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Sample records for integrating phage vhml

  1. The Linear Plasmid Prophage Vp58.5 of Vibrio parahaemolyticus Is Closely Related to the Integrating Phage VHML and Constitutes a New Incompatibility Group of Telomere Phages▿ †

    PubMed Central

    Zabala, Beatriz; Hammerl, Jens A.; Espejo, Romilio T.; Hertwig, Stefan

    2009-01-01

    Vibrio parahaemolyticus O3:K6 pandemic strains recovered in Chile frequently possess a 42-kb plasmid which is the prophage of a myovirus. We studied the prototype phage VP58.5 and show that it does not integrate into the host cell chromosome but replicates as a linear plasmid (Vp58.5) with covalently closed ends (telomeres). The Vp58.5 replicon coexists with other plasmid prophages (N15, PY54, and ΦKO2) in the same cell and thus belongs to a new incompatibility group of telomere phages. We determined the complete nucleotide sequence (42,612 nucleotides) of the VP58.5 phage DNA and compared it with that of the plasmid prophage. The two molecules share the same nucleotide sequence but are 35% circularly permuted to each other. In contrast to the hairpin ends of the plasmid, VP58.5 phage DNA contains 5′-protruding ends. The VP58.5 sequence is 92% identical to the sequence of phage VHML, which was reported to integrate into the host chromosome. However, the gene order and termini of the phage DNAs are different. The VHML genome exhibits the same gene order as does the Vp58.5 plasmid. VHML phage DNA has been reported to contain terminal inverted repeats. This repetitive sequence is similar to the telomere resolution site (telRL) of VP58.5 which, after processing by the phage protelomerase, forms the hairpin ends of the Vp58.5 prophage. It is discussed why these closely related phages may be so different in terms of their genome ends and their lifestyle. PMID:19587034

  2. Replication and Maintenance of Linear Phage-Plasmid N15.

    PubMed

    Ravin, Nikolai V

    2015-02-01

    The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into the chromosome but is a linear plasmid molecule with covalently closed ends (telomeres). Upon infection, the phage DNA circularizes via cohesive ends, and then a special phage enzyme of the tyrosine recombinase family, protelomerase, cuts at another site and joins the ends, forming hairpin telomeres of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally, resulting in the formation of duplicated telomeres. The N15 protelomerase cuts them, generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by a partitioning operon similar to the F factor sop operon. Unlike the F centromere, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in the N15 genome regions involved in phage replication and control of lytic development, and binding of partition proteins at these sites regulates these processes. The family of N15-like linear phage-plasmids includes lambdoid phages ɸKO2 and pY54, as well as Myoviridae phages ΦHAP-1, VHML, VP882, Vp58.5, and vB_VpaM_MAR of marine gamma-proteobacteria. The genomes of these phages contain similar protelomerase genes, lysogeny control modules, and replication genes, suggesting that these phages may belong to a group diverged from a common ancestor.

  3. Complete nucleotide sequence of a new filamentous phage, Xf109, which integrates its genome into the chromosomal DNA of Xanthomonas oryzae.

    PubMed

    Yeh, Ting Y

    2017-02-01

    Unlike Ff-like coliphages, certain filamentous Inoviridae phages integrate their genomes into the host chromosome and enter a prophage state in their infectious cycle. This lysogenic life cycle was first reported for Xanthomonas citri Cf phage. However, except for the X. citri phages Cf and XacF1, complete genome sequence information about lysogenic Xanthomonas phages is not available to date. A proviral sequence of Xf109 phage was identified in the genome of Xanthomonas oryzae, the rice bacterial blight pathogen, and revived as infectious virions to lysogenize its host de novo. The genome of Xf109 phage is 7190 nucleotides in size and contains 12 predicted open reading frames in an organization similar to that of the Cf phage genome. Seven of the Xf109 proteins show significant sequence similarity to Cf and XacF1 phage proteins, while its ORF4 shares 92 % identity with the major coat protein of X. phage oryzae Xf. Integration of Xf109 phage DNA into the host genome is site-specific, and the attP/attB sequence contains the dif core sequence 5'-TATACATTATGCGAA-3', which is identical to that of Cf, XacF1, and Xanthomonas campestris phage ϕLf. To my knowledge, this is the first complete genome sequence of a filamentous bacteriophage that infects X. oryzae.

  4. A proposed integrated approach for the preclinical evaluation of phage therapy in Pseudomonas infections

    NASA Astrophysics Data System (ADS)

    Danis-Wlodarczyk, Katarzyna; Vandenheuvel, Dieter; Jang, Ho Bin; Briers, Yves; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Drabik, Marcin; Higgins, Gerard; Tyrrell, Jean; Harvey, Brian J.; Noben, Jean-Paul; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2016-06-01

    Bacteriophage therapy is currently resurging as a potential complement/alternative to antibiotic treatment. However, preclinical evaluation lacks streamlined approaches. We here focus on preclinical approaches which have been implemented to assess bacteriophage efficacy against Pseudomonas biofilms and infections. Laser interferometry and profilometry were applied to measure biofilm matrix permeability and surface geometry changes, respectively. These biophysical approaches were combined with an advanced Airway Surface Liquid infection model, which mimics in vitro the normal and CF lung environments, and an in vivo Galleria larvae model. These assays have been implemented to analyze KTN4 (279,593 bp dsDNA genome), a type-IV pili dependent, giant phage resembling phiKZ. Upon contact, KTN4 immediately disrupts the P. aeruginosa PAO1 biofilm and reduces pyocyanin and siderophore production. The gentamicin exclusion assay on NuLi-1 and CuFi-1 cell lines revealed the decrease of extracellular bacterial load between 4 and 7 logs and successfully prevents wild-type Pseudomonas internalization into CF epithelial cells. These properties and the significant rescue of Galleria larvae indicate that giant KTN4 phage is a suitable candidate for in vivo phage therapy evaluation for lung infection applications.

  5. A proposed integrated approach for the preclinical evaluation of phage therapy in Pseudomonas infections

    PubMed Central

    Danis-Wlodarczyk, Katarzyna; Vandenheuvel, Dieter; Jang, Ho Bin; Briers, Yves; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Drabik, Marcin; Higgins, Gerard; Tyrrell, Jean; Harvey, Brian J.; Noben, Jean-Paul; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2016-01-01

    Bacteriophage therapy is currently resurging as a potential complement/alternative to antibiotic treatment. However, preclinical evaluation lacks streamlined approaches. We here focus on preclinical approaches which have been implemented to assess bacteriophage efficacy against Pseudomonas biofilms and infections. Laser interferometry and profilometry were applied to measure biofilm matrix permeability and surface geometry changes, respectively. These biophysical approaches were combined with an advanced Airway Surface Liquid infection model, which mimics in vitro the normal and CF lung environments, and an in vivo Galleria larvae model. These assays have been implemented to analyze KTN4 (279,593 bp dsDNA genome), a type-IV pili dependent, giant phage resembling phiKZ. Upon contact, KTN4 immediately disrupts the P. aeruginosa PAO1 biofilm and reduces pyocyanin and siderophore production. The gentamicin exclusion assay on NuLi-1 and CuFi-1 cell lines revealed the decrease of extracellular bacterial load between 4 and 7 logs and successfully prevents wild-type Pseudomonas internalization into CF epithelial cells. These properties and the significant rescue of Galleria larvae indicate that giant KTN4 phage is a suitable candidate for in vivo phage therapy evaluation for lung infection applications. PMID:27301427

  6. Phage therapy pharmacology phage cocktails.

    PubMed

    Chan, Benjamin K; Abedon, Stephen T

    2012-01-01

    Phage therapy is the clinical or veterinary application of bacterial viruses (bacteriophages) as antibacterial "drugs." More generally, phages can be used as biocontrol agents against plant as well as foodborne pathogens. In this chapter, we consider the therapeutic use of phage cocktails, which is the combining of two or more phage types to produce more pharmacologically diverse formulations. The primary motivation for the use of cocktails is their broader spectra of activity in comparison to individual phage isolates: they can impact either more bacterial types or achieve effectiveness under a greater diversity of conditions. The combining of phages can also facilitate better targeting of multiple strains making up individual bacterial species or covering multiple species that might be responsible for similar disease states, in general providing, relative to individual phage isolates, a greater potential for presumptive or empirical treatment. Contrasting the use of phage banks, or even phage isolation against specific etiologies that have been obtained directly from patients under treatment, here we consider the utility as well as potential shortcomings associated with the use of phage cocktails as therapeutic antibacterial agents.

  7. Advance in phage display technology for bioanalysis.

    PubMed

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

    2016-06-01

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

  8. The origin of phage virology.

    PubMed

    Pennazio, Sergio

    2006-01-01

    The history of bacteriophage (phage) had its start in 1915, when Twort isolated an unusual filterable and infectious agent from excrete of patients struck by diarrhoea; this discovery was followed by an analogous, and probably independent, finding of d'Hérelle in 1917. For several years phage research made scant progress but great attention was paid to the question of phage nature, which saw the contrast between d'Hérelle and Bordet's views (living against chemical nature, respectively). This situation changed with the independent discovery of lysogeny, in 1925, thanks to Bordet and Bail: this phenomenon was considered of genetical origin, a view that Wollman interpreted by assimilating the properties of phage to those of gene (according to a previous idea of Muller). In the 1930s, Burnet's work opened a new era by demonstrating the occurrence of several species of phages and their antigenic property. In the same period, the physical and chemical characteristics of these viruses were disclosed thanks, in particular, to the work of Schlesinger, who first demonstrated that a virus (phage) was constituted of nucleoproteins. The peculiarity of phage was finally shown after the invention of electron microscope: H. Ruska, in 1940, and Anderson and Luria in the next years, obtained the first images of tailed phages, a finding that strongly helped the investigation on the first steps of the infection process. The decisive impulse to phage virology came from Delbrück, a physicist who entered biology giving it a new arrangement. The so-called "phage group" assembled brilliant minds (Luria, Hershey and Delbrück himself, and later a dozen of other scientists): this group faced three fundamental questions of phage virology, i.e., the mechanisms of attack, multiplication and lysis. In ten years' time, phage virology became an integrant part of molecular biology, also thanks to the discovery of the genetical properties of DNA: in such scientific context, Delbrück, Luria and

  9. Phage therapy pharmacology: calculating phage dosing.

    PubMed

    Abedon, Stephen

    2011-01-01

    Phage therapy, which can be described as a phage-mediated biocontrol of bacteria (or, simply, biocontrol), is the application of bacterial viruses-also bacteriophages or phages-to reduce densities of nuisance or pathogenic bacteria. Predictive calculations for phage therapy dosing should be useful toward rational development of therapeutic as well as biocontrol products. Here, I consider the theoretical basis of a number of concepts relevant to phage dosing for phage therapy including minimum inhibitory concentration (but also "inundation threshold"), minimum bactericidal concentration (but also "clearance threshold"), decimal reduction time (D value), time until bacterial eradication, threshold bacterial density necessary to support phage population growth ("proliferation threshold"), and bacterial density supporting half-maximal phage population growth rates (K(B)). I also address the concepts of phage killing titers, multiplicity of infection, and phage peak densities. Though many of the presented ideas are not unique to this chapter, I nonetheless provide variations on derivations and resulting formulae, plus as appropriate discuss relative importance. The overriding goal is to present a variety of calculations that are useful toward phage therapy dosing so that they may be found in one location and presented in a manner that allows facile appreciation, comparison, and implementation. The importance of phage density as a key determinant of the phage potential to eradicate bacterial targets is stressed throughout the chapter.

  10. Designing phage therapeutics.

    PubMed

    Goodridge, Lawrence D

    2010-01-01

    Phage therapy is the application of phages to bodies, substances, or environments to effect the biocontrol of pathogenic or nuisance bacteria. To be effective, phages, minimally, must be capable of attaching to bacteria (adsorption), killing those bacteria (usually associated with phage infection), and otherwise surviving (resisting decay) until they achieve attachment and subsequent killing. While a strength of phage therapy is that phages that possess appropriate properties can be chosen from a large diversity of naturally occurring phages, a more rational approach to phage therapy also can include post-isolation manipulation of phages genetically, phenotypically, or in terms of combining different products into a single formulation. Genetic manipulation, especially in these modern times, can involve genetic engineering, though a more traditional approach involves the selection of spontaneously occurring phage mutants during serial transfer protocols. While genetic modification typically is done to give rise to phenotypic changes in phages, phage phenotype alone can also be modified in vitro, prior to phage application for therapeutic purposes, as for the sake of improving phage lethality (such as by linking phage virions to antibacterial chemicals such as chloramphenicol) or survival capabilities (e.g., via virion PEGylation). Finally, phages, both naturally occurring isolates or otherwise modified constructs, can be combined into cocktails which provide collectively enhanced capabilities such as expanded overall host range. Generally these strategies represent different routes towards improving phage therapy formulations and thereby efficacy through informed design.

  11. Engineered phages for electronics.

    PubMed

    Cui, Yue

    2016-11-15

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

  12. N15: the linear phage-plasmid.

    PubMed

    Ravin, Nikolai V

    2011-03-01

    The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into chromosome but is a linear plasmid molecule with covalently closed ends. Upon infection the phage DNA circularises via cohesive ends, then phage-encoded enzyme, protelomerase, cuts at an inverted repeat site and forms hairpin ends (telomeres) of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally resulting in formation of duplicated telomeres. Then the N15 protelomerase cuts duplicated telomeres generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by partitioning operon similar to the F factor sop operon. Unlike F sop, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in N15 genome regions involved in phage replication and control of lysogeny, and binding of partition proteins at these sites regulates these processes. Two N15-related lambdoid Siphoviridae phages, φKO2 in Klebsiella oxytoca and pY54 in Yersinia enterocolitica, also lysogenize their hosts as linear plasmids, as well as Myoviridae marine phages VP882 and VP58.5 in Vibrio parahaemolyticus and ΦHAP-1 in Halomonas aquamarina. The genomes of all these phages contain similar protelomerase genes, lysogeny modules and replication genes, as well as plasmid-partitioning genes, suggesting that these phages may belong to a group diverged from a common ancestor.

  13. Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by streptomyces phage phiC31 integrase

    PubMed Central

    2013-01-01

    Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5′ and 3′ junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the

  14. An integrated microfluidic system for screening of phage-displayed peptides specific to colon cancer cells and colon cancer stem cells.

    PubMed

    Che, Yu-Jui; Wu, Huei-Wen; Hung, Lien-Yu; Liu, Ching-Ann; Chang, Hwan-You; Wang, Kuan; Lee, Gwo-Bin

    2015-09-01

    Affinity reagents recognizing biomarkers specifically are essential components of clinical diagnostics and target therapeutics. However, conventional methods for screening of these reagents often have drawbacks such as large reagent consumption, the labor-intensive or time-consuming procedures, and the involvement of bulky or expensive equipment. Alternatively, microfluidic platforms could potentially automate the screening process within a shorter period of time and reduce reagent and sample consumption dramatically. It has been demonstrated recently that a subpopulation of tumor cells known as cancer stem cells possess high drug resistance and proliferation potential and are regarded as the main cause of metastasis. Therefore, a peptide that recognizes cancer stem cells and differentiates them from other cancer cells will be extremely useful in early diagnosis and target therapy. This study utilized M13 phage display technology to identify peptides that bind, respectively, to colon cancer cells and colon cancer stem cells using an integrated microfluidic system. In addition to positive selection, a negative selection process was integrated on the chip to achieve the selection of peptides of high affinity and specificity. We successfully screened three peptides specific to colon cancer cells and colon cancer stem cells, namely, HOLC-1, HOLC-2, and COLC-1, respectively, and their specificity was measured by the capture rate between target, control, and other cell lines. The capture rates are 43.40 ± 7.23%, 45.16 ± 7.12%, and 49.79 ± 5.34% for colon cancer cells and colon cancer stem cells, respectively, showing a higher specificity on target cells than on control and other cell lines. The developed technique may be promising for early diagnosis of cancer cells and target therapeutics.

