applying suitable membrane control strategies are required. As the most abundant species in water and wastewater, bacteriophages have shown great potential to be employed in membrane processes as (1) indicators to assess membrane performance considering their similar properties to human pathogenic waterborne viruses; (2) surrogate particles to monitor membrane integrity due to their nano-sized nature; and (3) biological agents to alleviate membrane fouling because of their antimicrobial properties. This study aims to provide a comprehensive review on the roles of bacteriophages in membrane-based water and wastewater treatment processes, with focuses on their uses for membrane performance examination, membrane integrity monitoring, and membrane biofouling control. The advantages, limitations, and influencing factors for bacteriophage-based applications are reported. Finally, the challenges and prospects of bacteriophage-based applications in membrane processes for water treatment are highlighted. Copyright © 2016 Elsevier Ltd. All rights reserved.

Elucidation of the mechanisms of action of Bacteriophage K/nano-emulsion formulations against S. aureus via measurement of particle size and zeta potential.

PubMed

Esteban, Patricia Perez; Jenkins, A Toby A; Arnot, Tom C

2016-03-01

In earlier work we have demonstrated the effect that nano-emulsions have on bacterial growth, and most importantly the enhanced bacteriophage infectivity against Staphylococcus aureus in planktonic culture when phage are carried in nano-emulsions. However, the mechanisms of enhancement of the bacteriophage killing effect are not specifically understood. This work focuses on the investigation of the possible interactions between emulsion droplets and bacterial cells, between emulsion droplets and bacteriophages, and finally interactions between all three components: nano-emulsion droplets, bacteria, and bacteriophages. The first approach consists of simple calculations to determine the spatial distribution of the components, based on measurements of particle size. It was found that nano-emulsion droplets are much more numerous than bacteria or bacteriophage, and due to their size and surface area they must be covering the surface of both cells and bacteriophage particles. Stabilisation of bacteriophages due to electrostatic forces and interaction with nano-emulsion droplets is suspected, since bacteriophages may be protected against inactivation due to 'charge shielding'. Zeta potential was measured for the individual components in the system, and for all of them combined. It was concluded that the presence of nano-emulsions could be reducing electrostatic repulsion between bacterial cells and bacteriophage, both of which are very negatively 'charged'. Moreover, nano-emulsions lead to more favourable interaction between bacteriophages and bacteria, enhancing the anti-microbial or killing effect. These findings are relevant since the physicochemical properties of nano-emulsions (i.e. particle size distribution and zeta potential) are key in determining the efficacy of the formulation against infection in the context of responsive burn wound dressings-which is the main target for this work. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Inhibiting the Growth of Escherichia coli O157:H7 in Beef, Pork, and Chicken Meat using a Bacteriophage

PubMed Central

Seo, Jina; Seo, Dong Joo; Oh, Hyejin; Jeon, Su Been; Oh, Mi-Hwa; Choi, Changsun

2016-01-01

This study aimed to inhibit Escherichia coli (E. coli) O157:H7 artificially contaminated in fresh meat using bacteriophage. Among 14 bacteriophages, the highly lytic bacteriophage BPECO19 strain was selected to inhibit E. coli O157:H7 in artificially contaminated meat samples. Bacteriophage BPECO19 significantly reduced E. coli O157:H7 bacterial load in vitro in a multiplicity of infection (MOI)-dependent manner. E. coli O157:H7 was completely inhibited only in 10 min in vitro by the treatment of 10,000 MOI BPECO19. The treatment of BPECO19 at 100,000 MOI completely reduced 5 Log CFU/cm2 E. coli O157:H7 bacterial load in beef and pork at 4 and 8h, respectively. In chicken meat, a 4.65 log reduction of E. coli O157:H7 was observed at 4 h by 100,000 MOI. The treatment of single bacteriophage BPECO19 was an effective method to control E. coli O157:H7 in meat samples. PMID:27194926

Abundance of Antibiotic Resistance Genes in Bacteriophage following Soil Fertilization with Dairy Manure or Municipal Biosolids, and Evidence for Potential Transduction.

PubMed

Ross, Joseph; Topp, Edward

2015-11-01

Animal manures and municipal biosolids recycled onto crop production land carry antibiotic-resistant bacteria that can influence the antibiotic resistome of agricultural soils, but little is known about the contribution of bacteriophage to the dissemination of antibiotic resistance genes (ARGs) in this context. In this work, we quantified a set of ARGs in the bacterial and bacteriophage fractions of agricultural soil by quantitative PCR. All tested ARGs were present in both the bacterial and phage fractions. We demonstrate that fertilization of soil with dairy manure or human biosolids increases ARG abundance in the bacterial fraction but not the bacteriophage fraction and further show that pretreatment of dairy manure can impact ARG abundance in the bacterial fraction. Finally, we show that purified bacteriophage can confer increased antibiotic resistance to soil bacteria when combined with selective pressure. The results indicate that soilborne bacteriophage represents a substantial reservoir of antibiotic resistance and that bacteriophage could play a significant role in the horizontal transfer of resistance genes in the context of an agricultural soil microbiome. Overall, our work reinforces the advisability of composting or digesting fecal material prior to field application and suggests that application of some antibiotics at subclinical concentrations can promote bacteriophage-mediated horizontal transfer of ARGs in agricultural soil microbiomes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

PubMed Central

Tavis, John E.; Totten, Michael; Cao, Feng; Michailidis, Eleftherios; Aurora, Rajeev; Meyers, Marvin J.; Jacobsen, E. Jon; Parniak, Michael A.; Sarafianos, Stefan G.

2013-01-01

Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development. PMID

Bacteriophages and Their Role in Food Safety

PubMed Central

Sillankorva, Sanna M.; Oliveira, Hugo; Azeredo, Joana

2012-01-01

The interest for natural antimicrobial compounds has increased due to alterations in consumer positions towards the use of chemical preservatives in foodstuff and food processing surfaces. Bacteriophages fit in the class of natural antimicrobial and their effectiveness in controlling bacterial pathogens in agro-food industry has led to the development of different phage products already approved by USFDA and USDA. The majority of these products are to be used in farm animals or animal products such as carcasses, meats and also in agricultural and horticultural products. Treatment with specific phages in the food industry can prevent the decay of products and the spread of bacterial diseases and ultimately promote safe environments in animal and plant food production, processing, and handling. This is an overview of recent work carried out with phages as tools to promote food safety, starting with a general introduction describing the prevalence of foodborne pathogens and bacteriophages and a more detailed discussion on the use of phage therapy to prevent and treat experimentally induced infections of animals against the most common foodborne pathogens, the use of phages as biocontrol agents in foods, and also their use as biosanitizers of food contact surfaces. PMID:23316235

Plasma-mediated transfection of RPE

NASA Astrophysics Data System (ADS)

Palanker, D.; Chalberg, T.; Vankov, A.; Huie, P.; Molnar, F. E.; Butterwick, A.; Calos, M.; Marmor, M.; Blumenkranz, M. S.

2006-02-01

A major obstacle in applying gene therapy to clinical practice is the lack of efficient and safe gene delivery techniques. Viral delivery has encountered a number of serious problems including immunological reactions and malignancy. Non-viral delivery methods (liposomes, sonoporation and electroporation) have either low efficiency in-vivo or produce severe collateral damage to ocular tissues. We discovered that tensile stress greatly increases the susceptibility of cellular membranes to electroporation. For synchronous application of electric field and mechanical stress, both are generated by the electric discharge itself. A pressure wave is produced by rapid vaporization of the medium. To prevent termination of electric current by the vapor cavity it is ionized thus restoring its electric conductivity. For in-vivo experiments with rabbits a plasmid DNA was injected into the subretinal space, and RPE was treated trans-sclerally with an array of microelectodes placed outside the eye. Application of 250-300V and 100-200 μs biphasic pulses via a microelectrode array resulted in efficient transfection of RPE without visible damage to the retina. Gene expression was quantified and monitored using bioluminescence (luciferase) and fluorescence (GFP) imaging. Transfection efficiency of RPE with this new technique exceeded that of standard electroporation by a factor 10,000. Safe and effective non-viral DNA delivery to the mammalian retina may help to materialize the enormous potential of the ocular gene therapy. Future experiments will focus on continued characterization of the safety and efficacy of this method and evaluation of long-term transgene expression in the presence of phiC31 integrase.

Bacteriophages as Weapons Against Bacterial Biofilms in the Food Industry.

PubMed

Gutiérrez, Diana; Rodríguez-Rubio, Lorena; Martínez, Beatriz; Rodríguez, Ana; García, Pilar

2016-01-01

Microbiological contamination in the food industry is often attributed to the presence of biofilms in processing plants. Bacterial biofilms are complex communities of bacteria attached to a surface and surrounded by an extracellular polymeric material. Their extreme resistance to cleaning and disinfecting processes is related to a unique organization, which implies a differential bacterial growth and gene expression inside the biofilm. The impact of biofilms on health, and the economic consequences, has promoted the development of different approaches to control or remove biofilm formation. Recently, successful results in phage therapy have boosted new research in bacteriophages and phage lytic proteins for biofilm eradication. In this regard, this review examines the environmental factors that determine biofilm development in food-processing equipment. In addition, future perspectives for the use of bacteriophage-derived tools as disinfectants are discussed.

Biotinylation of environmentally isolated Shiga toxin-producing Escherichia coli (STEC) – specific bacteriophages for biosensor and biocontrol applications

USDA-ARS?s Scientific Manuscript database

Like common bacteriophages, Shiga toxin-producing Escherichia coli (STEC) bacteriophages are viruses that recognize and bind to specific bacterial host (STEC) for propagation. They co-exist with STEC hosts, which cause epidemic food and waterborne illnesses, but may act as host populations limiting ...

Bacteriophages as Biological Control Agents of Enteric Bacteria Contaminating Edible Oysters.

PubMed

Le, Tuan Son; Southgate, Paul C; O'Connor, Wayne; Poole, Sue; Kurtbӧke, D Ipek

2018-05-01

Bacterial contamination on seafood resulting from unhygienic food-handling practices causes foodborne diseases and significant revenue losses. Moreover, control measures are complicated by a high prevalence of antibiotic-resistant bacteria. Alternative measures such as the phage therapy, therefore, is considered as an environmental and consumer-friendly biological control strategy for controlling such bacterial contamination. In this study, we determined the effectiveness of a bacteriophage cocktail in controlling E. coli strains [JM 109, ATCC 13706 and the, extended spectrum beta-lactamase resistant strain (ATCC BAA 196)] and S. enterica subsp. enterica (ATCC 13311) as single and combined contaminants of the edible oysters. Five different E. coli-specific phages (belonging to the Siphoviridae family) and a Salmonella phage (belonging to the Tectiviridae family) were successfully isolated from sewage water samples taken from a local sewage treatment plan in the Sunshine Coast region of Australia. Phage treatments applied to the pathogens when they were presented on the oysters as either single or combined hosts, resulted in significant decrease of the number of these bacteria on edible oysters. Results obtained indicated that bacteriophages could have beneficial applications in oyster-processing plants in controlling pathogenic bacterial infestations. This study thus contributes towards ongoing international efforts into the effective use of bacteriophages for biological control purposes.

Drug delivery vectors based on filamentous bacteriophages and phage-mimetic nanoparticles.

PubMed

Ju, Zhigang; Sun, Wei

2017-11-01

With the development of nanomedicine, a mass of nanocarriers have been exploited and utilized for targeted drug delivery, including liposomes, polymers, nanoparticles, viruses, and stem cells. Due to huge surface bearing capacity and flexible genetic engineering property, filamentous bacteriophage and phage-mimetic nanoparticles are attracting more and more attentions. As a rod-like bio-nanofiber without tropism to mammalian cells, filamentous phage can be easily loaded with drugs and directly delivered to the lesion location. In particular, chemical drugs can be conjugated on phage surface by chemical modification, and gene drugs can also be inserted into the genome of phage by recombinant DNA technology. Meanwhile, specific peptides/proteins displayed on the phage surface are able to conjugate with nanoparticles which will endow them specific-targeting and huge drug-loading capacity. Additionally, phage peptides/proteins can directly self-assemble into phage-mimetic nanoparticles which may be applied for self-navigating drug delivery nanovehicles. In this review, we summarize the production of phage particles, the identification of targeting peptides, and the recent applications of filamentous bacteriophages as well as their protein/peptide for targeting drug delivery in vitro and in vivo. The improvement of our understanding of filamentous bacteriophage and phage-mimetic nanoparticles will supply new tools for biotechnological approaches.

Lytic bacteriophages reduce Escherichia coli O157

PubMed Central

Ferguson, Sean; Roberts, Cheryl; Handy, Eric; Sharma, Manan

2013-01-01

The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield™) or a control (phosphate buffered saline, PBS) was applied to lettuce by either; (1) immersion of lettuce in 500 ml of EcoShield™ 8.3 log PFU/ml or 9.8 log PFU/ml for up to 2 min before inoculation with E. coli O157:H7; (2) spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculation with E. coli O157:H7 (4.10 CFU/cm2) following exposure to 50 μg/ml chlorine for 30 sec. After immersion studies, lettuce was spot-inoculated with E. coli O157:H7 (2.38 CFU/cm2). Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzed for E. coli O157:H7 populations for up to 7 d. Immersion of lettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p < 0.05) reduced E. coli O157:H7 populations after 24 h when stored at 4°C compared with controls. Immersion of lettuce in suspensions containing high concentrations of EcoShield™ (9.8 log PFU/ml) resulted in the deposition of high concentrations (7.8 log log PFU/cm2) of bacteriophages on the surface of fresh cut lettuce, potentially contributing to the efficacy of the lytic phages on lettuce. Spraying phages on to inoculated fresh cut lettuce after being washed in hypochlorite solution was significantly more effective in reducing E. coli O157:H7 populations (2.22 log CFU/cm2) on day 0 compared with control treatments (4.10 log CFU/cm2). Both immersion and spray treatments provided protection from E. coli O157:H7 contamination on lettuce, but spray application of lytic bacteriophages to lettuce was more effective in immediately reducing E. coli O157:H7 populations fresh cut lettuce. PMID:23819106

Interaction of colicin E7 with the major coat protein (g8p) may confer limited protection on colicinogenic Escherichia coli against M13 bacteriophage infection.