  15. Estimating richness from phage metagenomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  16. Phage therapy: present and future

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

  17. Phage Therapy: Future Inquiries

    PubMed Central

    Wu, Sijia; Zachary, Elisabeth; Wells, Keenan; Loc-Carrillo, Catherine

    2016-01-01

    Western scientists have steadily been gaining interest in phage therapy since the mid-1980’s due to the rising problem of antibiotic resistance. Its introduction in the 20th century by Felix d’Herelle marked the beginning for the uses of bacteriophages as antibacterial agents. However, a lack in understanding phage biology, as well as the arrival of broad-spectrum antibiotics deprioritized using phage therapy to treat bacterial infections in the West. With the advent of molecular biology, we are now better able to understand the predator-prey relationships with which phage co-evolve with their hosts as well as the specificity of phage-host interactions which could lend itself into personalized treatments for infection. These discoveries give us greater insights on how to most effectively use bacteriophage as potential therapeutic agents. It is encouraging to note that bacteriophages are used as food additives in the U.S., suggesting that the FDA acknowledges the positive potential of bacteriophages for human applications. Unfortunately, there are only a few examples to date of bacteriophages used on humans in controlled clinical trials. Rigorous studies in-vitro and especially in-vivo are critically important to avoid the mishaps of our predecessors. Phage biologists must strive to meet regulatory standards and to design thorough, rugged studies in order to establish a substantiated need for phage therapy in health care.

  18. Phage treatment of human infections

    PubMed Central

    Abedon, Stephen T; Kuhl, Sarah J; Blasdel, Bob G

    2011-01-01

    Phages as bactericidal agents have been employed for 90 years as a means of treating bacterial infections in humans as well as other species, a process known as phage therapy. In this review we explore both the early historical and more modern use of phages to treat human infections. We discuss in particular the little-reviewed French early work, along with the Polish, US, Georgian and Russian historical experiences. We also cover other, more modern examples of phage therapy of humans as differentiated in terms of disease. In addition, we provide discussions of phage safety, other aspects of phage therapy pharmacology, and the idea of phage use as probiotics. PMID:22334863

  19. Phage choice, isolation, and preparation for phage therapy.

    PubMed

    Gill, Jason J; Hyman, Paul

    2010-01-01

    Phage therapy is the use of bacteriophages--viruses that use bacteria as their host cells--as biocontrol agents of bacteria. Currently, phage therapy is garnering renewed interest as bacterial resistance to antibiotics becomes widespread. Historically, phage therapy was largely abandoned in the West in the 1940s due to the advent of chemical antibiotics, and the unreliability of phage-based treatments when compared to antibiotics. The choice of phage strain and the methods of phage preparation are now thought to have been critical to the success or failure of phage therapy trials. Insufficiently virulent phages, especially against actual target bacteria, allow bacteria to survive treatment while poorly prepared phage stocks, even if of sufficiently virulent phages, lack the numbers of viable phages required for adequate treatment. In this review we discuss the factors that determine the methods of isolation, analysis, and identification of phage species for phage therapy. We go on to discuss the various methods available for purifying phages as well as considerations of the degree of purification which is sufficient for various applications. Lastly, we review the current practices used to prepare commercial phage therapy products.

  20. Temperate phages both mediate and drive adaptive evolution in pathogen biofilms

    PubMed Central

    Davies, Emily V.; James, Chloe E.; Williams, David; O’Brien, Siobhan; Fothergill, Joanne L.; Haldenby, Sam; Paterson, Steve; Winstanley, Craig

    2016-01-01

    Temperate phages drive genomic diversification in bacterial pathogens. Phage-derived sequences are more common in pathogenic than nonpathogenic taxa and are associated with changes in pathogen virulence. High abundance and mobilization of temperate phages within hosts suggests that temperate phages could promote within-host evolution of bacterial pathogens. However, their role in pathogen evolution has not been experimentally tested. We experimentally evolved replicate populations of Pseudomonas aeruginosa with or without a community of three temperate phages active in cystic fibrosis (CF) lung infections, including the transposable phage, ɸ4, which is closely related to phage D3112. Populations grew as free-floating biofilms in artificial sputum medium, mimicking sputum of CF lungs where P. aeruginosa is an important pathogen and undergoes evolutionary adaptation and diversification during chronic infection. Although bacterial populations adapted to the biofilm environment in both treatments, population genomic analysis revealed that phages altered both the trajectory and mode of evolution. Populations evolving with phages exhibited a greater degree of parallel evolution and faster selective sweeps than populations without phages. Phage ɸ4 integrated randomly into the bacterial chromosome, but integrations into motility-associated genes and regulators of quorum sensing systems essential for virulence were selected in parallel, strongly suggesting that these insertional inactivation mutations were adaptive. Temperate phages, and in particular transposable phages, are therefore likely to facilitate adaptive evolution of bacterial pathogens within hosts. PMID:27382184

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

    PubMed

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

    2011-01-01

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

  2. Phages in nature

    PubMed Central

    Millard, Andrew D; Letarov, Andrey V; Heaphy, Shaun

    2011-01-01

    Bacteriophages or phages are the most abundant organisms in the biosphere and they are a ubiquitous feature of prokaryotic existence. A bacteriophage is a virus which infects a bacterium. Archaea are also infected by viruses, whether these should be referred to as ‘phages’ is debatable, but they are included as such in the scope this article. Phages have been of interest to scientists as tools to understand fundamental molecular biology, as vectors of horizontal gene transfer and drivers of bacterial evolution, as sources of diagnostic and genetic tools and as novel therapeutic agents. Unraveling the biology of phages and their relationship with their hosts is key to understanding microbial systems and their exploitation. In this article we describe the roles of phages in different host systems and show how modeling, microscopy, isolation, genomic and metagenomic based approaches have come together to provide unparalleled insights into these small but vital constituents of the microbial world. PMID:21687533

  3. Extensive phage dynamics in Staphylococcus aureus contributes to adaptation to the human host during infection.

    PubMed

    Goerke, Christiane; Wirtz, Christiane; Flückiger, Ursula; Wolz, Christiane

    2006-09-01

    Bacteriophages serve as a driving force in microbial evolution, adaptation to new environments and the pathogenesis of human bacterial infections. In Staphylococcus aureus phages encoding immune evasion molecules (SAK, SCIN, CHIPS), which integrate specifically into the beta-haemolysin (Hlb) gene, are widely distributed. When comparing S. aureus strain collections from infectious and colonizing situations we could detect a translocation of sak-encoding phages to atypical genomic integration sites in the bacterium only in the disease-related isolates. Additionally, significantly more Hlb producing strains were detected in the infectious strain collection. Extensive phage dynamics (intragenomic translocation, duplication, transfer between hosts, recombination events) during infection was shown by analysing cocolonizing and consecutive isolates of patients. This activity leads to the splitting of the strain population into various subfractions exhibiting different virulence potentials (Hlb-production and/or production of immune evasion molecules). Thus, phage-inducing conditions and strong selection for survival of the bacterial host after phage movement are typical for the infectious situation. Further in vitro characterization of phages revealed that: (i) SAK is encoded not only on serogroup F phages showing a conserved tropism for hlb but also on serogroup B phages which always integrate in a distinct intergenic region, (ii) the level of sak transcription correlates to phage inducibility but is independent of the phage localization in the chromosome, and (iii) phages can be stabilized extra-chromosomally during their life cycle.

  4. Phage genomics: small is beautiful.

    PubMed

    Brüssow, Harald; Hendrix, Roger W

    2002-01-11

    The Age of Genomics dawned only gradually for bacteriophages. It was 1977 when the genome of phage phi X174 was published and 1983 when the "large" genome of phage lambda hit the streets. More recently, the pace has quickened, so that we now have over 100 complete phage genomes and can expect thousands in a very few years. These sequences have been marvelously informative for the biology of the individual phages, but with the advent of high volume sequencing technology, the real excitement for phage biology is that it is now possible to analyze the sequences together and thereby address--for the first time at whole genome resolution--a set of fundamental biological questions related to populations: What is the structure of the global phage population? What are its dynamics? How do phages evolve? This is Comparative Genomics with a capital "C".

  5. A novel fluorescent probe: europium complex hybridized T7 phage.

    PubMed

    Liu, Chin-Mei; Jin, Qiaoling; Sutton, April; Chen, Liaohai

    2005-01-01

    We report on the creation of a novel fluorescent probe of europium-complex hybridized T7 phage. It was made by filling a ligand-displayed T7 ghost phage with a fluorescent europium complex particle. The structure of the hybridized phage, which contains a fluorescent inorganic core surrounded by a ligand-displayed capsid shell, was confirmed by electron microscope, energy-dispersive X-ray analysis (EDX), bioassays, and fluorescence spectrometer. More importantly, as a benefit of the phage display technology, the hybridized phage has the capability to integrate an affinity reagent against virtually any target molecules. The approach provides an original method to fluorescently "tag" a bioligand and/or to "biofunctionalize" a fluorophore particle. By using other types of materials such as radioactive or magnetic particles to fill the ghost phage, we envision that the hybridized phages represent a new class of fluorescent, magnetic, or radioprobes for imaging and bioassays and could be used both in vitro and in vivo.

  6. Phage neutralization by sera of patients receiving phage therapy.

    PubMed

    Łusiak-Szelachowska, Marzanna; Zaczek, Maciej; Weber-Dąbrowska, Beata; Międzybrodzki, Ryszard; Kłak, Marlena; Fortuna, Wojciech; Letkiewicz, Sławomir; Rogóż, Paweł; Szufnarowski, Krzysztof; Jończyk-Matysiak, Ewa; Owczarek, Barbara; Górski, Andrzej

    2014-08-01

    The aim of our investigation was to verify whether phage therapy (PT) can induce antiphage antibodies. The antiphage activity was determined in sera from 122 patients from the Phage Therapy Unit in Wrocław with bacterial infections before and during PT, and in sera from 30 healthy volunteers using a neutralization test. Furthermore, levels of antiphage antibodies were investigated in sera of 19 patients receiving staphylococcal phages and sera of 20 healthy volunteers using enzyme-linked immunosorbent assay. The phages were administered orally, locally, orally/locally, intrarectally, or orally/intrarectally. The rate of phage inactivation (K) estimated the level of phages' neutralization by human sera. Low K rates were found in sera of healthy volunteers (K ≤ 1.73). Low K rates were detected before PT (K ≤ 1.64). High antiphage activity of sera K > 18 was observed in 12.3% of examined patients (n = 15) treated with phages locally (n = 13) or locally/orally (n = 2) from 15 to 60 days of PT. High K rates were found in patients treated with some Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis phages. Low K rates were observed during PT in sera of patients using phages orally (K ≤ 1.04). Increased inactivation of phages by sera of patients receiving PT decreased after therapy. These results suggest that the antiphage activity in patients' sera depends on the route of phage administration and phage type. The induction of antiphage activity of sera during or after PT does not exclude a favorable result of PT.

  7. Phage Neutralization by Sera of Patients Receiving Phage Therapy

    PubMed Central

    Żaczek, Maciej; Weber-Dąbrowska, Beata; Międzybrodzki, Ryszard; Kłak, Marlena; Fortuna, Wojciech; Letkiewicz, Sławomir; Rogóż, Paweł; Szufnarowski, Krzysztof; Jończyk-Matysiak, Ewa; Owczarek, Barbara; Górski, Andrzej

    2014-01-01

    Abstract The aim of our investigation was to verify whether phage therapy (PT) can induce antiphage antibodies. The antiphage activity was determined in sera from 122 patients from the Phage Therapy Unit in Wrocław with bacterial infections before and during PT, and in sera from 30 healthy volunteers using a neutralization test. Furthermore, levels of antiphage antibodies were investigated in sera of 19 patients receiving staphylococcal phages and sera of 20 healthy volunteers using enzyme-linked immunosorbent assay. The phages were administered orally, locally, orally/locally, intrarectally, or orally/intrarectally. The rate of phage inactivation (K) estimated the level of phages' neutralization by human sera. Low K rates were found in sera of healthy volunteers (K≤1.73). Low K rates were detected before PT (K≤1.64). High antiphage activity of sera K>18 was observed in 12.3% of examined patients (n=15) treated with phages locally (n=13) or locally/orally (n=2) from 15 to 60 days of PT. High K rates were found in patients treated with some Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis phages. Low K rates were observed during PT in sera of patients using phages orally (K≤1.04). Increased inactivation of phages by sera of patients receiving PT decreased after therapy. These results suggest that the antiphage activity in patients' sera depends on the route of phage administration and phage type. The induction of antiphage activity of sera during or after PT does not exclude a favorable result of PT. PMID:24893003

  8. Synergy as a rationale for phage therapy using phage cocktails

    PubMed Central

    Schmerer, Matthew; Molineux, Ian J.

    2014-01-01

    Where phages are used to treat bacterial contaminations and infections, multiple phages are typically applied at once as a cocktail. When two or more phages in the cocktail attack the same bacterium, the combination may produce better killing than any single phage (synergy) or the combination may be worse than the best single phage (interference). Synergy is of obvious utility, especially if it can be predicted a priori, but it remains poorly documented with few examples known. This study addresses synergy in which one phage improves adsorption by a second phage. It first presents evidence of synergy from an experimental system of two phages and a mucoid E. coli host. The synergy likely stems from a tailspike enzyme produced by one of the phages. We then offer mathematical models and simulations to understand the dynamics of synergy and the enhanced magnitude of bacterial control possible. The models and observations complement each other and suggest that synergy may be of widespread utility and may be predictable from easily observed phenotypes. PMID:25279269

  9. Drugs derived from phage display

    PubMed Central

    Nixon, Andrew E; Sexton, Daniel J; Ladner, Robert C

    2014-01-01

    Phage display, one of today’s fundamental drug discovery technologies, allows identification of a broad range of biological drugs, including peptides, antibodies and other proteins, with the ability to tailor critical characteristics such as potency, specificity and cross-species binding. Further, unlike in vivo technologies, generating phage display-derived antibodies is not restricted by immunological tolerance. Although more than 20 phage display-derived antibody and peptides are currently in late-stage clinical trials or approved, there is little literature addressing the specific challenges and successes in the clinical development of phage-derived drugs. This review uses case studies, from candidate identification through clinical development, to illustrate the utility of phage display as a drug discovery tool, and offers a perspective for future developments of phage display technology. PMID:24262785

  10. Highly efficient integration of the viral portal proteins from different types of phages into planar bilayers for the black lipid membrane analysis.