PubMed

Chen, Yuh-Ren; Yang, Tsung-Yeh; Lei, Guang-Sheng; Liao, Chen-Chung; Chak, Kin-Fu

2010-11-01

Colicin release provides producer strains with a competitive advantage under certain circumstances. We found that propagation of M13 bacteriophage in cells producing colicin E7 is impaired, without alteration in the efficiency of bacteriophage adsorption, as compared with non-producing cells. In contrast to the protective effect of the colicin against M13 bacteriophage infection, the endogenously expressed colicin does not confer limited protection against transfection with M13 bacteriophage DNA. Furthermore, it was found that the translocation-receptor-binding domain and toxicity domain of the colicin are able to interact with the M13 major coat protein, g8p, during bacteriophage infection. Based on these observations, we propose that interaction between colicin E7 and g8p during infection interferes with g8p depolymerizing into the cytoplasmic membrane during bacteriophage DNA penetration, thus resulting in the limited protection against M13 bacteriophage infection.

Ferrocenyl chalcone difluoridoborates inhibit HIV-1 integrase and display low activity towards cancer and endothelial cells.

PubMed

Monserrat, Jean-Philippe; Al-Safi, Rasha I; Tiwari, Keshri Nath; Quentin, Lionel; Chabot, Guy G; Vessières, Anne; Jaouen, Gérard; Neamati, Nouri; Hillard, Elizabeth A

2011-10-15

We report here the discovery of a potent series of HIV-1 integrase (IN) inhibitors based on the ferrocenyl chalcone difluoridoborate structure. Ten new compounds have been synthesized and were generally found to have similar inhibitory activities against the IN 3' processing and strand transfer (ST) processes. IC(50) values were found to be in the low micromolar range, and significantly lower than those found for the non-coordinated ferrocenyl chalcones and other ferrocene molecules. The ferrocenyl chalcone difluoridoborates furthermore exhibited low cytotoxicity against cancer cells and low morphological activity against epithelial cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

[POSSIBILITIES OF APPLICATION OF MALDI-TOF MASS-SPECTROMETRY FOR STUDY OF CARBOHYDRATE-SPECIFIC RECEPTORS FOR DIAGNOSTIC BACTERIOPHAGE EL TOR].

PubMed

Telesmanich, N R; Goncharenko, E V; Chaika, S O; Chaika, I A; Telicheva, V O

2016-01-01

Study mechanisms of interaction of diagnostic bacteriophage El Tor with sensitive strain Vibrio cholerae El Tor 18507 using direct protein profiling, identification of constant and variable proteins, taking part in interaction of the phage and cell, as well as carbohydrate-specific phage receptors. . A commercial preparation of cholera diagnostic bacteriophage El Tor, strain V. cholerae El Tor 18507 were used. Effect of carbohydrates on bacteriophage activity was determined in experiments with phage by a classic and modified by us method. Protein profiles of the studied objects were studied using MSP-analysis method. Sucrose was shown to inhibit lytic activity of bacteriophage. Proteome profiles of El Tor bacteriophage and sensitive indicator strains were studied, identification of constant and variable proteins of the studied objects by MSP Peak-list program was carried out. Analysis of changes of profiles of phage and microbial cell during interaction with sucrose gave a basis for assuming, that sucrose in the mixture of culture-phage enters interaction namely with phage protein receptors, blocking receptors specific for cholera vibrio, that subsequently manifests in a sharp decrease of phage activity against the sensitive strain.

Gene transfer of Hodgkin cell lines via multivalent anti-CD30 scFv displaying bacteriophage.

PubMed

Chung, Yoon-Suk A; Sabel, Katja; Krönke, Martin; Klimka, Alexander

2008-04-16

The display of binding ligands, such as recombinant antibody fragments, on the surface of filamentous phage makes it possible to specifically attach these phage particles to target cells. After uptake of the phage, their internal single-stranded DNA is processed by the host cell, which allows transient expression of an encoded eukaryotic gene cassette. This opens the possibility to use bacteriophage as vectors for targeted gene therapy, although the transduction efficiency is very low. Here we demonstrate the display of an anti-CD30 single chain variable fragment fused to the major coat protein pVIII on the surface of bacteriophage. These phage particles showed an improved binding and transduction efficiency of CD30 positive Hodgkin-lymphoma cells, compared to bacteriophage with the anti-CD30 single chain variable fragment fused to the minor coat protein pIII. We can conclude from the results that the postulated multivalency of the anti-CD30-pVIII displaying bacteriophage combined with disseminated display of the anti-CD30 scFv on the whole particle surface is responsible for the improved gene transfer rate. These results mark an important step towards the use of phage particles as a cheap and safe gene transfer vehicle for the gene delivery of the desired target cells via their specific surface receptors.

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection.

PubMed

Schooley, Robert T; Biswas, Biswajit; Gill, Jason J; Hernandez-Morales, Adriana; Lancaster, Jacob; Lessor, Lauren; Barr, Jeremy J; Reed, Sharon L; Rohwer, Forest; Benler, Sean; Segall, Anca M; Taplitz, Randy; Smith, Davey M; Kerr, Kim; Kumaraswamy, Monika; Nizet, Victor; Lin, Leo; McCauley, Melanie D; Strathdee, Steffanie A; Benson, Constance A; Pope, Robert K; Leroux, Brian M; Picel, Andrew C; Mateczun, Alfred J; Cilwa, Katherine E; Regeimbal, James M; Estrella, Luis A; Wolfe, David M; Henry, Matthew S; Quinones, Javier; Salka, Scott; Bishop-Lilly, Kimberly A; Young, Ry; Hamilton, Theron

2017-10-01

Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

PubMed Central

Biswas, Biswajit; Gill, Jason J.; Hernandez-Morales, Adriana; Lancaster, Jacob; Lessor, Lauren; Barr, Jeremy J.; Reed, Sharon L.; Rohwer, Forest; Benler, Sean; Segall, Anca M.; Taplitz, Randy; Smith, Davey M.; Kerr, Kim; Kumaraswamy, Monika; Nizet, Victor; Lin, Leo; McCauley, Melanie D.; Strathdee, Steffanie A.; Benson, Constance A.; Pope, Robert K.; Leroux, Brian M.; Picel, Andrew C.; Mateczun, Alfred J.; Cilwa, Katherine E.; Regeimbal, James M.; Estrella, Luis A.; Wolfe, David M.; Henry, Matthew S.; Quinones, Javier; Salka, Scott; Bishop-Lilly, Kimberly A.; Young, Ry; Hamilton, Theron

2017-01-01

ABSTRACT Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii. We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted. PMID:28807909

Characterization of a new ViI-like Erwinia amylovora bacteriophage phiEa2809.

PubMed

Lagonenko, Alexander L; Sadovskaya, Olga; Valentovich, Leonid N; Evtushenkov, Anatoly N

2015-04-01

Erwinia amylovora is a Gram-negative plant pathogenic bacteria causing fire blight disease in many Rosaceae species. A novel E. amylovora bacteriophage, phiEa2809, was isolated from symptomless apple leaf sample collected in Belarus. This phage was also able to infect Pantoea agglomerans strains. The genome of phiEa2809 is a double-stranded linear DNA 162,160 bp in length, including 145 ORFs and one tRNA gene. The phiEa2809 genomic sequence is similar to the genomes of the Serratia plymutica phage MAM1, Shigella phage AG-3, Dickeya phage vB DsoM LIMEstone1 and Salmonella phage ViI and lacks similarity to described E. amylovora phage genomes. Based on virion morphology (an icosahedral head, long contractile tail) and genome structure, phiEa2809 was classified as a member of Myoviridae, ViI-like bacteriophages group. PhiEa2809 is the firstly characterized ViI-like bacteriophage able to lyse E. amylovora. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Chemical modification of M13 bacteriophage and its application in cancer cell imaging.

PubMed

Li, Kai; Chen, Yi; Li, Siqi; Nguyen, Huong Giang; Niu, Zhongwei; You, Shaojin; Mello, Charlene M; Lu, Xiaobing; Wang, Qian

2010-07-21

The M13 bacteriophage has been demonstrated to be a robust scaffold for bionanomaterial development. In this paper, we report on the chemical modifications of three kinds of reactive groups, i.e., the amino groups of lysine residues or N-terminal, the carboxylic acid groups of aspartic acid or glutamic acid residues, and the phenol group of tyrosine residues, on M13 surface. The reactivity of each group was identified through conjugation with small fluorescent molecules. Furthermore, the regioselectivity of each reaction was investigated by HPLC-MS-MS. By optimizing the reaction condition, hundreds of fluorescent moieties could be attached to create a highly fluorescent M13 bacteriophage. In addition, cancer cell targeting motifs such as folic acid could also be conjugated onto the M13 surface. Therefore, dual-modified M13 particles with folic acid and fluorescent molecules were synthesized via the selective modification of two kinds of reactive groups. Such dual-modified M13 particles showed very good binding affinity to human KB cancer cells, which demonstrated the potential applications of M13 bacteriophage in bioimaging and drug delivery.

Environmental Augmentation with Bacteriophage Prevents Colibacillosis in Broiler Chickens

USDA-ARS?s Scientific Manuscript database

Bacteriophages are viruses that kill bacteria. They are plentiful in nature, are safe having no known activity to human or animal cells, and are an attractive alternative to antibiotics. The objectives of this research were to establish an experimental model of colibacillosis induced by indirect e...

Successful Prevention of Transmission of Integrase Resistance in the Swiss HIV Cohort Study.

PubMed

Scherrer, Alexandra U; Yang, Wan-Lin; Kouyos, Roger D; Böni, Jürg; Yerly, Sabine; Klimkait, Thomas; Aubert, Vincent; Cavassini, Matthias; Battegay, Manuel; Hauser, Christoph; Calmy, Alexandra; Schmid, Patrick; Bernasconi, Enos; Günthard, Huldrych F

2016-08-01

The prevalence of integrase strand transfer inhibitor (INSTI)-transmitted drug resistance (TDR) may increase with the increasing use of INSTIs. We analyzed the prevalence of INSTI TDR in the Swiss HIV Cohort Study (2008-2014). In 1 of 1316 drug-naive samples (0.1%), a major INSTI TDR mutation was detected. Prevalence was stable, although INSTIs were increasingly used. We showed that this is in contrast to the introduction of previous drug classes, in which more treatment failures with resistant strains occurred and TDR was observed more rapidly. We demonstrated on a population-level that it is possible to avoid TDR to a new drug class for years. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Complete genome sequence of IME15, the first T7-like bacteriophage lytic to pan-antibiotic-resistant Stenotrophomonas maltophilia.

PubMed

Huang, Yong; Fan, Huahao; Pei, Guangqian; Fan, Hang; Zhang, Zhiyi; An, Xiaoping; Mi, Zhiqiang; Shi, Taoxing; Tong, Yigang

2012-12-01

T7-like bacteriophages are a class of virulent bacteriophages which have a clearer genetic background and smaller genomes than other phages. In addition, it grows faster and is easier to culture than other phages. At present, the numbers of available T7-like bacteriophage genomes and Stenotrophomonas maltophilia genomes are small, and IME15 is the first T7-like virulent Stenotrophomonas phage whose sequence has been reported. It shows effective lysis of S. maltophilia. Here we announce its complete genome, and major findings from its annotation are described.

Novel Podoviridae Family Bacteriophage Infecting Weissella cibaria Isolated from Kimchi

PubMed Central

Holo, Helge; Jeon, Sang-Rok; Nes, Ingolf F.; Yoon, Sung-Sik

2012-01-01

The first complete genome sequence of a phage infecting Weissella cibaria (Weissella kimchii) is presented. The bacteriophage ϕYS61 was isolated from kimchi, a Korean fermented vegetable dish. Bacteriophages are recognized as a serious problem in industrial fermentations; however, ϕYS61 differed from many virulent phages associated with food fermentations since it was difficult to propagate and was very susceptible to resistance development. Sequence analysis revealed that ϕYS61 resembles Podoviridae of the subfamily Picovirinae. Within the subfamily Picovirinae, the ϕ29-like phages have been extensively studied, and their terminal protein-primed DNA replication is well characterized. Our data strongly suggest that ϕYS61 also replicates by a protein-primed mechanism. Weissella phage ϕYS61 is, however, markedly different from members of the Picovirinae with respect to genome size and morphology. Picovirinae are characterized by small (approximately 20-kb) genomes which contrasts with the 33,594-bp genome of ϕYS61. Based on electron microscopy analysis, ϕYS61 was classified as a member of the Podoviridae of morphotype C2, similar to the ϕ29-like phages, but its capsid dimensions are significantly larger than those reported for these phages. The novelty of ϕYS61 was also emphasized by the low number of open reading frames (ORFs) showing significant similarity to database sequences. We propose that the bacteriophage ϕYS61 should represent a new subfamily within the family Podoviridae. PMID:22885743

Bacteriophages as Weapons Against Bacterial Biofilms in the Food Industry

PubMed Central

Gutiérrez, Diana; Rodríguez-Rubio, Lorena; Martínez, Beatriz; Rodríguez, Ana; García, Pilar

2016-01-01

Microbiological contamination in the food industry is often attributed to the presence of biofilms in processing plants. Bacterial biofilms are complex communities of bacteria attached to a surface and surrounded by an extracellular polymeric material. Their extreme resistance to cleaning and disinfecting processes is related to a unique organization, which implies a differential bacterial growth and gene expression inside the biofilm. The impact of biofilms on health, and the economic consequences, has promoted the development of different approaches to control or remove biofilm formation. Recently, successful results in phage therapy have boosted new research in bacteriophages and phage lytic proteins for biofilm eradication. In this regard, this review examines the environmental factors that determine biofilm development in food-processing equipment. In addition, future perspectives for the use of bacteriophage-derived tools as disinfectants are discussed. PMID:27375566

Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses.

PubMed

Pritham, Ellen J; Putliwala, Tasneem; Feschotte, Cédric

2007-04-01

We previously identified a group of atypical mobile elements designated Mavericks from the nematodes Caenorhabditis elegans and C. briggsae and the zebrafish Danio rerio. Here we present the results of comprehensive database searches of the genome sequences available, which reveal that Mavericks are widespread in invertebrates and non-mammalian vertebrates but show a patchy distribution in non-animal species, being present in the fungi Glomus intraradices and Phakopsora pachyrhizi and in several single-celled eukaryotes such as the ciliate Tetrahymena thermophila, the stramenopile Phytophthora infestans and the trichomonad Trichomonas vaginalis, but not detectable in plants. This distribution, together with comparative and phylogenetic analyses of Maverick-encoded proteins, is suggestive of an ancient origin of these elements in eukaryotes followed by lineage-specific losses and/or recurrent episodes of horizontal transmission. In addition, we report that Maverick elements have amplified recently to high copy numbers in T. vaginalis where they now occupy as much as 30% of the genome. Sequence analysis confirms that most Mavericks encode a retroviral-like integrase, but lack other open reading frames typically found in retroelements. Nevertheless, the length and conservation of the target site duplication created upon Maverick insertion (5- or 6-bp) is consistent with a role of the integrase-like protein in the integration of a double-stranded DNA transposition intermediate. Mavericks also display long terminal-inverted repeats but do not contain ORFs similar to proteins encoded by DNA transposons. Instead, Mavericks encode a conserved set of 5 to 9 genes (in addition to the integrase) that are predicted to encode proteins with homology to replication and packaging proteins of some bacteriophages and diverse eukaryotic double-stranded DNA viruses, including a DNA polymerase B homolog and putative capsid proteins. Based on these and other structural similarities, we

Bacteriophages carrying antibiotic resistance genes in fecal waste from cattle, pigs, and poultry.

PubMed

Colomer-Lluch, Marta; Imamovic, Lejla; Jofre, Juan; Muniesa, Maite

2011-10-01

This study evaluates the occurrence of bacteriophages carrying antibiotic resistance genes in animal environments. bla(TEM), bla(CTX-M) (clusters 1 and 9), and mecA were quantified by quantitative PCR in 71 phage DNA samples from pigs, poultry, and cattle fecal wastes. Densities of 3 to 4 log(10) gene copies (GC) of bla(TEM), 2 to 3 log(10) GC of bla(CTX-M), and 1 to 3 log(10) GC of mecA per milliliter or gram of sample were detected, suggesting that bacteriophages can be environmental vectors for the horizontal transfer of antibiotic resistance genes.

Learning from Bacteriophages - Advantages and Limitations of Phage and Phage-Encoded Protein Applications

PubMed Central

Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grażyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob

2012-01-01

The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application. PMID:23305359

Bacteriophage removal efficiency as a validation and operational monitoring tool for virus reduction in wastewater reclamation: Review.

PubMed

Amarasiri, Mohan; Kitajima, Masaaki; Nguyen, Thanh H; Okabe, Satoshi; Sano, Daisuke

2017-09-15

The multiple-barrier concept is widely employed in international and domestic guidelines for wastewater reclamation and reuse for microbiological risk management, in which a wastewater reclamation system is designed to achieve guideline values of the performance target of microbe reduction. Enteric viruses are one of the pathogens for which the target reduction values are stipulated in guidelines, but frequent monitoring to validate human virus removal efficacy is challenging in a daily operation due to the cumbersome procedures for virus quantification in wastewater. Bacteriophages have been the first choice surrogate for this task, because of the well-characterized nature of strains and the presence of established protocols for quantification. Here, we performed a meta-analysis to calculate the average log 10 reduction values (LRVs) of somatic coliphages, F-specific phages, MS2 coliphage and T4 phage by membrane bioreactor, activated sludge, constructed wetlands, pond systems, microfiltration and ultrafiltration. The calculated LRVs of bacteriophages were then compared with reported human enteric virus LRVs. MS2 coliphage LRVs in MBR processes were shown to be lower than those of norovirus GII and enterovirus, suggesting it as a possible validation and operational monitoring tool. The other bacteriophages provided higher LRVs compared to human viruses. The data sets on LRVs of human viruses and bacteriophages are scarce except for MBR and conventional activated sludge processes, which highlights the necessity of investigating LRVs of human viruses and bacteriophages in multiple treatment unit processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

A bacteriophages journey through the human body.

PubMed

Barr, Jeremy J

2017-09-01

The human body is colonized by a diverse collective of microorganisms, including bacteria, fungi, protozoa and viruses. The smallest entity of this microbial conglomerate are the bacterial viruses. Bacteriophages, or phages for short, exert significant selective pressure on their bacterial hosts, undoubtedly influencing the human microbiome and its impact on our health and well-being. Phages colonize all niches of the body, including the skin, oral cavity, lungs, gut, and urinary tract. As such our bodies are frequently and continuously exposed to diverse collections of phages. Despite the prevalence of phages throughout our bodies, the extent of their interactions with human cells, organs, and immune system is still largely unknown. Phages physically interact with our mucosal surfaces, are capable of bypassing epithelial cell layers, disseminate throughout the body and may manipulate our immune system. Here, I establish the novel concept of an "intra-body phageome," which encompasses the collection of phages residing within the classically "sterile" regions of the body. This review will take a phage-centric view of the microbiota, human body, and immune system with the ultimate goal of inspiring a greater appreciation for both the indirect and direct interactions between bacteriophages and their mammalian hosts. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bacteriophages as indicators of faecal pollution and enteric virus removal

EPA Science Inventory

Bacteriophages are an attractive alternative to fecal indicator bacteria (FIB), particularly as surrogates of enteric virus fate and transport due to their closer morphological and biological properties compared to FIB. Based on a meta-analysis of published data, we summarize con...

Reduction of antibiotic resistome and integron-integrase genes in laboratory-scale photobioreactors treating municipal wastewater.

PubMed

Nõlvak, Hiie; Truu, Marika; Oopkaup, Kristjan; Kanger, Kärt; Krustok, Ivo; Nehrenheim, Emma; Truu, Jaak

2018-06-08

Wastewater treatment systems receiving municipal wastewater are major dissemination nodes of antibiotic resistance genes (ARGs) between anthropogenic and natural environments. This study examined the fate of antibiotic resistome and class 1-3 integron-integrase genes in photobioreactors that were treating municipal wastewater diluted (70/30) with lake or tap water for the algal biomass production. A combined approach of metagenomic and quantitative (qPCR) analysis was undertaken. Municipal wastewater treatment in the photobioreactors led to reduced antibiotic resistome proportion, number of ARG subtypes, and abundances of individual ARGs in the bacterial community. The ARGs and intI1 gene abundances and relative abundances in the discharges of the photobioreactors were either comparable or lower than the respective values in the effluents of conventional wastewater treatment plants. The reduction of the resistome proved to be strongly related to the changes in the bacterial community composition during the wastewater treatment process as it was responding to rising pH levels caused by intense algal growth. Several bacterial genera (e.g., Azoarcus, Dechloromonas, and Sulfuritalea) were recognized as potential hosts of multiple antibiotic resistance types. Although the lake water contributed a diverse and abundant resistome and intI genes profile to the treatment system, it proved to be considerably more beneficial for wastewater dilution than the tap water. The diversity (number of detected resistance types and subtypes) and proportion of the antibiotic resistome, the amount of plasmid borne integron-integrase gene reads, and the abundances and relative abundances of the majority of quantified ARGs (aadA, sul1, tetQ, tetW, qnrS, ermB, blaOXA2-type) and intI1 gene as well as the amount of multi-resistance determinants were significantly lower in the discharges of photobioreactors where lake water was used to dilute wastewater. Copyright © 2018. Published by Elsevier

Magic-angle spinning NMR of intact bacteriophages: Insights into the capsid, DNA and their interface

NASA Astrophysics Data System (ADS)

Abramov, Gili; Morag, Omry; Goldbourt, Amir

2015-04-01

Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid-DNA interface, describe capsid structure and dynamics and extract structural differences between viruses.

Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory, comparative study.

PubMed

Harwood, Valerie J; Boehm, Alexandria B; Sassoubre, Lauren M; Vijayavel, Kannappan; Stewart, Jill R; Fong, Theng-Theng; Caprais, Marie-Paule; Converse, Reagan R; Diston, David; Ebdon, James; Fuhrman, Jed A; Gourmelon, Michele; Gentry-Shields, Jennifer; Griffith, John F; Kashian, Donna R; Noble, Rachel T; Taylor, Huw; Wicki, Melanie

2013-11-15

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10-50 gene copies or plaques × 50 ml(-1)) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40-70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent. Copyright �

Complete Genome Sequences of 38 Gordonia sp. Bacteriophages

PubMed Central

Montgomery, Matthew T.; Bonilla, J. Alfred; Dejong, Randall; Garlena, Rebecca A.; Guerrero Bustamante, Carlos; Klyczek, Karen K.; Russell, Daniel A.; Wertz, John T.; Jacobs-Sera, Deborah; Hatfull, Graham F.

2017-01-01

ABSTRACT We report here the genome sequences of 38 newly isolated bacteriophages using Gordonia terrae 3612 (ATCC 25594) and Gordonia neofelifaecis NRRL59395 as bacterial hosts. All of the phages are double-stranded DNA (dsDNA) tail phages with siphoviral morphologies, with genome sizes ranging from 17,118 bp to 93,843 bp and spanning considerable nucleotide sequence diversity. PMID:28057748

Genetic Map of Bacteriophage φX174

PubMed Central

Benbow, R. M.; Hutchison, C. A.; Fabricant, J. D.; Sinsheimer, R. L.

1971-01-01

Bacteriophage φX174 temperature-sensitive and nonsense mutations in eight cistrons were mapped by using two-, three-, and four-factor genetic crosses. The genetic map is circular with a total length of 24 × 10−4wt recombinants per progeny phage. The cistron order is D-E-F-G-H-A-B-C. High negative interference is seen, consistent with a small closed circular deoxyribonucleic acid molecule as a genome. PMID:16789129

Recent advances in M13 bacteriophage-based optical sensing applications.

PubMed

Kim, Inhong; Moon, Jong-Sik; Oh, Jin-Woo

2016-01-01

Recently, M13 bacteriophage has started to be widely used as a functional nanomaterial for various electrical, chemical, or optical applications, such as battery components, photovoltaic cells, sensors, and optics. In addition, the use of M13 bacteriophage has expanded into novel research, such as exciton transporting. In these applications, the versatility of M13 phage is a result of its nontoxic, self-assembling, and specific binding properties. For these reasons, M13 phage is the most powerful candidate as a receptor for transducing chemical or optical phenomena of various analytes into electrical or optical signal. In this review, we will overview the recent progress in optical sensing applications of M13 phage. The structural and functional characters of M13 phage will be described and the recent results in optical sensing application using fluorescence, surface plasmon resonance, Förster resonance energy transfer, and surface enhanced Raman scattering will be outlined.

Recent advances in M13 bacteriophage-based optical sensing applications

NASA Astrophysics Data System (ADS)

Kim, Inhong; Moon, Jong-Sik; Oh, Jin-Woo

2016-10-01

Recently, M13 bacteriophage has started to be widely used as a functional nanomaterial for various electrical, chemical, or optical applications, such as battery components, photovoltaic cells, sensors, and optics. In addition, the use of M13 bacteriophage has expanded into novel research, such as exciton transporting. In these applications, the versatility of M13 phage is a result of its nontoxic, self-assembling, and specific binding properties. For these reasons, M13 phage is the most powerful candidate as a receptor for transducing chemical or optical phenomena of various analytes into electrical or optical signal. In this review, we will overview the recent progress in optical sensing applications of M13 phage. The structural and functional characters of M13 phage will be described and the recent results in optical sensing application using fluorescence, surface plasmon resonance, Förster resonance energy transfer, and surface enhanced Raman scattering will be outlined.

Recent Advances in the Development of Small-Molecular Inhibitors Target HIV Integrase-LEDGF/p75 Interaction.

PubMed

Zhao, Yu; Luo, Zaigang

2015-01-01

Lens epithelium-derived growth factor (LEDGF/p75) plays an essential role in the HIV-1 replication. It acts by tethering integrase (IN) into the host cellular chromatin. Due to its significance of the IN-LEDGF/p75 interaction affords a novel therapeutic approach for the design of new classes of antiretroviral agents. To date, many small molecules have been found to be the inhibitors of INLEDGF/ p75 interaction. This review summarizes recent advances in the development of potential structure-based IN-LEDGF/p75 interaction inhibitors. The work will be helpful to shed light on the antiretroviral drug development pipeline in the next future.