    PubMed

    Jing, Peng; Paraiso, Hallel; Burris, Benjamin

    2016-02-01

    The planar lipid bilayer technology is a technique that yields incredibly useful structural function information about a single channel protein. It is also currently actively utilized as a powerful platform using biological protein nanopores for the development of single-molecule nanopore sensing technology, as well as ultrafast DNA sequencing technology. The portal protein, GP10, from the bacteriophage Φ29 was the first phage portal protein shown to be successfully inserted into planar bilayer membranes, thereby it may inspire more researchers to apply the techniques to portal proteins from the other bacteriophages. However, the technology is far from perfect since the insertion of the channel proteins into planar bilayer membranes is not only technically difficult but also time-consuming. For the fusion of phage portal proteins, vesicles are typically needed to be reconstituted with the portal proteins to form proteoliposomes. However, most of the phage portal proteins have low solubility, and may self-aggregate during the preparation of the proteoliposomes. Furthermore, the fusion of the formed proteoliposomes is sporadic, unpredictable and varied from person to person. Due to the lack of experimental consistency between labs, the results from different methodologies reported for generating fusible proteoliposomes are highly variable. In this research, we propose a new method for the preparation of the fusible proteoliposomes containing portal proteins from bacteriophages, to circumvent the problems aforementioned. Compared to the conventional methods, this method was able to avoid the protein aggregation issues during the vesicle preparation by eliminating the need for detergents and the subsequent time-consuming step for detergent removal. The proteoliposomes prepared by the method were shown to be more efficiently and rapidly inserted into planar bilayer membranes bathed in different conducting buffer solutions including those with nonelectrolytes such as

  11. Clostridium difficile phages: still difficult?

    PubMed Central

    Hargreaves, Katherine R.; Clokie, Martha R. J.

    2014-01-01

    Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however, developing suitable phages is challenging. In this review we summarize the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics. Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage–host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution. No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using “whole-phages” are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem-free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen. PMID:24808893

  12. Phage cocktails and the future of phage therapy.

    PubMed

    Chan, Benjamin K; Abedon, Stephen T; Loc-Carrillo, Catherine

    2013-06-01

    Viruses of bacteria, known as bacteriophages or phages, were discovered nearly 100 years ago. Their potential as antibacterial agents was appreciated almost immediately, with the first 'phage therapy' trials predating Fleming's discovery of penicillin by approximately a decade. In this review, we consider phage therapy that can be used for treating bacterial infections in humans, domestic animals and even biocontrol in foods. Following an overview of the topic, we explore the common practice - both experimental and, in certain regions of the world, clinical - of mixing therapeutic phages into cocktails consisting of multiple virus types. We conclude with a discussion of the commercial and medical context of phage cocktails as therapeutic agents. In comparing off-the-shelf versus custom approaches, we consider the merits of a middle ground, which we deem 'modifiable'. Finally, we explore a regulatory framework for such an approach based on an influenza vaccine model.

  13. Phages targeting infected tissues: novel approach to phage therapy.

    PubMed

    Górski, Andrzej; Dąbrowska, Krystyna; Hodyra-Stefaniak, Katarzyna; Borysowski, Jan; Międzybrodzki, Ryszard; Weber-Dąbrowska, Beata

    2015-01-01

    While the true efficacy of phage therapy still requires formal confirmation in clinical trials, it continues to offer realistic potential treatment in patients in whom antibiotics have failed. Novel developments and approaches are therefore needed to ascertain that future clinical trials would evaluate the therapy in its optimal form thus allowing for reliable conclusions regarding the true value of phage therapy. In this article, we present our vision to develop and establish a bank of phages specific to most threatening pathogens and armed with homing peptides enabling their localization in infected tissues in densities assuring efficient and stable eradication of infection.

  14. Phage therapy of pulmonary infections

    PubMed Central

    Abedon, Stephen T

    2015-01-01

    It is generally agreed that a bacteriophage-associated phenomenon was first unambiguously observed one-hundred years ago with the findings of Twort in 1915. This was independently followed by complementary observations by d'Hérelle in 1917. D'Hérelle's appreciation of the bacteriophage phenomenon appears to have directly led to the development of phages as antibacterial agents within a variety of contexts, including medical and agricultural. Phage use to combat nuisance bacteria appears to be especially useful where targets are sufficiently problematic, suitably bactericidal phages exist, and alternative approaches are lacking in effectiveness, availability, safety, or cost effectiveness, etc. Phage development as antibacterial agents has been strongest particularly when antibiotics have been less available or useful, e.g., such as in the treatment of chronic infections by antibiotic-resistant bacteria. One relatively under-explored or at least not highly reported use of phages as therapeutic agents has been to combat bacterial infections of the lungs and associated tissues. These infections are diverse in terms of their etiologies, manifestations, and also in terms of potential strategies of phage delivery. Here I review the literature considering the phage therapy of pulmonary and pulmonary-related infections, with emphasis on reports of clinical treatment along with experimental treatment of pulmonary infections using animal models. PMID:26442188

  15. Targeting mammalian organelles with internalizing phage (iPhage) libraries

    PubMed Central

    Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

  16. Kinetics of filamentous phage assembly

    NASA Astrophysics Data System (ADS)

    Ploss, Martin; Kuhn, Andreas

    2010-12-01

    Filamentous phages release their progeny particles by a secretory process without lysing the bacterial cell. By this process about 6 viral particles per min are secreted from each cell. We show here that when the major coat protein (gp8) is provided from a plasmid we observe a phage progeny production rate depending on the induction of gp8 by IPTG. We also show that a transfection of Escherichia coli lacking F-pili is observed using a mutant of M13 that carries an ampicillin resistance gene, and phage particles are secreted in the absence of an F-plasmid. Extruding phage was visualized by atomic force microscopy (AFM) and by transmission electron microscopy (TEM) using gold-labeled antibodies to the major coat protein.

  17. Bacteria-phage interactions in natural environments.

    PubMed

    Díaz-Muñoz, Samuel L; Koskella, Britt

    2014-01-01

    Phages are considered the most abundant and diverse biological entities on Earth and are notable not only for their sheer abundance, but also for their influence on bacterial hosts. In nature, bacteria-phage relationships are complex and have far-reaching consequences beyond particular pairwise interactions, influencing everything from bacterial virulence to eukaryotic fitness to the carbon cycle. In this review, we examine bacteria and phage distributions in nature first by highlighting biogeographic patterns and nonhost environmental influences on phage distribution, then by considering the ways in which phages and bacteria interact, emphasizing phage life cycles, bacterial responses to phage infection, and the complex patterns of phage host specificity. Finally, we discuss phage impacts on bacterial abundance, genetics, and physiology, and further aim to clarify distinctions between current theoretical models and point out areas in need of future research.

  18. Current insights into phage biodiversity and biogeography.

    PubMed

    Thurber, Rebecca Vega

    2009-10-01

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

  19. A giant Pseudomonas phage from Poland.

    PubMed

    Drulis-Kawa, Zuzanna; Olszak, Tomasz; Danis, Katarzyna; Majkowska-Skrobek, Grazyna; Ackermann, Hans-W

    2014-03-01

    A novel giant phage of the family Myoviridae is described. Pseudomonas phage PA5oct was isolated from a sewage sample from an irrigated field near Wroclaw, Poland. The virion morphology indicates that PA5oct differs from known giant phages. The phage has a head of about 131 nm in diameter and a tail of 136 × 19 nm. Phage PA5oct contains a genome of approximately 375 kbp and differs in size from any tailed phages known. PA5oct was further characterized by determination of its latent period and burst size and its sensitivity to heating, chloroform, and pH.

  20. Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine

    PubMed Central

    Cao, Binrui; Yang, Mingying; Mao, Chuanbin

    2016-01-01

    CONSPECTUS Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (~900 nm long and ~8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic–inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their

  1. Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine.

    PubMed

    Cao, Binrui; Yang, Mingying; Mao, Chuanbin

    2016-06-21

    Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (∼900 nm long and ∼8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic-inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their osteogenic

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

    PubMed

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

    2015-02-01

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

  3. φ29 Family of Phages

    PubMed Central

    Meijer, Wilfried J. J.; Horcajadas, José A.; Salas, Margarita

    2001-01-01

    Continuous research spanning more than three decades has made the Bacillus bacteriophage φ29 a paradigm for several molecular mechanisms of general biological processes, such as DNA replication, regulation of transcription, phage morphogenesis, and phage DNA packaging. The genome of bacteriophage φ29 consists of a linear double-stranded DNA (dsDNA), which has a terminal protein (TP) covalently linked to its 5′ ends. Initiation of DNA replication, carried out by a protein-primed mechanism, has been studied in detail and is considered to be a model system for the protein-primed DNA replication that is also used by most other linear genomes with a TP linked to their DNA ends, such as other phages, linear plasmids, and adenoviruses. In addition to a continuing progress in unraveling the initiation of DNA replication mechanism and the role of various proteins involved in this process, major advances have been made during the last few years, especially in our understanding of transcription regulation, the head-tail connector protein, and DNA packaging. Recent progress in all these topics is reviewed. In addition to φ29, the genomes of several other Bacillus phages consist of a linear dsDNA with a TP molecule attached to their 5′ ends. These φ29-like phages can be divided into three groups. The first group includes, in addition to φ29, phages PZA, φ15, and BS32. The second group comprises B103, Nf, and M2Y, and the third group contains GA-1 as its sole member. Whereas the DNA sequences of the complete genomes of φ29 (group I) and B103 (group II) are known, only parts of the genome of GA-1 (group III) were sequenced. We have determined the complete DNA sequence of the GA-1 genome, which allowed analysis of differences and homologies between the three groups of φ29-like phages, which is included in this review. PMID:11381102

  4. Clinical aspects of phage therapy.

    PubMed

    Międzybrodzki, Ryszard; Borysowski, Jan; Weber-Dąbrowska, Beata; Fortuna, Wojciech; Letkiewicz, Sławomir; Szufnarowski, Krzysztof; Pawełczyk, Zdzisław; Rogóż, Paweł; Kłak, Marlena; Wojtasik, Elżbieta; Górski, Andrzej

    2012-01-01

    Phage therapy (PT) is a unique method of treatment of bacterial infections using bacteriophages (phages)-viruses that specifically kill bacteria, including their antibiotic-resistant strains. Over the last decade a marked increase in interest in the therapeutic use of phages has been observed, which has resulted from a substantial rise in the prevalence of antibiotic resistance of bacteria, coupled with an inadequate number of new antibiotics. The first, and so far the only, center of PT in the European Union is the Phage Therapy Unit (PTU) established at the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland in 2005. This center continues the rich tradition of PT in Poland, which dates from the early 1920s. The main objective of this chapter is to present a detailed retrospective analysis of the results of PT of 153 patients with a wide range of infections resistant to antibiotic therapy admitted for treatment at the PTU between January 2008 and December 2010. Analysis includes the evaluation of both the efficacy and the safety of PT. In general, data suggest that PT can provide good clinical results in a significant cohort of patients with otherwise untreatable chronic bacterial infections and is essentially well tolerated. In addition, the whole complex procedure employed to obtain and characterize therapeutic phage preparations, as well as ethical aspects of PT, is discussed.

  5. The Phage Shock Protein Response.

    PubMed

    Flores-Kim, Josué; Darwin, Andrew J

    2016-09-08

    The phage shock protein (Psp) system was identified as a response to phage infection in Escherichia coli, but rather than being a specific response to a phage, it detects and mitigates various problems that could increase inner-membrane (IM) permeability. Interest in the Psp system has increased significantly in recent years due to appreciation that Psp-like proteins are found in all three domains of life and because the bacterial Psp response has been linked to virulence and other important phenotypes. In this article, we summarize our current understanding of what the Psp system detects and how it detects it, how four core Psp proteins form a signal transduction cascade between the IM and the cytoplasm, and current ideas that explain how the Psp response keeps bacterial cells alive. Although recent studies have significantly improved our understanding of this system, it is an understanding that is still far from complete.

  6. Phage therapy--constraints and possibilities.

    PubMed

    Nilsson, Anders S

    2014-05-01

    The rise of antibiotic-resistant bacterial strains, causing intractable infections, has resulted in an increased interest in phage therapy. Phage therapy preceded antibiotic treatment against bacterial infections and involves the use of bacteriophages, bacterial viruses, to fight bacteria. Virulent phages are abundant and have proven to be very effective in vitro, where they in most cases lyse any bacteria within the hour. Clinical trials on animals and humans show promising results but also that the treatments are not completely effective. This is partly due to the studies being carried out with few phages, and with limited experimental groups, but also the fact that phage therapy has limitations in vivo. Phages are large compared with small antibiotic molecules, and each phage can only infect one or a few bacterial strains. A very large number of different phages are needed to treat infections as these are caused by genetically different strains of bacteria. Phages are effective only if enough of them can reach the bacteria and increase in number in situ. Taken together, this entails high demands on resources for the construction of phage libraries and the testing of individual phages. The effectiveness and host range must be characterized, and immunological risks must be assessed for every single phage.

  7. Who went into phage research?

    PubMed Central

    2012-01-01

    A total of 30,000 phage papers, books, or book chapters, published between 1965 and 2010, were analyzed for the ethnic origins of 14,429 first authors. Their names represent 40 linguistic domains or geographic areas and at least 70 languages. British and German names predominate. Results broadly concur with statistics on the frequency of publications by country and show the growing role of Third-World countries in phage research. Irish and Jewish scientists are prominent. Historical and societal factors appear to be very important elements in the advancement of science. PMID:22666657

  8. Comparative genomics of phages and prophages in lactic acid bacteria.

    PubMed

    Desiere, Frank; Lucchini, Sacha; Canchaya, Carlos; Ventura, Marco; Brüssow, Harald

    2002-08-01

    Comparative phage genomics has become possible due to the availability of more than 100 complete phage genome sequences and the development of powerful bioinformatics tools. This technology, profiting from classical molecular-biology knowledge, has opened avenues of research for topics, which were difficult to address in the past. Now, it is possible to retrace part of the evolutionary history of phage modules by comparative genomics. The diagnosis of relatedness is hereby not uniquely based on sequence similarity alone, but includes topological considerations of genome organization. Detailed transcription maps have allowed in silico predictions of genome organization to be verified and refined. This comparative knowledge is providing the basis for a new taxonomic classification concept for bacteriophages infecting low G + C-content Gram-positive bacteria based on the genetic organization of the structural gene module. An Sfi21-like and an Sfi11-like genus of Siphoviridae is proposed. The gene maps of many phages show remarkable synteny in their structural genes defining a lambda super-group within Siphoviridae. A hierarchy of relatedness within the lambda super-group suggests elements of vertical evolution in Siphoviridae. Tailed phages are the result of both vertical and horizontal evolution and are thus fascinating objects for the study of molecular evolution. Prophage sequences integrated into the genomes of their bacterial host present theoretical challenges for evolutionary biologists. Prophages represent up to 10% of the genome in some LAB. In pathogenic streptococci prophages confer genes of selective value for the lysogenic cell. The lysogenic conversion genes are located between the lysin gene and the right phage attachment site. Non-attributed genes were found at the same genome position of prophages from lactic streptococci. These genes belong to the few prophage genes transcribed in the lysogen. Prophages from dairy bacteria might therefore also

  9. Phage lytic enzymes: a history.

    PubMed

    Trudil, David

    2015-02-01

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

  10. Thioredoxin is required for filamentous phage assembly.

    PubMed Central

    Russel, M; Model, P

    1985-01-01

    Sequence comparisons show that the fip gene product of Escherichia coli, which is required for filamentous phage assembly, is thioredoxin. Thioredoxin serves as a cofactor for reductive processes in many cell types and is a constituent of phage T7 DNA polymerase. The fip-1 mutation makes filamentous phage and T7 growth temperature sensitive in cells that carry it. The lesion lies within a highly conserved thioredoxin active site. Thioredoxin reductase (NADPH), as well as thioredoxin, is required for efficient filamentous phage production. Mutant phages defective in phage gene I are particularly sensitive to perturbations in the fip-thioredoxin system. A speculative model is presented in which thioredoxin reductase, thioredoxin, and the gene I protein interact to drive an engine for filamentous phage assembly. Images PMID:3881756

  11. The habits of highly effective phages: population dynamics as a framework for identifying therapeutic phages

    PubMed Central

    Bull, James J.; Gill, Jason J.