Productive performance of weanling piglets was improved by administration of a mixture of bacteriophages, targeted to control Coliforms and Clostridium spp. shedding in a challenging environment.

PubMed

Hosseindoust, A R; Lee, S H; Kim, J S; Choi, Y H; Kwon, I K; Chae, B J

2017-10-01

This study was conducted to investigate the effects of bacteriophages in different environments on growth performance, digestibility, ileal and caecal microbiota, gut morphology and immunity of weanling pigs. Two hundred piglets were randomly assigned to four treatment groups with five replicate pens with 10 pigs per pen. A 2 × 2 factorial arrangement of treatments was used to investigate the response of weanling pigs to supplemental bacteriophages (0 and 1.0 g/kg of diet) in contaminated or hygienic environments. Bacteriophages supplementation did not affect average daily gain (ADG), average daily feed intake (ADFI) and gain:feed in phases I and III; however, there was a significant improvement in ADG and gain:feed in phase II. The supplementation of bacteriophages increased the overall gain:feed of pigs. The overall result showed a greater ADG and ADFI in hygienic room. There were reductions in population of both ileal (p < 0.05) and caecal (p < 0.01) Clostridium spp. and ileal coliforms (p < 0.01) with the inclusion of bacteriophages in the diet. Bacteriophages increased ileal Lactobacillus and caecal Bifidobacterium and tended to increase ileal Bifidobacterium (p = 0.08). Contaminated environment decreased ileal Lactobacillus and caecal Bifidobacterium and tended to increase ileal Clostridium (p = 0.08) and coliforms (p = 0.08). Total anaerobic bacteria was tended to decrease (p = 0.06) in contaminated environment. Jejunal villus height increased in pigs received bacteriophages, but they did not affect other morphological items. The interaction between bacteriophages and environment tended to be significant (p = 0.06) for ileal villus height and ileal villus height to crypt depth ratio. The overall faecal score was significantly greater in hygienic environment and bacteriophages groups. The present findings indicate that there is an interactive effect on feed efficiency between bacteriophages and contaminated environment. In addition

Experimental bacteriophage treatment of honeybees (Apis mellifera) infected with Paenibacillus larvae, the causative agent of American Foulbrood Disease

PubMed Central

Yost, Diane G.; Tsourkas, Philippos; Amy, Penny S.

2016-01-01

ABSTRACT American Foulbrood Disease (AFB) is an infection of honeybees caused by the bacterium Paenibacillus larvae. One potential remedy involves using biocontrol, such as bacteriophages (phages) to lyse P. larvae. Therefore, bacteriophages specific for P. larvae were isolated to determine their efficacy in lysing P. larvae cells. Samples from soil, beehive materials, cosmetics, and lysogenized P. larvae strains were screened; of 157 total samples, 28 were positive for at least one P. larvae bacteriophage, with a total of 30. Newly isolated bacteriophages were tested for the ability to lyse each of 11 P. larvae strains. Electron microscopy demonstrated that the phage isolates were from the family Siphoviridae. Seven phages with the broadest host ranges were combined into a cocktail for use in experimental treatments of infected bee larvae; both prophylactic and post-infection treatments were conducted. Results indicated that although both pre- and post-treatments were effective, prophylactic administration of the phages increased the survival of larvae more than post-treatment experiments. These preliminary experiments demonstrate the likelihood that phage therapy could be an effective method to control AFB. PMID:27144085

Evolutionary adaptation of an RNA bacteriophage to the simultaneous increase in the within-host and extracellular temperatures.

PubMed

Lázaro, Ester; Arribas, María; Cabanillas, Laura; Román, Ismael; Acosta, Esther

2018-05-24

Bacteriophages are the most numerous biological entities on Earth. They are on the basis of most ecosystems, regulating the diversity and abundance of bacterial populations and contributing to the nutrient and energy cycles. Bacteriophages have two well differentiated phases in their life cycle, one extracellular, in which they behave as inert particles, and other one inside their hosts, where they replicate to give rise to a progeny. In both phases they are exposed to environmental conditions that often act as selective pressures that limit both their survival in the environment and their ability to replicate, two fitness traits that frequently cannot be optimised simultaneously. In this study we have analysed the evolutionary ability of an RNA bacteriophage, the bacteriophage Qβ, when it is confronted with a temperature increase that affects both the extracellular and the intracellular media. Our results show that Qβ can optimise its survivability when exposed to short-term high temperature extracellular heat shocks, as well as its replicative ability at higher-than-optimal temperature. Mutations responsible for simultaneous adaptation were the same as those selected when adaptation to each condition proceeded separately, showing the absence of important trade-offs between survival and reproduction in this virus.

Inorganic and organic fertilizers impact the abundance and proportion of antibiotic resistance and integron-integrase genes in agricultural grassland soil.

PubMed

Nõlvak, Hiie; Truu, Marika; Kanger, Kärt; Tampere, Mailiis; Espenberg, Mikk; Loit, Evelin; Raave, Henn; Truu, Jaak

2016-08-15

Soil fertilization with animal manure or its digestate may facilitate an important antibiotic resistance dissemination route from anthropogenic sources to the environment. This study examines the effect of mineral fertilizer (NH4NO3), cattle slurry and cattle slurry digestate amendment on the abundance and proportion dynamics of five antibiotic resistance genes (ARGs) and two classes of integron-integrase genes (intI1 and intI2) in agricultural grassland soil. Fertilization was performed thrice throughout one vegetation period. The targeted ARGs (sul1, tetA, blaCTX-M, blaOXA2 and qnrS) encode resistance to several major antibiotic classes used in veterinary medicine such as sulfonamides, tetracycline, cephalosporins, penicillin and fluoroquinolones, respectively. The non-fertilized grassland soil contained a stable background of tetA, blaCTX-M and sul1 genes. The type of applied fertilizer significantly affected ARGs and integron-integrase genes abundances and proportions in the bacterial community (p<0.001 in both cases), explaining 67.04% of the abundance and 42.95% of the proportion variations in the grassland soil. Both cattle slurry and cattle slurry digestate proved to be considerable sources of ARGs, especially sul1, as well as integron-integrases. Sul1, intI1 and intI2 levels in grassland soil were elevated in response to each organic fertilizer's application event, but this increase was followed by a stage of decrease, suggesting that microbes possessing these genes were predominantly entrained into soil via cattle slurry or its digestate application and had somewhat limited survival potential in a soil environment. However, the abundance of these three target genes did not decrease to a background level by the end of the study period. TetA was most abundant in mineral fertilizer treated soil and blaCTX-M in cattle slurry digestate amended soil. Despite significantly different abundances, the abundance dynamics of bacteria possessing these genes were

Magic-angle spinning NMR of intact bacteriophages: insights into the capsid, DNA and their interface.

PubMed

Abramov, Gili; Morag, Omry; Goldbourt, Amir

2015-04-01

Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid-DNA interface, describe capsid structure and dynamics and extract structural differences between viruses. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

Grose, Julianne H; Casjens, Sherwood R

2014-11-01

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.

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

PubMed Central

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

Modified Filamentous Bacteriophage as a Scaffold for Carbon Nanofiber.

PubMed

Szot-Karpińska, Katarzyna; Golec, Piotr; Leśniewski, Adam; Pałys, Barbara; Marken, Frank; Niedziółka-Jönsson, Joanna; Węgrzyn, Grzegorz; Łoś, Marcin

2016-12-21

With the advent of nanotechnology, carbon nanomaterials such as carbon nanofibers (CNF) have aroused substantial interest in various research fields, including energy storage and sensing. Further improvement of their properties might be achieved via the application of viral particles such as bacteriophages. In this report, we present a filamentous M13 bacteriophage with a point mutation in gene VII (pVII-mutant-M13) that selectively binds to the carbon nanofibers to form 3D structures. The phage-display technique was utilized for the selection of the pVII-mutant-M13 phage from the phage display peptide library. The properties of this phage make it a prospective candidate for a scaffold material for CNFs. The results for binding of CNF by mutant phage were compared with those for maternal bacteriophage (pVII-M13). The efficiency of binding between pVII-mutant-M13 and CNF is about 2 orders of magnitude higher compared to that of the pVII-M13. Binding affinity between pVII-mutant-M13 and CNF was also characterized using atomic force microscopy, scanning electron microscopy, and transmission electron microscopy, which confirmed the specificity of the interaction of the phage pVII-mutant-M13 and the CNF; the binding occurs via the phage's ending, where the mutated pVII protein is located. No similar behavior has been observed for other carbon nanomaterials such as graphite, reduced graphene oxide, single-walled carbon nanotubes, and multiwalled carbon nanotubes. Infrared spectra confirmed differences in the interaction with CNF between the pVII-mutant-M13 and the pVII-M13. Basing on conducted research, we hypothesize that the interactions are noncovalent in nature, with π-π interactions playing the dominant role. Herein, the new bioconjugate material is introduced.

Comparative docking and CoMFA analysis of curcumine derivatives as HIV-1 integrase inhibitors.

PubMed

Gupta, Pawan; Garg, Prabha; Roy, Nilanjan

2011-08-01

The docking studies and comparative molecular field analysis (CoMFA) were performed on highly active molecules of curcumine derivatives against 3' processing activity of HIV-1 integrase (IN) enzyme. The optimum CoMFA model was selected with statistically significant cross-validated r(2) value of 0.815 and non-cross validated r (2) value of 0.99. The common pharmacophore of highly active molecules was used for screening of HIV-1 IN inhibitors. The high contribution of polar interactions in pharmacophore mapping is well supported by docking and CoMFA results. The results of docking, CoMFA, and pharmacophore mapping give structural insights as well as important binding features of curcumine derivatives as HIV-1 IN inhibitors which can provide guidance for the rational design of novel HIV-1 IN inhibitors.

Application of zinc chloride precipitation method for rapid isolation and concentration of infectious Pectobacterium spp. and Dickeya spp. lytic bacteriophages from surface water and plant and soil extracts.

PubMed

Czajkowski, Robert; Ozymko, Zofia; Lojkowska, Ewa

2016-01-01

This is the first report describing precipitation of bacteriophage particles with zinc chloride as a method of choice to isolate infectious lytic bacteriophages against Pectobacterium spp. and Dickeya spp. from environmental samples. The isolated bacteriophages are ready to use to study various (ecological) aspects of bacteria-bacteriophage interactions. The method comprises the well-known precipitation of phages from aqueous extracts of the test material by addition of ZnCl2, resuscitation of bacteriophage particles in Ringer's buffer to remove the ZnCl2 excess and a soft agar overlay assay with the host bacterium to isolate infectious individual phage plaques. The method requires neither an enrichment step nor other steps (e. g., PEG precipitation, ultrafiltration, or ultracentrifugation) commonly used in other procedures and results in isolation of active viable bacteriophage particles.

The Baseplate of Lactobacillus delbrueckii Bacteriophage Ld17 Harbors a Glycerophosphodiesterase.

PubMed

Cornelissen, Anneleen; Sadovskaya, Irina; Vinogradov, Evgeny; Blangy, Stéphanie; Spinelli, Silvia; Casey, Eoghan; Mahony, Jennifer; Noben, Jean-Paul; Dal Bello, Fabio; Cambillau, Christian; van Sinderen, Douwe

2016-08-05

Glycerophosphodiester phosphodiesterases (GDPDs; EC 3.1.4.46) typically hydrolyze glycerophosphodiesters to sn-glycerol 3-phosphate (Gro3P) and their corresponding alcohol during patho/physiological processes in bacteria and eukaryotes. GDPD(-like) domains were identified in the structural particle of bacterial viruses (bacteriophages) specifically infecting Gram-positive bacteria. The GDPD of phage 17 (Ld17; GDPDLd17), representative of the group b Lactobacillus delbrueckii subsp. bulgaricus (Ldb)-infecting bacteriophages, was shown to hydrolyze, besides the simple glycerophosphodiester, two complex surface-associated carbohydrates of the Ldb17 cell envelope: the Gro3P decoration of the major surface polysaccharide d-galactan and the oligo(glycerol phosphate) backbone of the partially glycosylated cell wall teichoic acid, a minor Ldb17 cell envelope component. Degradation of cell wall teichoic acid occurs according to an exolytic mechanism, and Gro3P substitution is presumed to be inhibitory for GDPDLd17 activity. The presence of the GDPDLd17 homotrimer in the viral baseplate structure involved in phage-host interaction together with the dependence of native GDPD activity, adsorption, and efficiency of plating of Ca(2+) ions supports a role for GDPDLd17 activity during phage adsorption and/or phage genome injection. In contrast to GDPDLd17, we could not identify any enzymatic activity for the GDPD-like domain in the neck passage structure of phage 340, a 936-type Lactococcus lactis subsp. lactis bacteriophage. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Baseplate of Lactobacillus delbrueckii Bacteriophage Ld17 Harbors a Glycerophosphodiesterase*

PubMed Central

Cornelissen, Anneleen; Sadovskaya, Irina; Vinogradov, Evgeny; Blangy, Stéphanie; Spinelli, Silvia; Casey, Eoghan; Mahony, Jennifer; Noben, Jean-Paul; Dal Bello, Fabio; Cambillau, Christian; van Sinderen, Douwe

2016-01-01

Glycerophosphodiester phosphodiesterases (GDPDs; EC 3.1.4.46) typically hydrolyze glycerophosphodiesters to sn-glycerol 3-phosphate (Gro3P) and their corresponding alcohol during patho/physiological processes in bacteria and eukaryotes. GDPD(-like) domains were identified in the structural particle of bacterial viruses (bacteriophages) specifically infecting Gram-positive bacteria. The GDPD of phage 17 (Ld17; GDPDLd17), representative of the group b Lactobacillus delbrueckii subsp. bulgaricus (Ldb)-infecting bacteriophages, was shown to hydrolyze, besides the simple glycerophosphodiester, two complex surface-associated carbohydrates of the Ldb17 cell envelope: the Gro3P decoration of the major surface polysaccharide d-galactan and the oligo(glycerol phosphate) backbone of the partially glycosylated cell wall teichoic acid, a minor Ldb17 cell envelope component. Degradation of cell wall teichoic acid occurs according to an exolytic mechanism, and Gro3P substitution is presumed to be inhibitory for GDPDLd17 activity. The presence of the GDPDLd17 homotrimer in the viral baseplate structure involved in phage-host interaction together with the dependence of native GDPD activity, adsorption, and efficiency of plating of Ca2+ ions supports a role for GDPDLd17 activity during phage adsorption and/or phage genome injection. In contrast to GDPDLd17, we could not identify any enzymatic activity for the GDPD-like domain in the neck passage structure of phage 340, a 936-type Lactococcus lactis subsp. lactis bacteriophage. PMID:27268053

First steps of bacteriophage SPP1 entry into Bacillus subtilis.

PubMed

Jakutytė, Lina; Lurz, Rudi; Baptista, Catarina; Carballido-Lopez, Rut; São-José, Carlos; Tavares, Paulo; Daugelavičius, Rimantas

2012-01-20

The mechanism of genome transfer from the virion to the host cytoplasm is critical to understand and control the beginning of viral infection. The initial steps of bacteriophage SPP1 infection of the Gram-positive bacterium Bacillus subtilis were monitored by following changes in permeability of the cytoplasmic membrane (CM). SPP1 leads to a distinctively faster CM depolarization than the one caused by podovirus ϕ29 or myovirus SP01 during B. subtilis infection. Depolarization requires interaction of SPP1 infective virion to its receptor protein YueB. The amplitude of depolarization depends on phage input and concentration of YueB at the cell surface. Sub-millimolar concentrations of Ca(2+) are necessary and sufficient for SPP1 reversible binding to the host envelope and thus to trigger depolarization while DNA delivery to the cytoplasm depends on millimolar concentrations of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented. Copyright © 2011 Elsevier Inc. All rights reserved.

Genomic characterization of key bacteriophages to formulate the potential biocontrol agent to combat enteric pathogenic bacteria.

PubMed

Parmar, Krupa M; Dafale, Nishant A; Tikariha, Hitesh; Purohit, Hemant J

2018-05-01

Combating bacterial pathogens has become a global concern especially when the antibiotics and chemical agents are failing to control the spread due to its resistance. Bacteriophages act as a safe biocontrol agent by selectively lysing the bacterial pathogens without affecting the natural beneficial microflora. The present study describes the screening of prominent enteric pathogens NDK1, NDK2, NDK3, and NDK4 (Escherichia, Klebsiella, Enterobacter, and Serratia) mostly observed in domestic wastewater; against which KNP1, KNP2, KNP3, and KNP4 phages were isolated. To analyze their potential role in eradicating enteric pathogens and toxicity issue, these bacteriophages were sequenced using next-generation sequencing and characterized based on its genomic content. The isolated bacteriophages were homologous to Escherichia phage (KNP1), Klebsiella phage (KNP2), Enterobacter phage (KNP3), Serratia phage (KNP4), and belonged to Myoviridae family of Caudovirales except for the unclassified KNP4 phage. Draft genome analysis revealed the presence of lytic enzymes such as holing and lysozyme in KNP1 phage, endolysin in KNP2 phage, and endopeptidase with holin in KNP3 phage. The absence of any lysogenic and virulent genes makes this bacteriophage suitable candidate for preparation of phage cocktail to combat the pathogens present in wastewater. However, KNP4 contained a virulent gene rendering it unsuitable to be used as a biocontrol agent. These findings make the phages (KNP1-KNP3) as a promising alternative for the biocontrol of pathogens in wastewater which is the main culprit to spread these dominated pathogens in different natural water bodies. This study also necessitates for genomic screening of bacteriophages for lysogenic and virulence genes prior to its use as a biocontrol agent.

Application of a bacteriophage lysin to disrupt biofilms formed by the animal pathogen Streptococcus suis.

PubMed

Meng, Xiangpeng; Shi, Yibo; Ji, Wenhui; Meng, Xueling; Zhang, Jing; Wang, Hengan; Lu, Chengping; Sun, Jianhe; Yan, Yaxian

2011-12-01

Bacterial biofilms are crucial to the pathogenesis of many important infections and are difficult to eradicate. Streptococcus suis is an important pathogen of pigs, and here the biofilm-forming ability of 32 strains of this species was determined. Significant biofilms were completely formed by 10 of the strains after 60 h of incubation, with exopolysaccharide production in the biofilm significantly higher than that in the corresponding planktonic cultures. S. suis strain SS2-4 formed a dense biofilm, as revealed by scanning electron microscopy, and in this state exhibited increased resistance to a number of antibiotics (ampicillin, amoxicillin, ciprofloxacin, kanamycin, and rifampin) compared to that of planktonic cultures. A bacteriophage lysin, designated LySMP, was used to attack biofilms alone and in combination with antibiotics and bacteriophage. The results demonstrated that the biofilms formed by S. suis, especially strains SS2-4 and SS2-H, could be dispersed by LySMP and with >80% removal compared to a biofilm reduction by treatment with either antibiotics or bacteriophage alone of less than 20%; in addition to disruption of the biofilm structure, the S. suis cells themselves were inactivated by LySMP. The efficacy of LySMP was not dose dependent, and in combination with antibiotics, it acted synergistically to maximize dispersal of the S. suis biofilm and inactivate the released cells. These data suggest that bacteriophage lysin could form part of an effective strategy to treat S. suis infections and represents a new class of antibiofilm agents.

A model system for pathogen detection using a two-component bacteriophage/bioluminescent signal amplification assay

NASA Astrophysics Data System (ADS)

Bright, Nathan G.; Carroll, Richard J.; Applegate, Bruce M.

2004-03-01

Microbial contamination has become a mounting concern the last decade due to an increased emphasis of minimally processed food products specifically produce, and the recognition of foodborne pathogens such as Campylobacter jejuni, Escherichia coli O157:H7, and Listeria monocytogenes. This research investigates a detection approach utilizing bacteriophage pathogen specificity coupled with a bacterial bioluminescent bioreporter utilizing the quorum sensing molecule from Vibrio fischeri, N-(3-oxohexanoyl)-homoserine lactone (3-oxo-C6-HSL). The 3-oxo-C6-HSL molecules diffuse out of the target cell after infection and induce bioluminescence from a population of 3-oxo-C6-HSL bioreporters (ROLux). E. coli phage M13, a well-characterized bacteriophage, offers a model system testing the use of bacteriophage for pathogen detection through cell-to-cell communication via a LuxR/3-oxo-C6-HSL system. Simulated temperate phage assays tested functionality of the ROLux reporter and production of 3-oxo-C6-HSL by various test strains. These assays showed detection limits of 102cfu after 24 hours in a varietry of detection formats. Assays incorporating the bacteriophage M13-luxI with the ROLux reporter and a known population of target cells were subsequently developed and have shown consistent detection limits of 105cfu target organisms. Measurable light response from high concentrations of target cells was almost immediate, suggesting an enrichment step to further improve detection limits and reduce assay time.

Bacteriophages for treating urinary tract infections in patients undergoing transurethral resection of the prostate: a randomized, placebo-controlled, double-blind clinical trial.

PubMed

Leitner, Lorenz; Sybesma, Wilbert; Chanishvili, Nina; Goderdzishvili, Marina; Chkhotua, Archil; Ujmajuridze, Aleksandre; Schneider, Marc P; Sartori, Andrea; Mehnert, Ulrich; Bachmann, Lucas M; Kessler, Thomas M

2017-09-26

Urinary tract infections (UTI) are among the most prevalent microbial diseases and their financial burden on society is substantial. The continuing increase of antibiotic resistance worldwide is alarming. Thus, well-tolerated, highly effective therapeutic alternatives are urgently needed. Although there is evidence indicating that bacteriophage therapy may be effective and safe for treating UTIs, the number of investigated patients is low and there is a lack of randomized controlled trials. This study is the first randomized, placebo-controlled, double-blind trial investigating bacteriophages in UTI treatment. Patients planned for transurethral resection of the prostate are screened for UTIs and enrolled if in urine culture eligible microorganisms ≥10 4 colony forming units/mL are found. Patients are randomized in a double-blind fashion to the 3 study treatment arms in a 1:1:1 ratio to receive either: a) bacteriophage (i.e. commercially available Pyo bacteriophage) solution, b) placebo solution, or c) antibiotic treatment according to the antibiotic sensitivity pattern. All treatments are intended for 7 days. No antibiotic prophylaxes will be given to the double-blinded treatment arms a) and b). As common practice, the Pyo bacteriophage cocktail is subjected to periodic adaptation cycles during the study. Urinalysis, urine culture, bladder and pain diary, and IPSS questionnaire will be completed prior to and at the end of treatment (i.e. after 7 days) or at withdrawal/drop out from the study. Patients with persistent UTIs will undergo antibiotic treatment according to antibiotic sensitivity pattern. Based on the high lytic activity and the potential of resistance optimization by direct adaptation of bacteriophages, and considering the continuing increase of antibiotic resistance worldwide, bacteriophage therapy is a very promising treatment option for UTIs. Thus, our randomized controlled trial investigating bacteriophages for treating UTIs will provide

Chemostat studies of bacteriophage M13 infected Escherichia coli JM109 for continuous ssDNA production.

PubMed

Kick, Benjamin; Behler, Karl Lorenz; Severin, Timm Steffen; Weuster-Botz, Dirk

2017-09-20

Steady state studies in a chemostat enable the control of microbial growth rate at defined reaction conditions. The effects of bacteriophage M13 infection on maximum growth rate of Escherichia coli JM109 were studied in parallel operated chemostats on a milliliter-scale to analyze the steady state kinetics of phage production. The bacteriophage infection led to a decrease in maximum specific growth rate of 15% from 0.74h -1 to 0.63h -1 . Under steady state conditions, a constant cell specific ssDNA formation rate of 0.15±0.004 mg ssDNA g CDW -1 h -1 was observed, which was independent of the growth rate. Using the estimated kinetic parameters for E. coli infected with bacteriophage M13, the ssDNA concentration in the steady state could be predicted as function of the dilution rate and the glucose concentration in the substrate. Scalability of milliliter-scale data was approved by steady state studies on a liter-scale at a selected dilution rate. An ssDNA space-time yield of 5.7mgL -1 h -1 was achieved with increased glucose concentration in the feed at a dilution rate of 0.3h -1 , which is comparable to established fed-batch fermentation with bacteriophage M13 for ssDNA production. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural dynamics of native and V260E mutant C-terminal domain of HIV-1 integrase

NASA Astrophysics Data System (ADS)

Sangeetha, Balasubramanian; Muthukumaran, Rajagopalan; Amutha, Ramaswamy

2015-04-01

The C-terminal domain (CTD) of HIV-1 integrase is a five stranded β-barrel resembling an SH3 fold. Mutational studies on isolated CTD and full-length IN have reported V260E mutant as either homo-dimerization defective or affecting the stability and folding of CTD. In this study, molecular dynamics simulation techniques were used to unveil the effect of V260E mutation on isolated CTD monomer and dimer. Both monomeric and dimeric forms of wild type and V260E mutant are highly stable during the simulated period. However, the stabilizing π-stacking interaction between Trp243 and Trp243' at the dimer interface is highly disturbed in CTD-V260E (>6 Å apart). The loss in entropy for dimerization is -30 and -25 kcal/mol for CTD-wt and CTD-V260E respectively signifying a weak hydrophobic interaction and its perturbation in CTD-V260E. The mutant Glu260 exhibits strong attraction/repulsion with all the basic/acidic residues of CTD. In addition to this, the dynamics of CTD-wild type and V260E monomers at 498 K was analyzed to elucidate the effect of V260E mutation on CTD folding. Increase in SASA and reduction in the number of contacts in CTD-V260E during simulation highlights the instability caused by the mutation. In general, V260E mutation affects both multimerization and protein folding with a pronounced effect on protein folding rather than multimerization. This study emphasizes the importance of the hydrophobic nature and SH3 fold of CTD in proper functioning of HIV integrase and perturbing this nature would be a rational approach toward designing more selective and potent allosteric anti-HIV inhibitors.

Bacteriophage Infecting the Myxobacterium Chondrococcus columnaris

PubMed Central

Kingsbury, David T.; Ordal, Erling J.