    2014-01-01

    The use of bacteriophages as antibacterial agents is being actively researched on a global scale. Typically, the phages used are isolated from the wild by plating on the bacteria of interest, and a far larger set of candidate phages is often available than can be used in any application. When an excess of phages is available, how should the best phages be identified? Here we consider phage-bacterial population dynamics as a basis for evaluating and predicting phage success. A central question is whether the innate dynamical properties of phages are the determinants of success, or instead, whether extrinsic, indirect effects can be responsible. We address the dynamical perspective, motivated in part by the absence of dynamics in previously suggested principles of phage therapy. Current mathematical models of bacterial-phage dynamics do not capture the realities of in vivo dynamics, nor is this likely to change, but they do give insight to qualitative properties that may be generalizable. In particular, phage adsorption rate may be critical to treatment success, so understanding the effects of the in vivo environment on host availability may allow prediction of useful phages prior to in vivo experimentation. Principles for predicting efficacy may be derived by developing a greater understanding of the in vivo system, or such principles could be determined empirically by comparing phages with known differences in their dynamic properties. The comparative approach promises to be a powerful method of discovering the key to phage success. We offer five recommendations for future study: (i) compare phages differing in treatment efficacy to identify the phage properties associated with success, (ii) assay dynamics in vivo, (iii) understand mechanisms of bacterial escape from phages, (iv) test phages in model infections that are relevant to the intended clinical applications, and (v) develop new classes of models for phage growth in spatially heterogeneous environments

  12. Phage Therapy: Eco-Physiological Pharmacology

    PubMed Central

    Abedon, Stephen T.

    2014-01-01

    Bacterial virus use as antibacterial agents, in the guise of what is commonly known as phage therapy, is an inherently physiological, ecological, and also pharmacological process. Physiologically we can consider metabolic properties of phage infections of bacteria and variation in those properties as a function of preexisting bacterial states. In addition, there are patient responses to pathogenesis, patient responses to phage infections of pathogens, and also patient responses to phage virions alone. Ecologically, we can consider phage propagation, densities, distribution (within bodies), impact on body-associated microbiota (as ecological communities), and modification of the functioning of body “ecosystems” more generally. These ecological and physiological components in many ways represent different perspectives on otherwise equivalent phenomena. Comparable to drugs, one also can view phages during phage therapy in pharmacological terms. The relatively unique status of phages within the context of phage therapy as essentially replicating antimicrobials can therefore result in a confluence of perspectives, many of which can be useful towards gaining a better mechanistic appreciation of phage therapy, as I consider here. Pharmacology more generally may be viewed as a discipline that lies at an interface between organism-associated phenomena, as considered by physiology, and environmental interactions as considered by ecology. PMID:25031881

  13. Recombinant phage probes for Listeria monocytogenes

    NASA Astrophysics Data System (ADS)

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

    2007-10-01

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

  14. Characterization of two polyvalent phages infecting Enterobacteriaceae

    PubMed Central

    Hamdi, Sana; Rousseau, Geneviève M.; Labrie, Simon J.; Tremblay, Denise M.; Kourda, Rim Saïed; Ben Slama, Karim; Moineau, Sylvain

    2017-01-01

    Bacteriophages display remarkable genetic diversity and host specificity. In this study, we explore phages infecting bacterial strains of the Enterobacteriaceae family because of their ability to infect related but distinct hosts. We isolated and characterized two novel virulent phages, SH6 and SH7, using a strain of Shigella flexneri as host bacterium. Morphological and genomic analyses revealed that phage SH6 belongs to the T1virus genus of the Siphoviridae family. Conversely, phage SH7 was classified in the T4virus genus of the Myoviridae family. Phage SH6 had a short latent period of 16 min and a burst size of 103 ± 16 PFU/infected cell while the phage SH7 latent period was 23 min with a much lower burst size of 26 ± 5 PFU/infected cell. Moreover, phage SH6 was sensitive to acidic conditions (pH < 5) while phage SH7 was stable from pH 3 to 11 for 1 hour. Of the 35 bacterial strains tested, SH6 infected its S. flexneri host strain and 8 strains of E. coli. Phage SH7 lysed additionally strains of E. coli O157:H7, Salmonella Paratyphi, and Shigella dysenteriae. The broader host ranges of these two phages as well as their microbiological properties suggest that they may be useful for controlling bacterial populations. PMID:28091598

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

    PubMed

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

    2012-05-01

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

  16. Phage display: applications, innovations, and issues in phage and host biology.

    PubMed

    Wilson, D R; Finlay, B B

    1998-04-01

    In the 7 years since the first publications describing phage-displayed peptide libraries, phage display has been successfully employed in a variety of research. Innovations in vector design and methods to identify target clones account for much of this success. At the same time, not all ventures have been entirely successful and it appears that phage and host biology play important roles in this. A key issue concerns the role played by a displayed peptide or protein in its successful expression and incorporation into virions. While few studies have examined these issues specifically in context of phage display, the literature as a whole provides insight. Accordingly, we review phage biology, relevant aspects of host biology, and phage display applications with the goals of illustrating (i) relevant aspects of the interplay between phage-host biology and successful phage display and (ii) the limitations and considerable potential of this important technology.

  17. Control of Pierce's Disease by Phage

    PubMed Central

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

    2015-01-01

    Pierce’s Disease (PD) of grapevines, caused by Xylella fastidiosa subsp. fastidiosa (Xf), is a limiting factor in the cultivation of grapevines in the US. There are presently no effective control methods to prevent or treat PD. The therapeutic and prophylactic efficacy of a phage cocktail composed of four virulent (lytic) phages was evaluated for control of PD. Xf levels in grapevines were significantly reduced in therapeutically or prophylactically treated grapevines. PD symptoms ceased to progress one week post-therapeutic treatment and symptoms were not observed in prophylactically treated grapevines. Cocktail phage levels increased in grapevines in the presence of the host. No in planta phage-resistant Xf isolates were obtained. Moreover, Xf mutants selected for phage resistance in vitro did not cause PD symptoms. Our results indicate that phages have great potential for biocontrol of PD and other economically important diseases caused by Xylella. PMID:26107261

  18. Bacteriophages with potential to inactivate Salmonella Typhimurium: Use of single phage suspensions and phage cocktails.

    PubMed

    Pereira, Carla; Moreirinha, Catarina; Lewicka, Magdalena; Almeida, Paulo; Clemente, Carla; Cunha, Ângela; Delgadillo, Ivonne; Romalde, Jésus L; Nunes, Maria L; Almeida, Adelaide

    2016-07-15

    The aim of this study was to compare the dynamics of three previously isolated bacteriophages (or phages) individually (phSE-1, phSE-2 and phSE-5) or combined in cocktails of two or three phages (phSE-1/phSE-2, phSE-1/phSE-5, phSE-2/phSE-5 and phSE-1/phSE-2/phSE-5) to control Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) in order to evaluate their potential application during depuration. Phages were assigned to the family Siphoviridae and revealed identical restriction digest profiles, although they showed a different phage adsorption, host range, burst size, explosion time and survival in seawater. The three phages were effective against S. Typhimurium (reduction of ∼2.0 log CFU/mL after 4h treatment). The use of cocktails was not significantly more effective than the use of single phages. A big fraction of the remained bacteria are phage-resistant mutants (frequency of phage-resistant mutants 9.19×10(-5)-5.11×10(-4)) but phage- resistant bacterial mutants was lower for the cocktail phages than for the single phage suspensions and the phage phSE-1 presented the highest rate of resistance and phage phSE-5 the lowest one. The spectral changes of S. Typhimurium resistant and phage-sensitive cells were compared and revealed relevant differences for peaks associated to amide I (1620cm(-1)) and amide II (1515cm(-1)) from proteins and from carbohydrates and phosphates region (1080-1000cm(-1)). Despite the similar efficiency of individual phages, the development of lower resistance indicates that phage cocktails might be the most promising choice to be used during the bivalve depuration to control the transmission of salmonellosis.

  19. Two flagellotropic phages and one pilus-specific phage active against Asticcacaulis biprosthecum.

    PubMed

    Pate, J L; Petzold, S J; Umbreit, T H

    1979-04-15

    Three phages active against cells of Asticcacaulis biprosthecum attach to receptor sites located at the pole of the cell where pili, flagella, and holdfast are produced. Phage phiAcS2, a large phage with a prolate cylindrical head and flexible, noncontractile tail, attaches to flagella as well as to receptor sites at the pole of the cell. Attachment to flagella occurs at the region where head and tail of the phage are joined, leaving the distal end of the tail free for attachment to receptor sites at the cell surface. Phages phiAcM2 and phiAcM4, are identical in appearance to each other, possessing prolate cylindrical heads and flexible, noncontractile tails, and are smaller than phage phiAcS2. Phage phiAcM4, exhibits the same flagellotropic characteristic as described for phage phiAcS2, including the manner of attachment to flagella. Phage phiAcM2 has no affinity for flagella, but attaches by the distal end of the tail to pili and to receptor sites at the pole of the cell. Mechanical removal of flagella and pili protects against infection by all three phages. Studies with phage-resistant mutants and with KCN-treated cells suggest that pili are required for infection by both flagellotropic and pilus-specific phages.

  20. [Peptide phage display in biotechnology and biomedicine].

    PubMed

    Kuzmicheva, G A; Belyavskaya, V A

    2016-07-01

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

  1. Experimental Phage Therapy for Burkholderia pseudomallei Infection

    PubMed Central

    Leang-Chung, Choh; Vellasamy, Kumutha Malar; Mariappan, Vanitha; Li-Yen, Chang; Vadivelu, Jamuna

    2016-01-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen intrinsically resistant to a variety of antibiotics. Phages have been developed for use as an alternative treatment therapy, particularly for bacterial infections that do not respond to conventional antibiotics. In this study, we investigated the use of phages to treat cells infected with B. pseudomallei. Phage C34 isolated from seawater was purified and characterised on the basis of its host range and morphology using transmission electron microscopy (TEM). Phage C34 was able to lyse 39.5% of B. pseudomallei clinical strains. Due to the presence of contractile tail, phage C34 is classified as a member of the family Myoviridae, a tailed double-stranded DNA virus. When 2 × 105 A549 cells were exposed to 2 × 107 PFU of phage C34, 24 hours prior to infection with 2 × 106 CFU of B. pseudomallei, it was found that the survivability of the cells increased to 41.6 ± 6.8% as compared to 22.8 ± 6.0% in untreated control. Additionally, application of phage successfully rescued 33.3% of mice infected with B. pseudomallei and significantly reduced the bacterial load in the spleen of the phage-treated mice. These findings indicate that phage can be a potential antimicrobial agent for B. pseudomallei infections. PMID:27387381

  2. DNA libraries for the construction of phage libraries: statistical and structural requirements and synthetic methods.

    PubMed

    Lindner, Thomas; Kolmar, Harald; Haberkorn, Uwe; Mier, Walter

    2011-02-15

    Peptide-based molecular probes identified by bacteriophage (phage) display technology expand the peptide repertoire for in vivo diagnosis and therapy of cancer. Numerous peptides that bind cancer-associated antigens have been discovered by panning phage libraries. However, until now only few of the peptides selected by phage display have entered clinical applications. The success of phage derived peptides essentially depends on the quality of the library screened. This review summarizes the methods to achieve highly homogenous libraries that cover a maximal sequence space. Biochemical and chemical strategies for the synthesis of DNA libraries and the techniques for their integration into the viral genome are discussed in detail. A focus is set on the methods that enable the exclusion of disturbing sequences. In addition, the parameters that define the variability, the minimal numbers of copies per library and the use of alternating panning cycles to avoid the loss of selected hits are evaluated.

  3. Statistical structure of host-phage interactions.

    PubMed

    Flores, Cesar O; Meyer, Justin R; Valverde, Sergi; Farr, Lauren; Weitz, Joshua S

    2011-07-12

    Interactions between bacteria and the viruses that infect them (i.e., phages) have profound effects on biological processes, but despite their importance, little is known on the general structure of infection and resistance between most phages and bacteria. For example, are bacteria-phage communities characterized by complex patterns of overlapping exploitation networks, do they conform to a more ordered general pattern across all communities, or are they idiosyncratic and hard to predict from one ecosystem to the next? To answer these questions, we collect and present a detailed metaanalysis of 38 laboratory-verified studies of host-phage interactions representing almost 12,000 distinct experimental infection assays across a broad spectrum of taxa, habitat, and mode of selection. In so doing, we present evidence that currently available host-phage infection networks are statistically different from random networks and that they possess a characteristic nested structure. This nested structure is typified by the finding that hard to infect bacteria are infected by generalist phages (and not specialist phages) and that easy to infect bacteria are infected by generalist and specialist phages. Moreover, we find that currently available host-phage infection networks do not typically possess a modular structure. We explore possible underlying mechanisms and significance of the observed nested host-phage interaction structure. In addition, given that most of the available host-phage infection networks examined here are composed of taxa separated by short phylogenetic distances, we propose that the lack of modularity is a scale-dependent effect, and then, we describe experimental studies to test whether modular patterns exist at macroevolutionary scales.

  4. A Virulent Phage Infecting Lactococcus garvieae, with Homology to Lactococcus lactis Phages

    PubMed Central

    Eraclio, Giovanni; Tremblay, Denise M.; Lacelle-Côté, Alexia; Labrie, Simon J.; Fortina, Maria Grazia

    2015-01-01

    A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor. PMID:26407890

  5. Selection of phages and conditions for the safe phage therapy against Pseudomonas aeruginosa infections.

    PubMed

    Krylov, Victor; Shaburova, Olga; Pleteneva, Elena; Krylov, Sergey; Kaplan, Alla; Burkaltseva, Maria; Polygach, Olga; Chesnokova, Elena

    2015-02-01

    The emergence of multidrug-resistant bacterial pathogens forced us to consider the phage therapy as one of the possible alternative approaches to treatment. The purpose of this paper is to consider the conditions for the safe, long-term use of phage therapy against various infections caused by Pseudomonas aeruginosa. We describe the selection of the most suitable phages, their most effective combinations and some approaches for the rapid recognition of phages unsuitable for use in therapy. The benefits and disadvantages of the various different approaches to the preparation of phage mixtures are considered, together with the specific conditions that are required for the safe application of phage therapy in general hospitals and the possibilities for the development of personalized phage therapy.

  6. Methods for Selecting Phage Display Antibody Libraries.

    PubMed

    Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2016-01-01

    The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

  7. Epitope Mapping with Random Phage Display Library

    PubMed Central

    Midoro-Horiuti, Terumi; Goldblum, Randall M.