1966-01-01

Kingsbury, David T. (University of Washington, Seattle), and Erling J. Ordal. Bacteriophage infecting the myxobacterium Chondrococcus columnaris. J. Bacteriol. 91:1327–1332. 1966.—During a series of screening experiments, seven bacteriophages which infect the pathogenic myxobacterium Chondrococcus columnaris were isolated. Of these, one was chosen for detailed study. This phage has a wide host range among strains of C. columnaris, but does not infect other myxobacterial species tested. Morphologically, this phage resembles coliphage T2, though it is smaller. It has a head diameter of 600 A, a tail length of 1,000 A, and a tail width of 200 A. The head is attached to the tail by a well-defined neck. The turbid plaques produced by this phage are similar in appearance to those produced by coliphage λ, and average 1 mm in diameter. The phage has a latent period of 100 min, a rise period of an additional 90 min, and a burst size of 23. Calcium ions at a concentration of 0.004 m are required for adsorption. This requirement cannot be met by substitution of magnesium ions. A purified preparation of 2 × 1012 phage particles was extracted with phenol, and the nucleic acid was identified as deoxyribonucleic acid (DNA). Base ratios of the phage DNA and the DNA of two propagating strains were similar. Streptomycin at a concentration of 70 μg/ml inhibits phage infection at an early stage, probably by inhibiting injection of the phage DNA. Images PMID:5929758

Spray-dried respirable powders containing bacteriophages for the treatment of pulmonary infections.

PubMed

Matinkhoo, Sadaf; Lynch, Karlene H; Dennis, Jonathan J; Finlay, Warren H; Vehring, Reinhard

2011-12-01

Myoviridae bacteriophages were processed into a dry powder inhalable dosage form using a low-temperature spray-drying process. The phages were incorporated into microparticles consisting of trehalose, leucine, and optionally a third excipient (either a surfactant or casein sodium salt). The particles were designed to have high dispersibility and a respirable particle size, and to preserve the phages during processing. Bacteriophages KS4- M, KS14, and cocktails of phages ΦKZ/D3 and ΦKZ/D3/KS4-M were spray-dried with a processing loss ranging from 0.4 to 0.8 log pfu. The aerosol performance of the resulting dry powders as delivered from an Aerolizer® dry powder inhaler (DPI) exceeded the performance of commercially available DPIs; the emitted mass and the in vitro total lung mass of the lead formulation were 82.7% and 69.7% of filled capsule mass, respectively. The total lung mass had a mass median aerodynamic diameter of 2.5-2.8 µm. The total in vitro lung doses of the phages, delivered from a single actuation of the inhaler, ranged from 10(7) to 10(8) pfu, levels that are expected to be efficacious in vivo. Spray drying of bacteriophages into a respirable dry powder was found to be feasible. Copyright © 2011 Wiley-Liss, Inc.

Direct feeding of microencapsulated bacteriophages to reduce Salmonella colonization in pigs

USDA-ARS?s Scientific Manuscript database

Salmonella shedding often increases in pigs following pre-slaughter transportation and/or lairage. We previously showed that administering anti-Salmonella bacteriophages to pigs by gavage significantly reduced Salmonella colonization when the pigs were exposed to a Salmonella-contaminated pen. In ...

Effects of Bacteriophage Supplementation on Egg Performance, Egg Quality, Excreta Microflora, and Moisture Content in Laying Hens

PubMed Central

Zhao, P. Y.; Baek, H. Y.; Kim, I. H.

2012-01-01

An experiment was conducted to evaluate the effects of bacteriophage supplementation on egg performance, egg quality, excreta microflora, and moisture content in laying hens. A total of 288 Hy-line brown commercial laying hens (36-wk-old) were randomly allotted to 4 treatments in this 6-wk trial and dietary treatments included: i) CON, basal diet; ii) T1, CON+0.020% bacteriophage; iii) T2, CON+0.035% bacteriophage; iv) T3, CON+0.050% bacteriophage. There were 6 replicates for each treatment with 6 adjacent cages (2 hens/cage). Laying hens in T2 and T3 treatments had higher (p<0.05) egg production than those in CON and T1 treatments during wk 0 to 3. In addition, egg production in T1, T2, and T3 treatments was increased (p<0.05) compared with that in CON treatment during wk 4 to 6. At wk 4 and 5, birds in T2 group had higher (p<0.05) HU than those in CON. In addition, at wk 5 and 6, HU in birds fed T1 and T3 diets was greater (p<0.05) than those fed CON diet. E. coli and Salmonella spp. concentrations in excreta were decreased (p<0.05) by T1, T2, and T3 treatments. However, egg weight, egg shell color, yolk height, yolk color unit, egg shell strength, egg shell thickness, egg gravity, and excreta moisture content were not influenced by dietary treatments during the entire experimental period. In conclusion, bacteriophage supplementation has beneficial effects on egg production, egg albumen, and excreta microflora concentration in laying hens. PMID:25049658

Effect of bacterial growth rate on bacteriophage population growth rate.

PubMed

Nabergoj, Dominik; Modic, Petra; Podgornik, Aleš

2018-04-01

It is important to understand how physiological state of the host influence propagation of bacteriophages (phages), due to the potential higher phage production needs in the future. In our study, we tried to elucidate the effect of bacterial growth rate on adsorption constant (δ), latent period (L), burst size (b), and bacteriophage population growth rate (λ). As a model system, a well-studied phage T4 and Escherichia coli K-12 as a host was used. Bacteria were grown in a continuous culture operating at dilution rates in the range between 0.06 and 0.98 hr -1 . It was found that the burst size increases linearly from 8 PFU·cell -1 to 89 PFU·cell -1 with increase in bacteria growth rate. On the other hand, adsorption constant and latent period were both decreasing from 2.6∙10 -9  ml·min -1 and 80 min to reach limiting values of 0.5 × 10 -9  ml·min -1 and 27 min at higher growth rates, respectively. Both trends were mathematically described with Michaelis-Menten based type of equation and reasons for such form are discussed. By applying selected equations, a mathematical equation for prediction of bacteriophage population growth rate as a function of dilution rate was derived, reaching values around 8 hr -1 at highest dilution rate. Interestingly, almost identical description can be obtained using much simpler Monod type equation and possible reasons for this finding are discussed. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage

PubMed Central

Jurczak-Kurek, Agata; Gąsior, Tomasz; Nejman-Faleńczyk, Bożena; Bloch, Sylwia; Dydecka, Aleksandra; Topka, Gracja; Necel, Agnieszka; Jakubowska-Deredas, Magdalena; Narajczyk, Magdalena; Richert, Malwina; Mieszkowska, Agata; Wróbel, Borys; Węgrzyn, Grzegorz; Węgrzyn, Alicja

2016-01-01

A large scale analysis presented in this article focuses on biological and physiological variety of bacteriophages. A collection of 83 bacteriophages, isolated from urban sewage and able to propagate in cells of different bacterial hosts, has been obtained (60 infecting Escherichia coli, 10 infecting Pseudomonas aeruginosa, 4 infecting Salmonella enterica, 3 infecting Staphylococcus sciuri, and 6 infecting Enterococcus faecalis). High biological diversity of the collection is indicated by its characteristics, both morphological (electron microscopic analyses) and biological (host range, plaque size and morphology, growth at various temperatures, thermal inactivation, sensitivity to low and high pH, sensitivity to osmotic stress, survivability upon treatment with organic solvents and detergents), and further supported by hierarchical cluster analysis. By the end of the research no larger collection of phages from a single environmental source investigated by these means had been found. The finding was confirmed by whole genome analysis of 7 selected bacteriophages. Moreover, particular bacteriophages revealed unusual biological features, like the ability to form plaques at low temperature (4 °C), resist high temperature (62 °C or 95 °C) or survive in the presence of an organic solvents (ethanol, acetone, DMSO, chloroform) or detergent (SDS, CTAB, sarkosyl) making them potentially interesting in the context of biotechnological applications. PMID:27698408

Isolation and characterization of two lytic cold-active bacteriophages infecting Pseudomonas fluorescens from the Napahai plateau wetland.

PubMed

Xiang, Yingying; Wang, Shuang; Li, Jiankai; Wei, Yunlin; Zhang, Qi; Lin, Lianbing; Ji, Xiuling

2018-03-01

As the "kidneys of the Earth", wetlands play important roles as biodiversity reservoirs, in water purification, and in flood control. In this study, 2 lytic cold-active bacteriophages, named VW-6S and VW-6B, infecting Pseudomonas fluorescens W-6 cells from the Napahai plateau wetland in China were isolated and characterized. Electron microscopy showed that both VW-6S and VW-6B had an icosahedral head (66.7 and 61.1 nm, respectively) and a long tail (8.3 nm width × 233.3 nm length and 11.1 nm width × 166.7 nm length, respectively). The bacteriophages VW-6S and VW-6B were classified as Siphoviridae and had an approximate genome size of 30-40 kb. The latent and burst periods of VW-6S were 60 and 30 min, whereas those of VW-6B were 30 and 30 min, respectively. The optimal pH values for the bacteriophages VW-6S and VW-6B were 8.0 and 10.0, respectively, and their activity decreased rapidly at temperatures higher than 60 °C. These cold-active bacteriophages provide good materials for further study of cold-adaptation mechanisms and interaction with the host P. fluorescens.

Preparation of Single-Stranded Bacteriophage M13 DNA by Precipitation with Polyethylene Glycol.

PubMed

Green, Michael R; Sambrook, Joseph

2017-11-01

Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected bacteria into the surrounding medium. Several methods are available to purify the polymorphic filamentous particles. In this protocol, the particles are concentrated by precipitation with polyethylene glycol (PEG) in the presence of high salt. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. The resulting preparation is pure enough to be used as a template for DNA sequencing. A yield of 5-10 µg of single-stranded DNA/mL of infected cells may be expected from recombinant bacteriophages bearing inserts of 300-1000 nt. © 2017 Cold Spring Harbor Laboratory Press.

Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage [psi]29 tail

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang, Ye; Morais, Marc C.; Cohen, Daniel N.

2009-08-28

The small bacteriophage {phi}29 must penetrate the {approx}250-{angstrom} thick external peptidoglycan cell wall and cell membrane of the Gram-positive Bacillus subtilis, before ejecting its dsDNA genome through its tail into the bacterial cytoplasm. The tail of bacteriophage {phi}29 is noncontractile and {approx}380 {angstrom} long. A 1.8-{angstrom} resolution crystal structure of gene product 13 (gp13) shows that this tail protein has spatially well separated N- and C-terminal domains, whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively. CryoEM reconstructions of the WT bacteriophage and mutant bacteriophages missing some or most of gp13 shows that this enzyme is located at the distal endmore » of the {phi}29 tail knob. This finding suggests that gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall. Comparisons of the gp13{sup -} mutants with the {phi}29 mature and emptied phage structures suggest the sequence of events that occur during the penetration of the tail through the peptidoglycan layer.« less

Isolation and characterization of Yersinia-specific bacteriophages from pig stools in Finland.

PubMed

Salem, M; Virtanen, S; Korkeala, H; Skurnik, M

2015-03-01

Bacteriophages infect bacteria, and they are present everywhere in the world including the intestinal tracts of animals. Yersiniosis is a common foodborne infection caused by Yersinia enterocolitica and Yersinia pseudotuberculosis. As these bacteria are frequently isolated from pigs, we wanted to know whether Yersinia-specific bacteriophages are also present in the pig stools and, if so, whether there is a positive or negative association between the prevalence of the Yersinia phages and the pathogenic Yersinia in the stool samples. Altogether 793 pig stool samples collected between November 2010 and March 2012 from 14 Finnish pig farms were screened for the presence of bacteriophages able to infect Y. enterocolitica serotype O:3, O:5,27 or O:9 strains, or Y. pseudotuberculosis serotype O:1a, O:1b or O:3 strains. Yersinia phages were isolated from 90 samples from eight farms. Yersinia enterocolitica O:3 was infected by 59 phages, 28 phages infected serotypes O:3 and O:5,27, and eight phages infected serotypes O:3, O:5,27 and O:9, and Y. pseudotuberculosis O:1a by eight phages. Many phages originating from pigs in the same farm were identical based on their restriction enzyme digestion patterns; 20 clearly different phages were selected for further characterization. Host ranges of these phages were tested with 94 Yersinia strains. Six of the phages infected eight strains, 13 phages infected three strains, and one phage infected only one strain, indicating that the phages had a relatively narrow host range. There was a clear association between the presence of the host bacteria and specific phages in the stools. The isolated bacteriophages may have potential as biocontrol agents for yersiniosis in both humans and pigs in future, and as alternatives or in addition to antibiotics. To our knowledge, this is the first reported isolation of Yersinia-specific phages from pig stool samples. © 2014 The Society for Applied Microbiology.

Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships.

PubMed

Pope, Welkin H; Mavrich, Travis N; Garlena, Rebecca A; Guerrero-Bustamante, Carlos A; Jacobs-Sera, Deborah; Montgomery, Matthew T; Russell, Daniel A; Warner, Marcie H; Hatfull, Graham F

2017-08-15

The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. A collection of over 1,000 phages infecting Mycobacterium smegmatis reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on Gordonia shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are "singletons" with no closely related genomes. One group of six phages is closely related to Cluster A mycobacteriophages, but the other Gordonia phages are distant relatives and share only 10% of their genes with the mycobacteriophages. The Gordonia phage genomes vary in genome length (17.1 to 103.4 kb), percentage of GC content (47 to 68.8%), and genome architecture and contain a variety of features not seen in other phage genomes. Like the mycobacteriophages, the highly mosaic Gordonia phages demonstrate a spectrum of genetic relationships. We show this is a general property of bacteriophages and suggest that any barriers to genetic exchange are soft and readily violable. IMPORTANCE Despite the numerical dominance of bacteriophages in the biosphere, there is a dearth of complete genomic sequences. Current genomic information reveals that phages are highly diverse genomically and have mosaic architectures formed by extensive horizontal genetic exchange. Comparative analysis of 79 phages of Gordonia shows them to not only be highly diverse, but to present a spectrum of relatedness. Most are distantly related to phages of the phylogenetically proximal host Mycobacterium smegmatis , although one group of Gordonia phages is more closely related to mycobacteriophages than to the other Gordonia phages. Phage genome sequence space remains largely unexplored, but

Campylobacters and their bacteriophages from chicken liver: The prospect for phage biocontrol.

PubMed

Firlieyanti, Antung S; Connerton, Phillippa L; Connerton, Ian F

2016-11-21

Consumption of foods containing chicken liver has been associated with Campylobacter enteritis. Campylobacters can contaminate the surface of livers post-mortem but can also arise through systemic infection of colonising bacteria in live birds. The use of bacteriophage to reduce levels of Campylobacter entering the food chain is a promising intervention approach but most phages have been isolated from chicken excreta. This study examined the incidence and contamination levels of Campylobacter and their bacteriophage in UK retail chicken liver. Using enrichment procedures, 87% of 109 chicken livers were surface contaminated with Campylobacter and 83% contaminated within internal tissues. Direct plating on selective agar allowed enumeration of viable bacteria from 43% of liver samples with counts ranging from 1.8->3.8log 10 CFU/cm 2 for surface samples, and 3.0->3.8log 10 CFU/g for internal tissue samples. Three C. jejuni isolates recovered from internal liver tissues were assessed for their ability to colonise the intestines and extra-intestinal organs of broiler chickens following oral infection. All isolates efficiently colonised the chicken intestines but were variable in their abilities to colonise extra-intestinal organs. One isolate, CLB104, could be recovered by enrichment from the livers and kidneys of three of seven chickens. Campylobacter isolates remained viable within fresh livers stored at 4°C over 72h and frozen livers stored at -20°C over 7days in atmospheric oxygen, and therefore constitute a risk to human health. Only three Campylobacter-specific bacteriophages were isolated, and these exhibited a limited host range against the Camplylobacter chicken liver isolates. All were identified as group III virulent bacteriophage based on their genome size of 140kb. The application of broad host range group II virulent phages (8log 10 PFU/g) to liver homogenates containing C. jejuni strains of diverse origin at 4°C resulted in modest but significant

Space, time, and host evolution facilitate coexistence of competing bacteriophages: theory and experiment.

PubMed

Coberly, L Caitlin; Wei, Wei; Sampson, Koffi Y; Millstein, Jack; Wichman, Holly A; Krone, Stephen M

2009-04-01

We present a joint experimental/theoretical investigation into the roles of spatial structure and time in the competition between two pathogens for a single host. We suggest a natural mechanism by which competing pathogens can coexist when host evolution and competitive dynamics occur on similar timescales. Our experimental system consisted of a single bacterial host species and two competing bacteriophage strains grown on agar plates, with a serial transfer of samples of the bacteriophage population to fresh host populations after each incubation cycle. The experiments included two incubation times and two transfer protocols that either maintained or disrupted the spatial structure of the viruses at each transfer. The same bacteriophage acted as the dominant competitor under both transfer protocols. A striking difference between the treatments is that the weak competitor was able to persist in the long-incubation experiments but not in the short-incubation experiments. Mathematical and experimental evidence suggest that coexistence is due to the appearance of resistant mutant host cells that provide a transient "spatiotemporal refuge" for the weaker competitor. Our mathematical model is individual based, captures the stochastic spatial dynamics down to the level of individual cells, and helps to explain the differences in behavior under the various experimental conditions.

iPHLoc-ES: Identification of bacteriophage protein locations using evolutionary and structural features.

PubMed

Shatabda, Swakkhar; Saha, Sanjay; Sharma, Alok; Dehzangi, Abdollah

2017-12-21

Bacteriophage proteins are viruses that can significantly impact on the functioning of bacteria and can be used in phage based therapy. The functioning of Bacteriophage in the host bacteria depends on its location in those host cells. It is very important to know the subcellular location of the phage proteins in a host cell in order to understand their working mechanism. In this paper, we propose iPHLoc-ES, a prediction method for subcellular localization of bacteriophage proteins. We aim to solve two problems: discriminating between host located and non-host located phage proteins and discriminating between the locations of host located protein in a host cell (membrane or cytoplasm). To do this, we extract sets of evolutionary and structural features of phage protein and employ Support Vector Machine (SVM) as our classifier. We also use recursive feature elimination (RFE) to reduce the number of features for effective prediction. On standard dataset using standard evaluation criteria, our method significantly outperforms the state-of-the-art predictor. iPHLoc-ES is readily available to use as a standalone tool from: https://github.com/swakkhar/iPHLoc-ES/ and as a web application from: http://brl.uiu.ac.bd/iPHLoc-ES/. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bacteriophage significantly reduces Listeria monocytogenes on raw salmon fillet tissue

USDA-ARS?s Scientific Manuscript database

We have demonstrated the antilisterial activity of generally recognized as safe (GRAS) bacteriophage LISTEX P100 (phage P100) on the surface of raw salmon fillet tissue against Listeria monocytogenes serotypes 1/2a and 4b. In a broth model system, phage P100 completely inhibited L. monocytogenes gro...

Bacteriophages and Their Immunological Applications against Infectious Threats.

PubMed

Criscuolo, Elena; Spadini, Sara; Lamanna, Jacopo; Ferro, Mattia; Burioni, Roberto

2017-01-01

Bacteriophage therapy dates back almost a century, but the discovery of antibiotics led to a rapid decline in the interests and investments within this field of research. Recently, the novel threat of multidrug-resistant bacteria highlighted the alarming drop in research and development of new antibiotics: 16 molecules were discovered during 1983-87, 10 new therapeutics during the nineties, and only 5 between 2003 and 2007. Phages are therefore being reconsidered as alternative therapeutics. Phage display technique has proved to be extremely promising for the identification of effective antibodies directed against pathogens, as well as for vaccine development. At the same time, conventional phage therapy uses lytic bacteriophages for treatment of infections and recent clinical trials have shown great potential. Moreover, several other approaches have been developed in vitro and in vivo using phage-derived proteins as antibacterial agents. Finally, their use has also been widely considered for public health surveillance, as biosensor phages can be used to detect food and water contaminations and prevent bacterial epidemics. These novel approaches strongly promote the idea that phages and their proteins can be exploited as an effective weapon in the near future, especially in a world which is on the brink of a "postantibiotic era."

Langevin Dynamics Simulations of Genome Packing in Bacteriophage

PubMed Central

Forrey, Christopher; Muthukumar, M.

2006-01-01

We use Langevin dynamics simulations to study the process by which a coarse-grained DNA chain is packaged within an icosahedral container. We focus our inquiry on three areas of interest in viral packing: the evolving structure of the packaged DNA condensate; the packing velocity; and the internal buildup of energy and resultant forces. Each of these areas has been studied experimentally, and we find that we can qualitatively reproduce experimental results. However, our findings also suggest that the phage genome packing process is fundamentally different than that suggested by the inverse spool model. We suggest that packing in general does not proceed in the deterministic fashion of the inverse-spool model, but rather is stochastic in character. As the chain configuration becomes compressed within the capsid, the structure, energy, and packing velocity all become dependent upon polymer dynamics. That many observed features of the packing process are rooted in condensed-phase polymer dynamics suggests that statistical mechanics, rather than mechanics, should serve as the proper theoretical basis for genome packing. Finally we suggest that, as a result of an internal protein unique to bacteriophage T7, the T7 genome may be significantly more ordered than is true for bacteriophage in general. PMID:16617089

Langevin dynamics simulations of genome packing in bacteriophage.

PubMed

Forrey, Christopher; Muthukumar, M

2006-07-01

We use Langevin dynamics simulations to study the process by which a coarse-grained DNA chain is packaged within an icosahedral container. We focus our inquiry on three areas of interest in viral packing: the evolving structure of the packaged DNA condensate; the packing velocity; and the internal buildup of energy and resultant forces. Each of these areas has been studied experimentally, and we find that we can qualitatively reproduce experimental results. However, our findings also suggest that the phage genome packing process is fundamentally different than that suggested by the inverse spool model. We suggest that packing in general does not proceed in the deterministic fashion of the inverse-spool model, but rather is stochastic in character. As the chain configuration becomes compressed within the capsid, the structure, energy, and packing velocity all become dependent upon polymer dynamics. That many observed features of the packing process are rooted in condensed-phase polymer dynamics suggests that statistical mechanics, rather than mechanics, should serve as the proper theoretical basis for genome packing. Finally we suggest that, as a result of an internal protein unique to bacteriophage T7, the T7 genome may be significantly more ordered than is true for bacteriophage in general.

Bacteriophages and Their Immunological Applications against Infectious Threats

PubMed Central

Lamanna, Jacopo; Ferro, Mattia; Burioni, Roberto

2017-01-01

Bacteriophage therapy dates back almost a century, but the discovery of antibiotics led to a rapid decline in the interests and investments within this field of research. Recently, the novel threat of multidrug-resistant bacteria highlighted the alarming drop in research and development of new antibiotics: 16 molecules were discovered during 1983–87, 10 new therapeutics during the nineties, and only 5 between 2003 and 2007. Phages are therefore being reconsidered as alternative therapeutics. Phage display technique has proved to be extremely promising for the identification of effective antibodies directed against pathogens, as well as for vaccine development. At the same time, conventional phage therapy uses lytic bacteriophages for treatment of infections and recent clinical trials have shown great potential. Moreover, several other approaches have been developed in vitro and in vivo using phage-derived proteins as antibacterial agents. Finally, their use has also been widely considered for public health surveillance, as biosensor phages can be used to detect food and water contaminations and prevent bacterial epidemics. These novel approaches strongly promote the idea that phages and their proteins can be exploited as an effective weapon in the near future, especially in a world which is on the brink of a “postantibiotic era.” PMID:28484722

Valyl-tRNA synthetase modification-dependent restriction of bacteriophage T4.

PubMed Central

Olson, N J; Marchin, G L

1984-01-01

A strain of Escherichia coli, CP 790302, severely restricts the growth of wild-type bacteriophage T4. In broth culture, most infections of single cells are abortive, although a few infected cells exhibit reduced burst sizes. In contrast, bacteriophage T4 mutants impaired in the ability to modify valyl-tRNA synthetase develop normally on this strain. Biochemical evidence indicates that the phage-modified valyl-tRNA synthetase in CP 790302 is different from that previously described. Although the enzyme is able to support normal protein synthesis, a disproportionate amount of phage structural protein (serum blocking power) fails to mature into particles of the appropriate density. The results with host strain CP 790302 are consistent with either a gratuitous inhibition of phage assembly by faulty modification or abrogation of an unknown role that valyl-tRNA synthetase might normally play in viral assembly. PMID:6374167

Polar flagella rotation in Vibrio parahaemolyticus confers resistance to bacteriophage infection

PubMed Central

Zhang, Hui; Li, Lu; Zhao, Zhe; Peng, Daxin; Zhou, Xiaohui

2016-01-01

Bacteriophage has been recognized as a novel approach to treat bacterial infectious diseases. However, phage resistance may reduce the efficacy of phage therapy. Here, we described a mechanism of bacterial resistance to phage infections. In Gram-negative enteric pathogen Vibrio parahaemolyticus, we found that polar flagella can reduce the phage infectivity. Deletion of polar flagella, but not the lateral flagella, can dramatically promote the adsorption of phage to the bacteria and enhances the phage infectivity to V. parahaemolyticus, indicating that polar flagella play an inhibitory role in the phage infection. Notably, it is the rotation, not the physical presence, of polar flagella that inhibits the phage infection of V. parahaemolyticus. Strikingly, phage dramatically reduces the virulence of V. parahaemolyticus only when polar flagella were absent both in vitro and in vivo. These results indicated that polar flagella rotation is a previously unidentified mechanism that confers bacteriophage resistance. PMID:27189325

Production and Purification of Recombinant Filamentous Bacteriophages Displaying Immunogenic Heterologous Epitopes.

PubMed

Deng, Lei; Linero, Florencia; Saelens, Xavier

2016-01-01

Viruslike particles often combine high physical stability with robust immunogenicity. Furthermore, when such particles are based on bacteriophages, they can be produced in high amounts at minimal cost and typically will require only standard biologically contained facilities. We provide protocols for the characterization and purification of recombinant viruslike particles derived from filamentous bacteriophages. As an example, we focus on filamentous Escherichia coli fd phage displaying a conserved influenza A virus epitope that is fused genetically to the N-terminus of the major coat protein of this phage. A step-by-step procedure to obtain a high-titer, pure recombinant phage preparation is provided. We also describe a quality control experiment based on a biological readout of the purified fd phage preparation. These protocols together with the highlighted critical steps may facilitate generic implementation of the provided procedures for the display of other epitopes by recombinant fd phages.

A mechanical model of bacteriophage DNA ejection

NASA Astrophysics Data System (ADS)

Arun, Rahul; Ghosal, Sandip

2017-08-01

Single molecule experiments on bacteriophages show an exponential scaling for the dependence of mobility on the length of DNA within the capsid. It has been suggested that this could be due to the ;capstan mechanism; - the exponential amplification of friction forces that result when a rope is wound around a cylinder as in a ship's capstan. Here we describe a desktop experiment that illustrates the effect. Though our model phage is a million times larger, it exhibits the same scaling observed in single molecule experiments.

Computational determination of the effects of virulent Escherichia coli and salmonella bacteriophages on human gut.