    2017-01-01

    Random phage display library is used to map conformational as well as linear epitopes. These libraries are available in varying lengths and with circularization. We provide here a protocol conveying our experience using a commercially available peptide phage display library, which in our hands provides good results. PMID:24515483

  8. Aeromonas phages encode tRNAs for their overused codons.

    PubMed

    Prabhakaran, Ramanandan; Chithambaram, Shivapriya; Xia, Xuhua

    2014-01-01

    The GC-rich bacterial species, Aeromonas salmonicida, is parasitised by both GC-rich phages (Aeromonas phages - phiAS7 and vB_AsaM-56) and GC-poor phages (Aeromonas phages - 25, 31, 44RR2.8t, 65, Aes508, phiAS4 and phiAS5). Both the GC-rich Aeromonas phage phiAS7 and Aeromonas phage vB_AsaM-56 have nearly identical codon usage bias as their host. While all the remaining seven GC-poor Aeromonas phages differ dramatically in codon usage from their GC-rich host. Here, we investigated whether tRNA encoded in the genome of Aeromonas phages facilitate the translation of phage proteins. We found that tRNAs encoded in the phage genome correspond to synonymous codons overused in the phage genes but not in the host genes.

  9. Population Dynamics of Phage and Bacteria in Spatially Structured Habitats Using Phage λ and Escherichia coli

    PubMed Central

    Brown, Stanley; Sneppen, Kim

    2016-01-01

    ABSTRACT Bacteria living in physically structured habitats are exposed heterogeneously to both resources and different types of phages. While there have been numerous experimental approaches to examine spatially distributed bacteria exposed to phages, there is little theory to guide the design of these experiments, interpret their results, or expand the inferences drawn to a broader ecological and evolutionary context. Plaque formation provides a window into understanding phage-bacterium interactions in physically structured populations, including surfaces, semisolids, and biofilms. We develop models to address the plaque dynamics for a temperate phage and its virulent mutants. The models are compared with phage λ-Escherichia coli system to quantify their applicability. We found that temperate phages gave an increasing number of gradually smaller colonies as the distance increased from the plaque center. For low-lysogen frequency this resulted in plaques with most of the visible colonies at an intermediate distance between the center and periphery. Using spot inoculation, where phages in excess of bacteria were inoculated in a circular area, we measured the frequency and spatial distribution of lysogens. The spot morphology of cII-negative (cII−) and cIII− mutants of phage λ displays concentric rings of high-density lysogenic colonies. The simplest of these ring morphologies was reproduced by including multiplicity of infection (MOI) sensitivity in lysis-lysogeny decisions, but its failure to explain the occasional observation of multiple rings in cIII− mutant phages highlights unknown features of this phage. Our findings demonstrated advantages of temperate phages over virulent phages in exploiting limited resources in spatially distributed microbial populations. IMPORTANCE Phages are the most abundant organisms on earth, and yet little is known about how phages and bacterial hosts are influencing each other in density and evolution. Phages can be either

  10. Thinking about microcolonies as phage targets

    PubMed Central

    Abedon, Stephen T.

    2012-01-01

    Phage targets for adsorption can include: (1) individual bacteria; (2) bacterial cellular arrangements such as streptococci; (3) microcolonies consisting of bacterial clones as can make up bacterial lawns and biofilms; and (4) bacterial biofilms themselves. While much effort has gone into considering category 1, and some into category 4, substantially less has been put into the question of how bacterial association into clonal arrangements or microcolonies might affect phage-bacterial interactions. Recently I have been exploring just this issue—within a single-authored monograph published in 2011 and a theoretical article published in 2012 as part of a special issue of the journal, Viruses. For this commentary, I have been invited to summarize my thinking on how bacterial association into either cellular arrangements or microcolonies might affect their susceptibility to phages along with related issues of bacterial resistance to phages and phage propagation in the context of both plaques and biofilms. PMID:23275871

  11. Preparation and assay of phage lambda.

    PubMed

    Dale, J W; Greenaway, P J

    1985-01-01

    Lambda, a temperate bacteriophage of E. coli, has two alternative modes of replication in sensitive cells, known as the lytic and lysogenic cycles. In the lytic cycle, after the lambda DNA enters the cells, various phage functions are expressed that result in the production of a large number of mature phage particles and cell lysis. In the lysogenic mode, which normally occurs in only a small proportion of the infected cells, the phage forms a more or less stable relationship with the host bacterium; this stable state is known as lysogeny. In a lysogenic cell, phage DNA is normally incorporated into the chromosomal DNA via specific attachment sites on both the phage DNA and the host chromosome. Replication of lambda DNA then occurs only during replication of the host chromosome, and the phage genome is inherited by each daughter cell at cell division. The phage is maintained in this prophage state through the action of a repressor protein, coded for by the phage gene cl. This repressor protein turns off the expression of virtually the whole of the lambda genome. If the repressor is inactivated, the expression of phage genes is initiated. This leads to the excision of lambda DNA from the host chromosome and entry into the lytic cycle. The balance between the lytic and lysogenic modes of replication is a delicate and complex one in which a key factor is the concentration of the cl gene product. Some of the many sources of further information about the basic biology of lambda phage are listed in the references to this chapter.

  12. Phage display: concept, innovations, applications and future.

    PubMed

    Pande, Jyoti; Szewczyk, Magdalena M; Grover, Ashok K

    2010-01-01

    Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.

  13. Holding a grudge: persisting anti-phage CRISPR immunity in multiple human gut microbiomes.

    PubMed

    Mick, Eran; Stern, Adi; Sorek, Rotem

    2013-05-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system of bacteria and archaea constitutes a mechanism of acquired adaptive immunity against phages, which is based on genome-encoded markers of previously infecting phage sequences ("spacers"). As a repository of phage sequences, these spacers make the system particularly suitable for elucidating phage-bacteria interactions in metagenomic studies. Recent metagenomic analyses of CRISPRs associated with the human microbiome intriguingly revealed conserved "memory spacers" shared by bacteria in multiple unrelated, geographically separated individuals. Here, we discuss possible avenues for explaining this phenomenon by integrating insights from CRISPR biology and phage-bacteria ecology, with a special focus on the human gut. We further explore the growing body of evidence for the role of CRISPR/Cas in regulating the interplay between bacteria and lysogenic phages, which may be intimately related to the presence of memory spacers and sheds new light on the multifaceted biological and ecological modes of action of CRISPR/Cas.

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

    PubMed

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

    2015-01-01

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

  15. BREX is a novel phage resistance system widespread in microbial genomes.

    PubMed

    Goldfarb, Tamara; Sberro, Hila; Weinstock, Eyal; Cohen, Ofir; Doron, Shany; Charpak-Amikam, Yoav; Afik, Shaked; Ofir, Gal; Sorek, Rotem

    2015-01-13

    The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR-Cas and restriction-modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six-gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon-like protease, an alkaline phosphatase domain protein, a putative RNA-binding protein, a DNA methylase, an ATPase-domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non-palindromic TAGGAG motifs in the bacterial genome guides self/non-self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction-modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX-like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ~10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti-BREX mechanisms may have evolved in some phages as part of their arms race with bacteria.

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

    PubMed Central

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

    2016-01-01

    In this study, we investigated the humoral immune response (through the release of IgG, IgA, and IgM antiphage antibodies) to a staphylococcal phage cocktail in patients undergoing experimental phage therapy at the Phage Therapy Unit, Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław, Poland. We also evaluated whether occurring antiphage antibodies had neutralizing properties toward applied phages (K rate). Among 20 examined patients receiving the MS-1 phage cocktail orally and/or locally, the majority did not show a noticeably higher level of antiphage antibodies in their sera during phage administration. Even in those individual cases with an increased immune response, mostly by induction of IgG and IgM, the presence of antiphage antibodies did not translate into unsatisfactory clinical results of phage therapy. On the other hand, a negative outcome of the treatment occurred in some patients who showed relatively weak production of antiphage antibodies before and during treatment. This may imply that possible induction of antiphage antibodies is not an obstacle to the implementation of phage therapy and support our assumption that the outcome of the phage treatment does not primarily depend on the appearance of antiphage antibodies in sera of patients during therapy. These conclusions are in line with our previous findings. The confirmation of this thesis is of great interest as regards the efficacy of phage therapy in humans. PMID:27822205

  17. Development of transient phage resistance in Campylobacter coli against the group II phage CP84.

    PubMed

    Orquera, Stefanie; Hertwig, Stefan; Alter, Thomas; Hammerl, Jens A; Jirova, Alice; Gölz, Greta

    2015-01-01

    Recently, there is a growing interest in the use of bacteriophages for pre- and post-harvest applications to reduce foodborne pathogens (including Campylobacter) along the food chain. Quantitative Campylobacter reductions of up to three log10 units have been achieved by phage application. However, possible phage resistance might limit this approach. In Campylobacter (C.) jejuni, phage resistance mechanisms have been described in detail but data on these mechanisms in C. coli are still missing. To study phage resistance in C. coli, strain NCTC 12668 was infected with the lytic phage CP84, belonging to group II of Campylobacter phages. Resistant and sensitive clones were analysed using phenotypic and genotypic assays. C. coli clones acquired only transient resistance against CP84. The resistance led to cross-protection to one out of five other group II phages tested. Phage resistance was apparently neither caused by large genomic rearrangements nor by a CRISPR system. Binding assays demonstrated that CP84 could not adsorb to resistant C. coli clones suggesting a bacterial phage receptor to be involved in resistance. However, phage resistant C. coli clones did not reveal an altered motility or modified flaA sequence. Considering the loss of binding capacity and the reversion to a phage sensitive phenotype we hypothesize that acquired resistance depends on temporal phase variable switch-off modifications of the phage receptor genes, even though the resistance mechanism could not be elucidated in detail. We further speculate that even closely related phages of the same group use different bacterial receptors for binding on C. coli.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. 'Bioluminescent' reporter phage for the detection of Category A bacterial pathogens.

    PubMed

    Schofield, David A; Molineux, Ian J; Westwater, Caroline

    2011-07-08

    Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively. Although the natural occurrence of both diseases' is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease's inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management. Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant "light-tagged" reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens. For demonstration purposes, here we describe the method for the phage

  20. Genome Sequencing Reveals a Phage in Helicobacter pylori

    PubMed Central

    Lehours, Philippe; Vale, Filipa F.; Bjursell, Magnus K.; Melefors, Ojar; Advani, Reza; Glavas, Steve; Guegueniat, Julia; Gontier, Etienne; Lacomme, Sabrina; Alves Matos, António; Menard, Armelle; Mégraud, Francis; Engstrand, Lars; Andersson, Anders F.

    2011-01-01

    ABSTRACT Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population; in a subset of this population, its presence is associated with development of severe disease, such as gastric cancer. Genomic analysis of several strains has revealed an extensive H. pylori pan-genome, likely to grow as more genomes are sampled. Here we describe the draft genome sequence (63 contigs; 26× mean coverage) of H. pylori strain B45, isolated from a patient with gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The major finding was a 24.6-kb prophage integrated in the bacterial genome. The prophage shares most of its genes (22/27) with prophage region II of Helicobacter acinonychis strain Sheeba. After UV treatment of liquid cultures, circular DNA carrying the prophage integrase gene could be detected, and intracellular tailed phage-like particles were observed in H. pylori cells by transmission electron microscopy, indicating that phage production can be induced from the prophage. PCR amplification and sequencing of the integrase gene from 341 H. pylori strains from different geographic regions revealed a high prevalence of the prophage (21.4%). Phylogenetic reconstruction showed four distinct clusters in the integrase gene, three of which tended to be specific for geographic regions. Our study implies that phages may play important roles in the ecology and evolution of H. pylori. PMID:22086490

  1. Pros and cons of phage therapy

    PubMed Central

    Loc-Carrillo, Catherine

    2011-01-01

    Many publications list advantages and disadvantages associated with phage therapy, which is the use of bacterial viruses to combat populations of nuisance or pathogenic bacteria. The goal of this commentary is to discuss many of those issues in a single location. In terms of “Pros,” for example, phages can be bactericidal, can increase in number over the course of treatment, tend to only minimally disrupt normal flora, are equally effective against antibiotic-sensitive and antibiotic-resistant bacteria, often are easily discovered, seem to be capable of disrupting bacterial biofilms, and can have low inherent toxicities. In addition to these assets, we consider aspects of phage therapy that can contribute to its safety, economics, or convenience, but in ways that are perhaps less essential to the phage potential to combat bacteria. For example, autonomous phage transfer between animals during veterinary application could provide convenience or economic advantages by decreasing the need for repeated phage application, but is not necessarily crucial to therapeutic success. We also consider possible disadvantages to phage use as antibacterial agents. These “Cons,” however, tend to be relatively minor. PMID:22334867

  2. Inactivation and reactivation of B. megatherium phage.

    PubMed

    NORTHROP, J H

    1955-11-20

    Preparation of Reversibly Inactivated (R.I.) Phage.- If B. megatherium phage (of any type, or in any stage of purification) is suspended in dilute salt solutions at pH 5-6, it is completely inactivated; i.e., it does not form plaques, or give rise to more phage when mixed with a sensitive organism (Northrop, 1954). The inactivation occurs when the phage is added to the dilute salt solution. If a suspension of the inactive phage in pH 7 peptone is titrated to pH 5 and allowed to stand, the activity gradually returns. The inactivation is therefore reversible. Properties of R.I. Phage.- The R.I. phage is adsorbed by sensitive cells at about the same rate as the active phage. It kills the cells, but no active phage is produced. The R.I. phage therefore has the properties of phage "ghosts" (Herriott, 1951) or of colicines (Gratia, 1925), or phage inactivated by ultraviolet light (Luria, 1947). The R.I. phage is sedimented in the centrifuge at the same rate as active phage. It is therefore about the same size as the active phage. The R.I. phage is most stable in pH 7, 5 per cent peptone, and may be kept in this solution for weeks at 0 degrees C. The rate of digestion of R.I. phage by trypsin, chymotrypsin, or desoxyribonuclease is about the same as that of active phage (Northrop, 1955 a). Effect of Various Substances on the Formation of R.I. Phage.- There is an equilibrium between R.I. phage and active phage. The R.I. form is the stable one in dilute salt solution, pH 5 to 6.5 and at low temperature (<20 degrees C.). At pH >6.5, in dilute salt solution, the R.I. phage changes to the active form. The cycle, active right harpoon over left harpoon inactive phage, may be repeated many times at 0 degrees C. by changing the pH of the solution back and forth between pH 7 and pH 6. Irreversible inactivation is caused by distilled water, some heavy metals, concentrated urea or quanidine solutions, and by l-arginine. Reversible inactivation is prevented by all salts tested (except

  3. Rapid enumeration of phage in monodisperse emulsions.

    PubMed

    Tjhung, Katrina F; Burnham, Sean; Anany, Hany; Griffiths, Mansel W; Derda, Ratmir

    2014-06-17

    Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed.