PubMed

Mostafa, Marwa Mostafa; Nassef, Mohammad; Badr, Amr

2016-10-01

Salmonella and Escherichia coli are different types of bacteria that cause food poisoning in humans. In the elderly, infants and people with chronic conditions, it is very dangerous if Salmonella or E. coli gets into the bloodstream and then they must be treated by phage therapy. Treating Salmonella and E. coli by phage therapy affects the gut flora. This research paper presents a system for detecting the effects of virulent E. coli and Salmonella bacteriophages on human gut. A method based on Domain-Domain Interactions (DDIs) model is implemented in the proposed system to determine the interactions between the proteins of human gut bacteria and the proteins of bacteriophages that infect virulent E. coli and Salmonella. The system helps gastroenterologists to realize the effect of injecting bacteriophages that infect virulent E. coli and Salmonella on the human gut. By testing the system over Enterobacteria phage 933W, Enterobacteria phage VT2-Sa and Enterobacteria phage P22, it resulted in four interactions between the proteins of the bacteriophages that infect E. coli O157:H7, E. coli O104:H4 and Salmonella typhimurium and the proteins of human gut bacterium strains. Several effects were detected such as: antibacterial activity against a number of bacterial species in human gut, regulation of cellular differentiation and organogenesis during gut, lung, and heart development, ammonia assimilation in bacteria, yeasts, and plants, energizing defense system and its function in the detoxification of lipopolysaccharide, and in the prevention of bacterial translocation in human gut. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Functional Coupling between HIV-1 Integrase and the SWI/SNF Chromatin Remodeling Complex for Efficient in vitro Integration into Stable Nucleosomes

PubMed Central

Lesbats, Paul; Botbol, Yair; Chevereau, Guillaume; Vaillant, Cédric; Calmels, Christina; Arneodo, Alain; Andreola, Marie-Line; Lavigne, Marc; Parissi, Vincent

2011-01-01

Establishment of stable HIV-1 infection requires the efficient integration of the retroviral genome into the host DNA. The molecular mechanism underlying the control of this process by the chromatin structure has not yet been elucidated. We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase. Remodeling of the chromatinized template by the SWI/SNF complex, whose INI1 major component interacts with IN, restores and redirects the full-site integration into the stable nucleosome region. These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1. This suggests that the restoration process depends on the direct interaction between IN and the whole SWI/SNF complex, supporting a functional coupling between the remodeling and integration complexes. Furthermore, in silico comparison between more than 40,000 non-redundant cellular integration sites selected from literature and nucleosome occupancy predictions also supports that HIV-1 integration is promoted in the genomic region of weaker intrinsic nucleosome density in the infected cell. Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier. PMID:21347347

Antibacterial efficacy of nisin, bacteriophage P100 and sodium lactate against Listeria monocytogenes in ready-to-eat sliced pork ham.

PubMed

Figueiredo, Ana Cláudia L; Almeida, Rogeria C C

The effectiveness of bacteriophage P100, nisin and sodium lactate, individually and in combination, in inhibiting Listeria monocytogenes in ready-to-eat pork ham slices was assessed. The antimicrobials were applied to the surfaces of ready-to-eat pork ham slices, which were inoculated with a mixture of L. monocytogenes. Among the individual antimicrobial treatments, bacteriophage P100 was the most effective, decreasing L. monocytogenes to undetectable levels at zero and 72h post-infection. Sodium lactate was the least effective treatment. Treatment with nisin at zeroh significantly reduced initial cell density (p<0.05). However, this pattern was not observed at 72h of storage. A significant difference (p<0.05) existed between the results of separate bacteriophage and nisin treatments after refrigerated storage, but not immediately upon inoculation of the bacteria. The results showed that the use of bacteriophage P100 is the method of choice for the control of bacteria. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Bacteriophage 5' untranslated regions for control of plastid transgene expression.

PubMed

Yang, Huijun; Gray, Benjamin N; Ahner, Beth A; Hanson, Maureen R

2013-02-01

Expression of foreign proteins from transgenes incorporated into plastid genomes requires regulatory sequences that can be recognized by the plastid transcription and translation machinery. Translation signals harbored by the 5' untranslated region (UTR) of plastid transcripts can profoundly affect the level of accumulation of proteins expressed from chimeric transgenes. Both endogenous 5' UTRs and the bacteriophage T7 gene 10 (T7g10) 5' UTR have been found to be effective in combination with particular coding regions to mediate high-level expression of foreign proteins. We investigated whether two other bacteriophage 5' UTRs could be utilized in plastid transgenes by fusing them to the aadA (aminoglycoside-3'-adenyltransferase) coding region that is commonly used as a selectable marker in plastid transformation. Transplastomic plants containing either the T7g1.3 or T4g23 5' UTRs fused to Myc-epitope-tagged aadA were successfully obtained, demonstrating the ability of these 5' UTRs to regulate gene expression in plastids. Placing the Thermobifida fusca cel6A gene under the control of the T7g1.3 or T4g23 5' UTRs, along with a tetC downstream box, resulted in poor expression of the cellulase in contrast with high-level accumulation while using the T7g10 5' UTR. However, transplastomic plants with the bacteriophage 5' UTRs controlling the aadA coding region exhibited fewer undesired recombinant species than plants containing the same marker gene regulated by the Nicotiana tabacum psbA 5' UTR. Furthermore, expression of the T7g1.3 and T4g23 5' UTR::aadA fusions downstream of the cel6A gene provided sufficient spectinomycin resistance to allow selection of homoplasmic transgenic plants and had no effect on Cel6A accumulation.

Impairment of Human Immunodeficiency Virus Type-1 Integrase SUMOylation Correlates with an Early Replication Defect*

PubMed Central

Zamborlini, Alessia; Coiffic, Audrey; Beauclair, Guillaume; Delelis, Olivier; Paris, Joris; Koh, Yashuiro; Magne, Fabian; Giron, Marie-Lou; Tobaly-Tapiero, Joelle; Deprez, Eric; Emiliani, Stephane; Engelman, Alan; de Thé, Hugues; Saïb, Ali

2011-01-01

HIV-1 integrase (IN) orchestrates the integration of the reverse transcribed viral cDNA into the host cell genome and participates also in other steps of HIV-1 replication. Cellular and viral factors assist IN in performing its multiple functions, and post-translational modifications contribute to modulate its activities. Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO acceptor sites. Viruses harboring SUMOylation site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Because SUMOylation-defective IN mutants retained WT catalytic activity, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication. PMID:21454548

Removal of Surrogate Bacteriophages and Enteric Viruses from Seeded Environmental Waters Using a Semi-technical Ultrafiltration Unit.

PubMed

Frohnert, Anne; Kreißel, Katja; Lipp, Pia; Dizer, Halim; Hambsch, Beate; Szewzyk, Regine; Selinka, Hans-Christoph

2015-03-19

Experiments to determine the removal of viruses in different types of water (surface water from two reservoirs for drinking water treatment, treated groundwater and groundwater contaminated with either 5 or 30 % of wastewater) by ultrafiltration were performed with a semi-technical ultrafiltration unit. Concentrations of human adenoviruses (HAdVs), murine norovirus (MNV), and the bacteriophages MS2, ΦX174 and PRD1 were measured in the feed water and the filtrate, and log removal values were calculated. Bacteria added to the feed water were not detected in the filtrates. In contrast, in most cases viruses and bacteriophages were still present in the filtrates: log removal values were in the range of 1.4-6.3 depending on virus sizes and water qualities. Best removals were observed with bacteriophage PRD1 and HAdVs, followed by MNV and phages MS2 and ΦX174. Virus size, however, was not the only criterion for efficient removal. In diluted wastewater as compared to drinking water and uncontaminated environmental waters, virus removal was clearly higher for all viruses, most likely due to higher membrane fouling. For quality assessment purposes of membrane filtration efficiencies with regard to the elimination of human viruses the small bacteriophages MS2 and ΦX174 should be used as conservative viral indicators.

Bacteriophage Taxonomy: An Evolving Discipline.

PubMed

Tolstoy, Igor; Kropinski, Andrew M; Brister, J Rodney

2018-01-01

While taxonomy is an often-unappreciated branch of science it serves very important roles. Bacteriophage taxonomy has evolved from a mainly morphology-based discipline, characterized by the work of David Bradley and Hans-Wolfgang Ackermann, to the holistic approach that is taken today. The Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) takes a comprehensive approach to classifying prokaryote viruses measuring overall DNA and protein identity and phylogeny before making decisions about the taxonomic position of a new virus. The huge number of complete genomes being deposited with NCBI and other public databases has resulted in a reassessment of the taxonomy of many viruses, and the future will see the introduction of new viral families and higher orders.

The possible use of V. parahaemolyticus - specific bacteriophages for prevention and therapy of infections caused by V. parahaemolyticus.

PubMed

Tskhvediani, A; Khukhunashvili, T; Eliashvili, T; Tsertsvadze, G; Gachechiladze, N; Tediashvili, M

2014-06-01

Vibrio parahaemolyticus is the most common halophilic Vibrio species causing serious gastroenteritis in humans. The main source of infection is consumption of undercooked or raw seafood or exposure to contaminated water. The monitoring conducted in 2006-2008 demonstrated that warm, subtropical climate and low- to moderate salinity of water in the Black Sea coastal zone provides a favorable environment for growth and spread of V. parahaemolyticus bacteria. Antibiotics are commonly applied for control V.parahaemolyticus infections in humans. However, with the growing problem with bacterial antibiotic-resistance search for alternative biological anti-infectives, such as bacteriophages, becomes more actual. The aim of the presented work was characterization of V. parahamolyticus- specific bacteriophages in relation with their possible use for treatment and prevention of food and waterborne gastroenteritis in humans infected with V.parahaemolyticus. 69 bacteriophages specific to V.parahaemolyticus were isolated from different water sources and 5 of them were characterized according to their virion morphology, host-range, temperature and pH dependence. Stability of phages in different media and solutions, also susceptibility to action of a number of protolithic enzymes was studied as well. Obtained results showed that studied bacteriophages can be used for preparation of phage mixture as a potential therapeutic preparation against V.parahaemolyticus associated infections.

Quantification of the HIV-integrase inhibitor raltegravir (MK-0518) in human plasma by high-performance liquid chromatography with fluorescence detection.

PubMed

Poirier, Jean-Marie; Robidou, Pascal; Jaillon, Patrice

2008-05-15

A simple and sensitive HLPC method with fluorescence detection was developed for the accurate determination of the first licensed HIV integrase inhibitor raltegravir in human plasma. A 500-microL plasma sample was spiked with delavirdine as internal standard and subjected to liquid-liquid extraction based on a previously described assay i.e. using hexane/methylene chloride (1:1, v/v%) at pH 4.0. HPLC was performed using a Symmetry Shield RP18 column (150 mm x 4.6 mm), a gradient elution of acetonitrile -0.01% (v/v) triethylamine in water adjusted to pH 3.0 at a flow rate of 1 mL/min and a fluorimetric detector set at 299 and 396 nm as excitation and emission wavelengths, respectively. The retention time was 5.0 min for internal standard and 6.4 min for raltegravir. Calibration curves were linear in the range 5-1000 ng/mL and the accuracy of quality control samples in the range 10-750 ng/mL varied from 98.3 to 99.1% and 98.3 to 101.0% of the nominal concentrations for intra-day and day-to-day analysis, respectively with a precision of 6.3% or less. Among the other antiretroviral drugs which can be given in association to HIV-infected patients, none was found to interfere with internal standard or raltegravir. The described assay was developed for the purpose of therapeutic drug of this HIV integrase inhibitor.

The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku

PubMed Central

di Fagagna, Fabrizio d'Adda; Weller, Geoffrey R.; Doherty, Aidan J.; Jackson, Stephen P.

2003-01-01

Mu bacteriophage inserts its DNA into the genome of host bacteria and is used as a model for DNA transposition events in other systems. The eukaryotic Ku protein has key roles in DNA repair and in certain transposition events. Here we show that the Gam protein of phage Mu is conserved in bacteria, has sequence homology with both subunits of Ku, and has the potential to adopt a similar architecture to the core DNA-binding region of Ku. Through biochemical studies, we demonstrate that Gam and the related protein of Haemophilus influenzae display DNA binding characteristics remarkably similar to those of human Ku. In addition, we show that Gam can interfere with Ty1 retrotransposition in Saccharomyces cerevisiae. These data reveal structural and functional parallels between bacteriophage Gam and eukaryotic Ku and suggest that their functions have been evolutionarily conserved. PMID:12524520

Hydrodynamics of bacteriophages

NASA Astrophysics Data System (ADS)

Katsamba, Panayiota; Lauga, Eric

2017-11-01

Bacteriophage viruses, one of the most abundant entities in our planet, lack the ability to move independently. Instead, they crowd fluid environments in anticipation of a random encounter with bacteria. Once they 'land' on their victim's surface, they eject their genetic material inside the host cell. A big fraction of phage species, however, first attach to the flagella of bacteria. Being immotile, these so-called flagellotropic phages still manage to reach the cell body for infection, and the process by which they move up the flagellum has intrigued the scientific community for over four decades. In 1973 Berg and Anderson proposed the nut-and-bolt mechanism in which, just like a nut being rotated moves along a bolt, the phage wraps itself around a flagellum possessing helical grooves (due to the helical rows of flagellin molecules) and exploits the rotation of the flagellum in order to passively travel along it. We provide here a first-principle theoretical model for this nut-and-bolt mechanism and show that it is able to predict experiment observations.