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

    PubMed

    Chaturongakul, Soraya; Ounjai, Puey

    2014-01-01

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

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

    PubMed Central

    Dalmasso, Marion; Strain, Ronan; Neve, Horst; Franz, Charles M. A. P.; Cousin, Fabien J.; Ross, R. Paul; Hill, Colin

    2016-01-01

    With the emergence of multi-drug resistant bacteria the use of bacteriophages (phages) is gaining renewed interest as promising anti-microbial agents. The aim of this study was to isolate and characterize phages from human fecal samples. Three new coliphages, ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03, were isolated. Their phenotypic and genomic characteristics, and lytic activity against biofilm, and in combination with ciprofloxacin, were investigated. All three phages reduced the growth of E. coli strain DPC6051 at multiplicity of infection (MOI) between 10−3 and 105. A cocktail of all three phages completely inhibited the growth of E. coli. The phage cocktail also reduced biofilm formation and prevented the emergence of phage-resistant mutants which occurred with single phage. When combined with ciprofloxacin, phage alone or in cocktail inhibited the growth of E. coli and prevented the emergence of resistant mutants. These three new phages are promising biocontrol agents for E. coli infections. PMID:27280590

  6. Phage as a modulator of immune responses: practical implications for phage therapy.

    PubMed

    Górski, Andrzej; Międzybrodzki, Ryszard; Borysowski, Jan; Dąbrowska, Krystyna; Wierzbicki, Piotr; Ohams, Monika; Korczak-Kowalska, Grażyna; Olszowska-Zaremba, Natasza; Łusiak-Szelachowska, Marzena; Kłak, Marlena; Jończyk, Ewa; Kaniuga, Ewelina; Gołaś, Aneta; Purchla, Sylwia; Weber-Dąbrowska, Beata; Letkiewicz, Sławomir; Fortuna, Wojciech; Szufnarowski, Krzysztof; Pawełczyk, Zdzisław; Rogóż, Paweł; Kłosowska, Danuta

    2012-01-01

    Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations.

  7. Phage-Antibiotic Synergy (PAS): beta-lactam and quinolone antibiotics stimulate virulent phage growth.

    PubMed

    Comeau, André M; Tétart, Françoise; Trojet, Sabrina N; Prère, Marie-Françoise; Krisch, H M

    2007-08-29

    Although the multiplication of bacteriophages (phages) has a substantial impact on the biosphere, comparatively little is known about how the external environment affects phage production. Here we report that sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacterial cell's production of some virulent phage. For example, a low dosage of cefotaxime, a cephalosporin, increased an uropathogenic Escherichia coli strain's production of the phage PhiMFP by more than 7-fold. We name this phenomenon Phage-Antibiotic Synergy (PAS). A related effect was observed in diverse host-phage systems, including the T4-like phages, with beta-lactam and quinolone antibiotics, as well as mitomycin C. A common characteristic of these antibiotics is that they inhibit bacterial cell division and trigger the SOS system. We therefore examined the PAS effect within the context of the bacterial SOS and filamentation responses. We found that the PAS effect appears SOS-independent and is primarily a consequence of cellular filamentation; it is mimicked by cells that constitutively filament. The fact that completely unrelated phages manifest this phenomenon suggests that it confers an important and general advantage to the phages.

  8. An improved helper phage system for efficient isolation of specific antibody molecules in phage display.

    PubMed

    Baek, Hyunjung; Suk, Kyoung-ho; Kim, Yong-hwan; Cha, Sanghoon

    2002-03-01

    Phage display technology has been applied in many fields of biological and medical sciences to study molecular interactions and especially in the generation of monoclonal antibodies of human origin. However, extremely low display level of antibody molecules on the surface of phage is an intrinsic problem of a phagemid-based display system resulting in low success rate of isolating specific binding molecules. We show here that display of single-chain antibody fragment (scFv) generated with pIGT3 phagemid can be increased dramatically by using a genetically modified Ex-phage. Ex-phage has a mutant pIII gene that produces a functional wild-type pIII in suppressing Escherichia coli strains but does not make any pIII in non-suppressing E.coli strains. Packaging phagemids encoding antibody-pIII fusion in F+ non-suppressing E.coli strains with Ex-phage enhanced the display level of antibody fragments on the surfaces of recombinant phage particles resulting in an increase of antigen-binding reactivity >100-fold compared to packaging with M13KO7 helper phage. Thus, the Ex-phage and pIGT3 phagemid vector provides a system for the efficient enrichment of specific binding antibodies from a phage display library and, thereby, increases the chance of obtaining more diverse antibodies specific for target antigens.

  9. The phage-shock-protein response.

    PubMed

    Darwin, Andrew J

    2005-08-01

    The phage-shock-protein (Psp) system responds to extracytoplasmic stress that may reduce the energy status of the cell. It is conserved in many different bacteria and has been linked to several important phenotypes. Escherichia coli psp mutants have defects in maintenance of the proton-motive force, protein export by the sec and tat pathways, survival in stationary phase at alkaline pH, and biofilm formation. Yersinia enterocolitica psp mutants cannot grow when the secretin component of a type III secretion system is mislocalized, and have a severe virulence defect in animals. A Salmonella enterica psp mutation exacerbates some phenotypes of an rpoE null mutant and the psp genes of S. enterica and Shigella flexneri are highly induced during macrophage infection. PspA, the most abundant of the Psp proteins, is required for most of the phenotypes associated with the Psp system. Therefore, PspA is probably an effector that may play a role in maintaining cytoplasmic membrane integrity and/or the proton-motive force. However, PspA is not required for the ability to tolerate secretin mislocalization, which suggests an important physiological role for other Psp proteins. This article summarizes our current understanding of the Psp system: inducing signals, the underlying signal transduction mechanisms, the physiological roles it may play, and a genomic analysis of its conservation.

  10. Genome Sequence of Mycobacterium Phage Waterfoul

    PubMed Central

    Jackson, Paige N.; Embry, Ella K.; Johnson, Christa O.; Watson, Tiara L.; Weast, Sayre K.; DeGraw, Caroline J.; Douglas, Jessica R.; Sellers, J. Michael; D’Angelo, William A.

    2016-01-01

    Waterfoul is a newly isolated temperate siphovirus of Mycobacterium smegmatis mc2155. It was identified as a member of the K5 cluster of Mycobacterium phages and has a 61,248-bp genome with 95 predicted genes. PMID:27856585

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

    PubMed Central

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

    2016-01-01

    Authentic research experiences are valuable components of effective undergraduate education. Research experiences during the first years of college are especially critical to increase persistence in science, technology, engineering, and mathematics fields. The Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) model provides a high-impact research experience to first-year students but is usually available to a limited number of students, and its implementation is costly in faculty time and laboratory space. To offer a research experience to all students taking introductory biology at Gonzaga University (n = 350/yr), we modified the traditional two-semester SEA-PHAGES course by streamlining the first-semester Phage Discovery lab and integrating the second SEA-PHAGES semester into other courses in the biology curriculum. Because most students in the introductory course are not biology majors, the Phage Discovery semester may be their only encounter with research. To discover whether students benefit from the first semester alone, we assessed the effects of the one-semester Phage Discovery course on students’ understanding of course content. Specifically, students showed improvement in knowledge of bacteriophages, lab math skills, and understanding experimental design and interpretation. They also reported learning gains and benefits comparable with other course-based research experiences. Responses to open-ended questions suggest that students experienced this course as a true undergraduate research experience. PMID:27146160

  12. Genome analysis of the staphylococcal temperate phage DW2 and functional studies on the endolysin and tail hydrolase

    PubMed Central

    Keary, Ruth; McAuliffe, Olivia; Ross, R Paul; Hill, Colin; O’Mahony, Jim; Coffey, Aidan

    2014-01-01

    This study describes the genome of temperate Siphoviridae phage DW2, which is routinely propagated on Staphylococcus aureus DPC5246. The 41941 bp genome revealed an open reading frame (ORF1) which has a high level of homology with members of the resolvase subfamily of site-specific serine recombinase, involved in chromosomal integration and excision. In contrast, the majority of staphylococcal phages reported to date encode tyrosine recombinases. Two putative genes encoded by phage DW2 (ORF15 and ORF24) were highly homologous to the NWMN0273 and NWMN0280 genes encoding virulence factors carried on the genome of ϕNM4, a prophage in the genome of S. aureus Newman. Phage DW2 also encodes proteins highly homologous to two well-characterized Staphylococcus aureus pathogenicity island derepressors encoded by the staphylococcal helper phage 80α indicating that it may similarly act as a helper phage for mobility of pathogenicity islands in S. aureus. This study also focused on the enzybiotic potential of phage DW2. The structure of the putative endolysin and tail hydrolase were investigated and used as the basis for a cloning strategy to create recombinant peptidoglycan hydrolyzing proteins. After overexpression in E. coli, four of these proteins (LysDW2, THDW2, CHAPE1-153, and CHAPE1-163) were demonstrated to have hydrolytic activity against peptidoglycan of S. aureus and thus represent novel candidates for exploitation as enzybiotics. PMID:25105056

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

    PubMed

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

    2016-01-01

    Authentic research experiences are valuable components of effective undergraduate education. Research experiences during the first years of college are especially critical to increase persistence in science, technology, engineering, and mathematics fields. The Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) model provides a high-impact research experience to first-year students but is usually available to a limited number of students, and its implementation is costly in faculty time and laboratory space. To offer a research experience to all students taking introductory biology at Gonzaga University (n = 350/yr), we modified the traditional two-semester SEA-PHAGES course by streamlining the first-semester Phage Discovery lab and integrating the second SEA-PHAGES semester into other courses in the biology curriculum. Because most students in the introductory course are not biology majors, the Phage Discovery semester may be their only encounter with research. To discover whether students benefit from the first semester alone, we assessed the effects of the one-semester Phage Discovery course on students' understanding of course content. Specifically, students showed improvement in knowledge of bacteriophages, lab math skills, and understanding experimental design and interpretation. They also reported learning gains and benefits comparable with other course-based research experiences. Responses to open-ended questions suggest that students experienced this course as a true undergraduate research experience.

  14. Supersize me: Cronobacter sakazakii phage GAP32

    SciTech Connect

    Abbasifar, Reza; Griffiths, Mansel W.; Sabour, Parviz M.; Ackermann, Hans-Wolfgang; Vandersteegen, Katrien; Lavigne, Rob; Noben, Jean-Paul; Alanis Villa, Argentina; Abbasifar, Arash; Nash, John H.E.; Kropinski, Andrew M.

    2014-07-15

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

  15. Recombinant Phage Probes for Salmonella Typhimurium Detection

    DTIC Science & Technology

    2007-11-02

    food safety analysis that are slower, labor-intensive, and cost-inefficient. Confirmation of presence in food products can take as long as 48 hours by conventional culture. Current rapid detection initiatives include biosensors that routinely incorporate antibodies as the biorecognition unit. Although sensitive and specific, antibodies are costly and may degrade under unfavorable environmental conditions. We believe that a stable, inexpensive substitute for antibodies is filamentous phage manipulated through phage display technique then affinity selected for specificity to

  16. Quantitative Analysis of the Stability of Lysogenic State in Phage lambda

    NASA Astrophysics Data System (ADS)

    Ao, Ping

    2004-03-01

    mutations and parameter fluctuations. This talk is based on work done with L. Hood, C. Kwon, D.J. Thouless, L. Yin and X.-M. Zhu. References: 1. Zhu X-M, Yin L, Hood L, and Ao P; Calculating Biological Behaviors of Epigenetic States in Phage lambda Life Cycle, to appear in Functional and Integrative Genomics. 2. Ao P; Stochastic force defined evolution in dynamical systems. Submitted to Phys. Rev. Lett. (http://it/arXiv.org/find/physics/1/Ao/0/1/0/past/3/0)

  17. Diversity and censoring of landscape phage libraries

    PubMed Central

    Kuzmicheva, G.A.; Jayanna, P.K.; Sorokulova, I.B.; Petrenko, V.A.

    2009-01-01

    Libraries of random peptides displayed on the surface of filamentous phages are a valuable source for biospecific ligands. However, their successful use can be hindered by a disproportionate representation of different phage clones and fluctuation of their composition that arises during phage reproduction, which have potential to affect efficiency of selection of clones with an optimal binding. Therefore, there is a need to develop phage display libraries with extended and varied repertoires of displayed peptides. In this work, we compared the complexity, evolution and representation of two phage display libraries displaying foreign octamers and nonamers in 4000 copies as the N-terminal part of the major coat protein pVIII of phage fd–tet (landscape libraries). They were obtained by replacement of amino acids 2–4 and 2–5 of pVIII with random octa- and nonamers, respectively. Statistical analysis of the libraries revealed their dramatic censoring and evolution during amplification. Further, a survey of both libraries for clones that bind common selectors revealed the presence of different non-overlapping families of target-specific clones in each library justifying the concept that different landscape libraries cover different areas of a sequence space. PMID:18988692

  18. Characterization of Marine Temperate Phage-Host Systems Isolated from Mamala Bay, Oahu, Hawaii

    PubMed Central

    Jiang, Sunny C.; Kellogg, Christina A.; Paul, John H.

    1998-01-01

    To understand the ecological and genetic role of viruses in the marine environment, it is critical to know the infectivity of viruses and the types of interactions that occur between marine viruses and their hosts. We isolated four marine phages from turbid plaques by using four indigenous bacterial hosts obtained from concentrated water samples from Mamala Bay, Oahu, Hawaii. Two of the rod-shaped bacterial hosts were identified as Sphingomonas paucimobilis and Flavobacterium sp. All of the phage isolates were tailed phages and contained double-stranded DNA. Two of the phage isolates had morphologies typical of the family Siphoviridae, while the other two belonged to the families Myoviridae and Podoviridae. The head diameters of these viruses ranged from 47 to 70.7 nm, and the tail lengths ranged from 12 to 146 nm. The burst sizes ranged from 7.8 to 240 phage/bacterial cell, and the genome sizes, as determined by restriction digestion, ranged from 36 to 112 kb. The members of the Siphoviridae, T-φHSIC, and T-φD0, and the member of the Myoviridae, T-φD1B, were found to form lysogenic associations with their bacterial hosts, which were isolated from the same water samples. Hybridization of phage T-φHSIC probe with lysogenic host genomic DNA was observed in dot blot hybridization experiments, indicating that prophage T-φHSIC was integrated within the host genome. These phage-host systems are available for use in studies of marine lysogeny and transduction. PMID:9464390

  19. Phage Display Derived IgNAR V Region Binding Domains for Therapeutic Development.

    PubMed

    Ubah, Obinna C; Barelle, Caroline J; Buschhaus, Magdalena J; Porter, Andrew J

    2016-01-01

    Phage display technology has revolutionized the science of drug discovery by transforming the generation and manipulation of ligands, such as antibody fragments, enzymes, and peptides. The basis of this technology is the expression of recombinant proteins or peptides fused to a phage coat protein, and subsequent isolation of ligands based on a variety of catalytic, physicochemical/binding kinetic and/or biological characteristics. An incredible number of diagnostic and therapeutic domains have been successfully isolated using phage display technology. The variable domain of the New Antigen Receptors (VNAR) found in cartilaginous fish, is also amenable to phage display selection. Whilst not an antibody, VNARs are unquestionable the oldest (450 million years), and smallest antigen binding, single-domains so far identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established, enhancing our understanding of the evolutionary origins of humoral immunity and the unusual but divergent ancestry of the VNARs themselves. VNARs exhibit remarkable physicochemical properties, such as small size, stability in extreme conditions, solubility, molecular flexibility, high affinity and selectivity for target. The purpose of this review is to illustrate the important role phage display has played in the isolation and characterization of potent therapeutic and diagnostic VNAR domains.

  20. Self-assembling Phage-Quantum Dot Nanocomplexes for Quantitative Biodetection

    NASA Astrophysics Data System (ADS)

    Clarke, Matthew; Kang, Hyeonggon; Hwang, Jeeseong

    2010-03-01

    Colloidal quantum dots (QDs) have been used for many biodetection applications because of their brightness and broad spectral coverage in multiplexed approaches. QD surfaces can be functionalized for bio-conjugation to enable self-assembly with other nanomaterials and biomolecules using biological or bio-inspired processes. We demonstrate a model bacterial detection system using phage-QD nanocomplexes. To engineer the nanocomplexes, we genetically modified phage to express lysine residues on the capsid region, resulting in biotin labeling during replication inside the host cell. The biotinylated phages were conjugated with QDs and employed for detection. Bacteriophages have specificity to bacteria, enabling targeted detection of specific strains. Brightness of QDs enables high-throughput optical detection. The properties of nanocomplexes and detection limit/sensitivity were quantitatively evaluated using integrated differential interference contrast and fluorescence microscopy and automated image-based cytometry technique.

  1. Burkholderia cepacia complex Phage-Antibiotic Synergy (PAS): antibiotics stimulate lytic phage activity.

    PubMed

    Kamal, Fatima; Dennis, Jonathan J

    2015-02-01

    The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing.

  2. Burkholderia cepacia Complex Phage-Antibiotic Synergy (PAS): Antibiotics Stimulate Lytic Phage Activity

    PubMed Central

    Kamal, Fatima

    2014-01-01

    The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing. PMID:25452284

  3. Insights into the diversity of φRSM phages infecting strains of the phytopathogen Ralstonia solanacearum complex: regulation and evolution.

    PubMed

    Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2014-08-01

    The filamentous φRSM phages (φRSM1 and φRSM3) have integration/excision capabilities in the phytopathogenic bacterium Ralstonia solanacearum. In the present study, we further investigated φRSM-like sequences present in the genomes of R. solanacearum strains belonging to the four major evolutionary lineages (phylotypes I-IV). Based on bioinformatics and comparative genomic analyses, we found that φRSM homologs are highly diverse in R. solanacearum complex strains. We detected an open reading frame (ORF)15 located upstream of the gene for φRSM integrase, which exhibited amino acid sequence similarity to phage repressor proteins. ORF15-encoded protein (a putative repressor) was found to encode a 104-residue polypeptide containing a DNA-binding (helix-turn-helix) domain and was expressed in R. solanacearum lysogenic strains. This suggested that φRSM3-ORF15 might be involved in the establishment and maintenance of a lysogenic state, as well as in phage immunity. Comparison of the putative repressor proteins and their binding sites within φRSM-related prophages provides insights into how these regulatory systems of filamentous phages have evolved and diverged in the R. solanacearum complex. In conclusion, φRSM phages represent a unique group of filamentous phages that are equipped with innate integration/excision (ORF14) and regulatory systems (ORF15).

  4. Characterization of Two Virulent Phages of Lactobacillus plantarum

    PubMed Central

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

    2012-01-01

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

  5. Characterization of two virulent phages of Lactobacillus plantarum.

    PubMed

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

    2012-12-01

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

  6. The morphology and nucleotide composition of DNA of Citrobacter phages.

    PubMed

    Gabrilovich, I M; Kirillova, F M; Khakesheva, T A

    1987-01-01

    Citrobacter phages 38/37, 31/37, 40/1 and 8/5, isolated from lysogenic cultures, were concentrated and purified by 2 cycles of differential centrifugation. Electron microscopy of the phages has shown that their particles have similar morphology and that they relate to the morphological group A1. The heads of the phages are hexagonal, 50 +/- 2 nm in diameter. The tail of the phage is straight, 112-152 nm in length, with a contracting sheath 11.5-12.5 nm wide. The tails of the phages 38/37 and 40/1 were found to be slightly longer in comparison with the phages 31/37 and 8/5. Chromatographic investigation of DNA preparations of the phages revealed the presence of 4 nitrous bases. Identification of the latter permitted us to relate them to common nitrous bases. DNA of the phages is double-stranded and belongs to a weakly expressed guanine-cytosine type. The content of guanine and cytosine in DNA of the phage 38/37 amounts to 56.68%, that of the phage 31/37 to 56.75, of the phage 40/1 to 57.36% and of the phage 8/5 to 55.58%. No substantial variations were observed in the DNA composition of the phages.

  7. Network models of phage-bacteria coevolution

    NASA Astrophysics Data System (ADS)

    Rosvall, Martin; Dodd, Ian B.; Krishna, Sandeep; Sneppen, Kim

    2006-12-01

    Bacteria and their bacteriophages are the most abundant, widespread, and diverse groups of biological entities on the planet. In an attempt to understand how the interactions between bacteria, virulent phages, and temperate phages might affect the diversity of these groups, we developed a stochastic network model for examining the coevolution of these ecologies. In our approach, nodes represent whole species or strains of bacteria or phages, rather than individuals, with “speciation” and extinction modeled by duplication and removal of nodes. Phage-bacteria links represent host-parasite relationships and temperate-virulent phage links denote prophage-encoded resistance. The effect of horizontal transfer of genetic information between strains was also included in the dynamical rules. The observed networks evolved in a highly dynamic fashion but the ecosystems were prone to collapse (one or more entire groups going extinct). Diversity could be stably maintained in the model only if the probability of speciation was independent of the diversity. Such an effect could be achieved in real ecosystems if the speciation rate is primarily set by the availability of ecological niches.

  8. Targeting Enterococcus faecalis Biofilms with Phage Therapy

    PubMed Central

    Khalifa, Leron; Brosh, Yair; Gelman, Daniel; Coppenhagen-Glazer, Shunit; Beyth, Shaul; Poradosu-Cohen, Ronit; Que, Yok-Ai; Beyth, Nurit

    2015-01-01

    Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment. PMID:25662974

  9. Targeting Enterococcus faecalis biofilms with phage therapy.

    PubMed

    Khalifa, Leron; Brosh, Yair; Gelman, Daniel; Coppenhagen-Glazer, Shunit; Beyth, Shaul; Poradosu-Cohen, Ronit; Que, Yok-Ai; Beyth, Nurit; Hazan, Ronen

    2015-04-01

    Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.

  10. Phage diversity in a methanogenic digester.

    PubMed

    Park, M-O; Ikenaga, H; Watanabe, K

    2007-01-01

    It has been shown that phages are present in natural and engineered ecosystems and influence the structure and performance of prokaryotic communities. However, little has been known about phages occurring in anaerobic ecosystems, including those in methanogenic digesters for waste treatment. This study investigated phages produced in an upflow anaerobic sludge blanket methanogenic digester treating brewery wastes. Phage-like particles (PLPs) in the influent and effluent of the digester were concentrated and purified by sequential filtration and quantified and characterized by transmission electron microscopy (TEM), fluorescence assay, and field inversion gel electrophoresis (FIGE). Results indicate that numbers of PLPs in the effluent of the digester exceeded 1 x 10(9) L-1 and at least 10 times greater than those in the influent, suggesting that substantial amounts of PLPs were produced in the digester. A production rate of the PLPs was estimated at least 5.2 x 10(7) PLPs day-1 L-1. TEM and FIGE showed that a variety of phages were produced in the digester, including those affiliated with Siphoviridae, Myoviridae, and Cystoviridae.

  11. Aerosol phage therapy efficacy in Burkholderia cepacia complex respiratory infections.

    PubMed

    Semler, Diana D; Goudie, Amanda D; Finlay, Warren H; Dennis, Jonathan J

    2014-07-01

    Phage therapy has been suggested as a potential treatment for highly antibiotic-resistant bacteria, such as the species of the Burkholderia cepacia complex (BCC). To address this hypothesis, experimental B. cenocepacia respiratory infections were established in mice using a nebulizer and a nose-only inhalation device. Following infection, the mice were treated with one of five B. cenocepacia-specific phages delivered as either an aerosol or intraperitoneal injection. The bacterial and phage titers within the lungs were assayed 2 days after treatment, and mice that received the aerosolized phage therapy demonstrated significant decreases in bacterial loads. Differences in phage activity were observed in vivo. Mice that received phage treatment by intraperitoneal injection did not demonstrate significantly reduced bacterial loads, although phage particles were isolated from their lung tissue. Based on these data, aerosol phage therapy appears to be an effective method for treating highly antibiotic-resistant bacterial respiratory infections, including those caused by BCC bacteria.

  12. Genome Sequence of Escherichia coli Tailed Phage Utah

    PubMed Central

    Leavitt, Justin C.; Heitkamp, Alexandra J.; Bhattacharjee, Ananda S.; Gilcrease, Eddie B.

    2017-01-01

    ABSTRACT Escherichia coli bacteriophage Utah is a member of the chi-like tailed phage cluster in the Siphoviridae family. We report here the complete 59,024-bp sequence of the genome of phage Utah. PMID:28360173

  13. Current taxonomy of phages infecting lactic acid bacteria

    PubMed Central

    Mahony, Jennifer; van Sinderen, Douwe

    2013-01-01

    Phages infecting lactic acid bacteria have been the focus of significant research attention over the past three decades. Through the isolation and characterization of hundreds of phage isolates, it has been possible to classify phages of the dairy starter and adjunct bacteria Lactococus lactis, Streptococcus thermophilus, Leuconostoc spp., and Lactobacillus spp. Among these, phages of L. lactis have been most thoroughly scrutinized and serve as an excellent model system to address issues that arise when attempting taxonomic classification of phages infecting other LAB species. Here, we present an overview of the current taxonomy of phages infecting LAB genera of industrial significance, the methods employed in these taxonomic efforts and how these may be employed for the taxonomy of phages of currently underrepresented and emerging phage species. PMID:24478767

  14. Phage display—A powerful technique for immunotherapy

    PubMed Central

    Bazan, Justyna; Całkosiński, Ireneusz; Gamian, Andrzej

    2012-01-01

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

  15. Aerosol Phage Therapy Efficacy in Burkholderia cepacia Complex Respiratory Infections

    PubMed Central

    Semler, Diana D.; Goudie, Amanda D.; Finlay, Warren H.

    2014-01-01

    Phage therapy has been suggested as a potential treatment for highly antibiotic-resistant bacteria, such as the species of the Burkholderia cepacia complex (BCC). To address this hypothesis, experimental B. cenocepacia respiratory infections were established in mice using a nebulizer and a nose-only inhalation device. Following infection, the mice were treated with one of five B. cenocepacia-specific phages delivered as either an aerosol or intraperitoneal injection. The bacterial and phage titers within the lungs were assayed 2 days after treatment, and mice that received the aerosolized phage therapy demonstrated significant decreases in bacterial loads. Differences in phage activity were observed in vivo. Mice that received phage treatment by intraperitoneal injection did not demonstrate significantly reduced bacterial loads, although phage particles were isolated from their lung tissue. Based on these data, aerosol phage therapy appears to be an effective method for treating highly antibiotic-resistant bacterial respiratory infections, including those caused by BCC bacteria. PMID:24798268

  16. Development of a workflow for screening and identification of α-amylase inhibitory peptides from food source using an integrated Bioinformatics-phage display approach: Case study - Cumin seed.

    PubMed

    Siow, Hwee-Leng; Lim, Theam Soon; Gan, Chee-Yuen

    2017-01-01

    The main objective of this study was to develop an efficient workflow to discover α-amylase inhibitory peptides from cumin seed. A total of 56 unknown peptides was initially found in the cumin seed protein hydrolysate. They were subjected to 2 different in silico screenings and 6 peptides were shortlisted. The peptides were then subjected to in vitro selection using phage display technique and 3 clones (CSP3, CSP4 and CSP6) showed high affinity in binding α-amylase. These clones were subjected to the inhibitory test and only CSP4 and CSP6 exhibited high inhibitory activity. Therefore, these peptides were chemically synthesized for validation purposes. CSP4 exhibited inhibition of bacterial and human salivary α-amylases with IC50 values of 0.11 and 0.04μmol, respectively, whereas CSP6 was about 0.10 and 0.15μmol, respectively. Results showed that the strength of each protocol has been successfully combined as deemed fit to enhance the α-amylase inhibitor peptide discovery.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Mercanti, Diego J; Rousseau, Geneviève M; Capra, María L; Quiberoni, Andrea; Tremblay, Denise M; Labrie, Simon J; Moineau, Sylvain

    2015-10-16

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

  19. Phage therapy in the food industry.

    PubMed

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

    2014-01-01

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

  20. Lysogenic conversion and phage resistance development in phage exposed Escherichia coli biofilms.

    PubMed

    Moons, Pieter; Faster, David; Aertsen, Abram

    2013-01-11

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

  1. Genetic and Physical Structure of Salmonella-coli Phage Hybrids and Development of New Generalized Transducing Hybrid Phages for E. Coli.

    DTIC Science & Technology

    1984-05-01

    coli chromosome although the parental phage Mu80, is a specialized transducing phage for the E . coli trp operon. Phage P22 can recombine with coli...mutator phage Mu. Genetic analysis of Mu80immP22 hybrid genomes led us to isolate high specialized transducing phages for argF and proAB genes of the E

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

    PubMed

    Andrade-Domínguez, Andrés; Kolter, Roberto

    2016-08-25

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

  3. Diversity and geographical distribution of Flavobacterium psychrophilum isolates and their phages: patterns of susceptibility to phage infection and phage host range.

    PubMed

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

    2014-05-01

    Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that "enhanced infection" is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.

  4. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies

    PubMed Central

    Mu, Xihui; Tong, Zhaoyang; Huang, Qibin; Liu, Bing; Liu, Zhiwei; Hao, Lanqun; Dong, Hua; Zhang, Jinping; Gao, Chuan

    2016-01-01

    Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL) technique, Staphylococcus protein A (SPA) functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb) as magnetic capturing probes, and Ru(bpy)32+-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD) was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy)32+-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy)32+-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility. PMID:26927130

  5. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies.

    PubMed

    Mu, Xihui; Tong, Zhaoyang; Huang, Qibin; Liu, Bing; Liu, Zhiwei; Hao, Lanqun; Dong, Hua; Zhang, Jinping; Gao, Chuan

    2016-02-27

    Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL) technique, Staphylococcus protein A (SPA) functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb) as magnetic capturing probes, and Ru(bpy)₃(2+)-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD) was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy)₃(2+)-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy)₃(2+)-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility.

  6. Biological characterization of induced phages from Saccharopolyspora hirsuta 367 and comparison with phage JHJ-1.

    PubMed

    Gaudreau, L R; Lavoie, J M; Déry, C V

    1991-10-01

    Phages JHJ-2 and JHJ-3 were isolated from Saccharopolyspora hirsuta 367 UC 8106 following induction with mitomycin C and amplified on S. hirsuta NRRL B-5792. Their properties were compared with those of phage JHJ-1, isolated previously from S. hirsuta 367 NRRL 12045. The DNA restriction patterns appeared to be identical. One-step growth experiments showed no differences between the replication cycles. Burst sizes ranged from 100 to 110 p.f.u. per cell. However, the three phages showed some differences in their behaviour in different hosts. The host range of phage JHJ-1, on non-lysogenic strains, was emended to include all of the Saccharopolyspora strains tested; the host range of phage JHJ-2 was shown to be identical to JHJ-1. Phage JHJ-3 did not form detectable plaques on strains of S. rectivirgula or S. erythraea except S. erythraea NRRL 2359. Neither phage JHJ-2 nor JHJ-3 formed plaques on any lysogenic strains, while JHJ-1 formed plaques on all such strains except S. hirsuta 367 UC8106. Phage JHJ-3 was characterized as a temperate bacteriophage because it formed turbid, self-limiting plaques and lysogenized S. hirsuta NRRL B-5792. It was spontaneously released from UC8106. Both JHJ-1 and JHJ-2 formed clear and invasive (Inv+ phenotype: the property to grow on old mycelium) plaques on some Saccharopolyspora strains but clear and self-limiting plaques on others. Thus, the expression of the Inv+ phenotype encoded by JHJ-1 and JHJ-2 appears to be modulated by the host cell.

  7. Recognition of epoxy with phage displayed peptides.

    PubMed

    Swaminathan, Swathi; Cui, Yue

    2013-07-01

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

  8. 2004 ASM Conference on the New Phage Biology: the 'Phage Summit'.

    PubMed

    Adhya, Sankar; Black, Lindsay; Friedman, David; Hatfull, Graham; Kreuzer, Kenneth; Merril, Carl; Oppenheim, Amos; Rohwer, Forest; Young, Ry

    2005-03-01

    In August, more than 350 conferees from 24 countries attended the ASM Conference on the New Phage Biology, in Key Biscayne, Florida. This meeting, also called the Phage Summit, was the first major international gathering in decades devoted exclusively to phage biology. What emerged from the 5 days of the Summit was a clear perspective on the explosive resurgence of interest in all aspects of bacteriophage biology. The classic phage systems like lambda and T4, reinvigorated by structural biology, bioinformatics and new molecular and cell biology tools, remain model systems of unequalled power and facility for studying fundamental biological issues. In addition, the New Phage Biology is also populated by basic and applied scientists focused on ecology, evolution, nanotechnology, bacterial pathogenesis and phage-based immunologics, therapeutics and diagnostics, resulting in a heightened interest in bacteriophages per se, rather than as a model system. Besides constituting another landmark in the long history of a field begun by d'Herelle and Twort during the early 20th century, the Summit provided a unique venue for establishment of new interactive networks for collaborative efforts between scientists of many different backgrounds, interests and expertise.

  9. Thermophilic lactic acid bacteria phages isolated from Argentinian dairy industries.

    PubMed

    Suárez, V B; Quiberoni, A; Binetti, A G; Reinheimer, J A

    2002-10-01

    Sixty-one natural phages (59 of Streptococcus thermophilus and 2 of Lactobacillus delbrueckii subsp. bulgaricus) were isolated from Argentinian dairy plants from November 1994 to July 2000. Specifically, 17 yogurt samples (18% of all samples) and 26 cheese samples (79%) contained phages lytic to S. thermophilus strains. The number of viral particles found in samples ranged from 10(2) to 10(9) PFU/ml. The phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed high burst size values and remarkably short latent periods. The results of this study show that phages were found more frequently in cheesemaking processes than in yogurt-making processes. The commercial streptococcus strains appeared to propagate more phages, whereas the natural strains propagated fewer phage strains. These results suggest that the naturally occurring cultures are inherently more phage resistant.

  10. Phage display technology: clinical applications and recent innovations.

    PubMed

    Azzazy, Hassan M E; Highsmith, W Edward

    2002-09-01

    Phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. Phage display libraries permit the selection of peptides and proteins, including antibodies, with high affinity and specificity for almost any target. A crucial advantage of this technology is the direct link that exists between the experimental phenotype and its encapsulated genotype, which allows the evolution of the selected binders into optimized molecules. Phage display facilitates engineering of antibodies with regard to their size, valency, affinity, and effector functions. The selection of antibodies and peptides from libraries displayed on the surface of filamentous phage has proven significant for routine isolation of peptides and antibodies for diagnostic and therapeutic applications. This review serves as an introduction to phage display, antibody engineering, the development of phage-displayed peptides and antibody fragments into viable diagnostic reagents, and recent trends in display technology.

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

    SciTech Connect

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

    2013-01-01

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

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

    PubMed

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

    2013-07-30

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

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

    PubMed

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

    2015-12-20

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

  14. A phagemid vector using the E. coli phage shock promoter facilitates phage display of toxic proteins.

    PubMed

    Beekwilder, J; Rakonjac, J; Jongsma, M; Bosch, D

    1999-03-04

    Phage display is a powerful tool with which to adapt the specificity of protease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lower plasmid copy number, as compared to the canonical vector. Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation. A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression. The psp promoter is induced upon helper phage infection. A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host. Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display. The inhibitor library was introduced in both new vectors. Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions. This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E. coli.

  15. Therapeutic effect of Pseudomonas aeruginosa phage YH30 on mink hemorrhagic pneumonia.

    PubMed

    Gu, Jingmin; Li, Xinwei; Yang, Mei; Du, Chongtao; Cui, Ziyin; Gong, Pengjuan; Xia, Feifei; Song, Jun; Zhang, Lei; Li, Juecheng; Yu, Chuang; Sun, Changjiang; Feng, Xin; Lei, Liancheng; Han, Wenyu

    2016-07-15

    Hemorrhagic pneumonia caused by Pseudomonas aeruginosa remains one of the most costly infectious diseases among farmed mink and commonly leads to large economic losses during mink production. The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink. A broad-host-range phage from the Podoviridae family, YH30, was isolated using the mink-originating P. aeruginosa (serotype G) D7 strain as a host. The genome of YH30 was 72,192bp (54.92% G+C), contained 86 open reading frames and lacked regions encoding known virulence factors, integration-related proteins or antibiotic resistance determinants. These characteristics make YH30 eligible for use in phage therapy. The results of a curative treatment experiment demonstrated that a single intranasal administration of YH30 was sufficient to cure hemorrhagic pneumonia in mink. The mean colony count of P. aeruginosa in the blood and lung of YH30-protected mink was less than 10(3) CFU/mL (g) within 24h of bacterial challenge and ultimately became undetectable, whereas that in unprotected mink reached more than 10(8) CFU/mL (g). Additionally, YH30 dramatically improved the pathological manifestations of lung injury in mink with hemorrhagic pneumonia. Our work demonstrates the potential of phages to treat P. aeruginosa-caused hemorrhagic pneumonia in mink.

  16. [Dual promoters enhance heterologous enzyme production from bacterial phage based recombinant Bacillus subtilis].

    PubMed

    Liu, Gang; Zhang, Yan; Xing, Miao

    2006-03-01

    The effect of dual promoters on recombinant protein production from bacterial phage based Bacillus subtilis expression system was investigated. Alpha amylase (from Bacillus amyloliquefaciens) and penicillin acylase (from Bacillus megaterium) were selected as the indicating enzymes. Both the promoterless genes and the promoter-bearing genes were isolated through PCR amplification with properly designed primers, and were inserted into plasmid pSG703 that contains the lacZ-cat expression cartridge. The lysogenic B. subtilis (phi105 MU331) was transformed with the resultant recombinant plasmids, and the heterologous genes were thereby integrated into the chromosommal DNA of B. subtilis via homologous recombination. The transformants were designated as B. subtilis AMY1, B. subtilis AMY2, B. subtilis PA1, and B. subtilis PA2, respectively. In the recombinant B. subtilis strains, the inserted sequences were located down stream of a strong phage promoter that could be activated by thermal induction. In B. subtilis AMY1 and B. subtilis PA1, transcription of the heterologous genes was only initiated by the phage promoter after heat shock, whereas in B. subtilis AMY2 and B. subtilis PA2, transcription of the heterologous genes was initiated by dual promoters, the phage promoter and the native promoter. The application of dual promoters increased the productivity of both enzymes, with 133% enhancement for alpha-amylase production and 113% enhancement for penicillin acylase production.

  17. Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

    PubMed Central

    Brüssow, Harald; Canchaya, Carlos; Hardt, Wolf-Dietrich

    2004-01-01

    Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework. PMID:15353570

  18. Maximizing filamentous phage yield during computer-controlled fermentation.

    PubMed

    Grieco, Sung-Hye H; Lee, Seungil; Dunbar, W Scott; MacGillivray, Ross T A; Curtis, Susan B

    2009-10-01

    Filamentous phage such as M13 and fd consist of a circular, single-stranded DNA molecule surrounded by several different coat proteins. These phages have been used extensively as vectors in phage display where one of the phage coat proteins is genetically engineered to contain a unique peptide surface loop. Through these peptide sequences, a phage collection can be screened for individual phage that binds to different macromolecules or small organic and inorganic molecules. Here, we use computer-controlled bioreactors to produce large quantities of filamentous phage in the bacterial host Escherichia coli. By measuring phage yield and bacterial growth while changing the growth medium, pH and dissolved oxygen concentration, we found that the optimal conditions for phage yield were NZY medium with pH maintained at 7.4, the dO(2) held at 100% and agitation at 800 rpm. These computer-controlled fermentations result in a minimum of a tenfold higher filamentous phage production compared to standard shake flask conditions.

  19. Engineering resistance to phage GVE3 in Geobacillus thermoglucosidasius.

    PubMed

    van Zyl, Leonardo Joaquim; Taylor, Mark Paul; Trindade, Marla

    2016-02-01

    Geobacillus thermoglucosidasius is a promising platform organism for the production of biofuels and other metabolites of interest. G. thermoglucosidasius fermentations could be subject to bacteriophage-related failure and financial loss. We develop two strains resistant to a recently described G. thermoglucosidasius-infecting phage GVE3. The phage-encoded immunity gene, imm, was overexpressed in the host leading to phage resistance. A phage-resistant mutant was isolated following expression of a putative anti-repressor-like protein and phage challenge. A point mutation was identified in the polysaccharide pyruvyl transferase, csaB. A double crossover knockout mutation of csaB confirmed its role in the phage resistance phenotype. These resistance mechanisms appear to prevent phage DNA injection and/or lysogenic conversion rather than just reducing efficiency of plating, as no phage DNA could be detected in resistant bacteria challenged with GVE3 and no plaques observed even at high phage titers. Not only do the strains developed here shed light on the biological relationship between the GVE3 phage and its host, they could be employed by those looking to make use of this organism for metabolite production, with reduced occurrence of GVE3-related failure.

  20. Efficacy of two Staphylococcus aureus phage cocktails in cheese production.

    PubMed

    El Haddad, Lynn; Roy, Jean-Pierre; Khalil, Georges E; St-Gelais, Daniel; Champagne, Claude P; Labrie, Steve; Moineau, Sylvain

    2016-01-18

    Staphylococcus aureus is one of the most prevalent pathogenic bacteria contaminating dairy products. In an effort to reduce food safety risks, virulent phages are investigated as antibacterial agents to control foodborne pathogens. The aim of this study was to compare sets of virulent phages, design phage cocktails, and use them in a cocktail to control pathogenic staphylococci in cheese. Six selected phages belonging to the three Caudovirales families (Myoviridae, Siphoviridae, Podoviridae) were strictly lytic, had a broad host range, and did not carry genes coding for virulence traits in their genomes. However, they were sensitive to pasteurization. At MOI levels of 15, 45, and 150, two anti-S. aureus phage cocktails, each containing three phages, one from each of the three phage families, eradicated a 10(6)CFU/g S. aureus population after 14 days of Cheddar cheese curd ripening at 4°C. The use of these phages did not trigger over-production of S. aureus enterotoxin C. The use of phage cocktails and their rotation may prevent the emergence of phage resistant bacterial strains.

  1. Convergent evolution of pathogenicity islands in helper cos phage interference

    PubMed Central

    Manning, Keith A.; Dokland, Terje; Marina, Alberto

    2016-01-01

    Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution. This article is part of the themed issue ‘The new bacteriology’. PMID:27672154

  2. Assembling filamentous phage occlude pIV channels.

    PubMed

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

    2001-07-31

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

  3. Convergent evolution of pathogenicity islands in helper cos phage interference.

    PubMed

    Carpena, Nuria; Manning, Keith A; Dokland, Terje; Marina, Alberto; Penadés, José R

    2016-11-05

    Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution.This article is part of the themed issue 'The new bacteriology'.

  4. Precisely modulated pathogenicity island interference with late phage gene transcription.

    PubMed

    Ram, Geeta; Chen, John; Ross, Hope F; Novick, Richard P

    2014-10-07

    Having gone to great evolutionary lengths to develop resistance to bacteriophages, bacteria have come up with resistance mechanisms directed at every aspect of the bacteriophage life cycle. Most genes involved in phage resistance are carried by plasmids and other mobile genetic elements, including bacteriophages and their relatives. A very special case of phage resistance is exhibited by the highly mobile phage satellites, staphylococcal pathogenicity islands (SaPIs), which carry and disseminate superantigen and other virulence genes. Unlike the usual phage-resistance mechanisms, the SaPI-encoded interference mechanisms are carefully crafted to ensure that a phage-infected, SaPI-containing cell will lyse, releasing the requisite crop of SaPI particles as well as a greatly diminished crop of phage particles. Previously described SaPI interference genes target phage functions that are not required for SaPI particle production and release. Here we describe a SaPI-mediated interference system that affects expression of late phage gene transcription and consequently is required for SaPI and phage. Although when cloned separately, a single SaPI gene totally blocks phage production, its activity in situ is modulated accurately by a second gene, achieving the required level of interference. The advantage for the host bacteria is that the SaPIs curb excessive phage growth while enhancing their gene transfer activity. This activity is in contrast to that of the clustered regularly interspaced short palindromic repeats (CRISPRs), which totally block phage growth at the cost of phage-mediated gene transfer. In staphylococci the SaPI strategy seems to have prevailed during evolution: The great majority of Staphylococcus aureus strains carry one or more SaPIs, whereas CRISPRs are extremely rare.

  5. Progress in lactic acid bacterial phage research

    PubMed Central

    2014-01-01

    Research on lactic acid bacteria (LAB) has advanced significantly over the past number of decades and these developments have been driven by the parallel advances in technologies such as genomics, bioinformatics, protein expression systems and structural biology, combined with the ever increasing commercial relevance of this group of microorganisms. Some of the more significant and impressive outputs have been in the domain of bacteriophage-host interactions which provides a prime example of the cutting-edge model systems represented by LAB research. Here, we present a retrospective overview of the key advances in LAB phage research including phage-host interactions and co-evolution. We describe how in many instances this knowledge can be pivotal in creating real improvements in the application of LAB cultures in commercial practice. PMID:25185514

  6. Indirect Ultraviolet-Reactivation of Phage λ

    PubMed Central

    George, Jacqueline; Devoret, Raymond; Radman, Miroslav

    1974-01-01

    When an F- recipient Escherichia coli K12 bacterium receives Hfr or F-lac+ DNA from an ultraviolet-irradiated donor, its capacity to promote DNA repair and mutagenesis of ultraviolet-damaged phage λ is substantially increased. We call this phenomenon indirect ultraviolet-reactivation, since its features are essentially the same as those of ultraviolet-reactivation; this repair process occurs in pyrimidine dimer excision-deficient strains and produces clear plaque mutations of the restored phage. Moreover, this process is similar to indirect ultraviolet-induction of prophage λ, since it is promoted by conjugation. However, contrarily to indirect induction, it is produced by Hfr donors and occurs in recipients restricting the incoming ultraviolet-damaged donor DNA. The occurrence of indirect ultraviolet-reactivation provides evidence for the existence in E. coli of an inducible error-prone mechanism for the repair of DNA. PMID:4589889

  7. PHAGE FORMATION IN STAPHYLOCOCCUS MUSCAE CULTURES

    PubMed Central

    Price, Winston H.

    1952-01-01

    1. Under a variety of conditions in which cells are infected with one or a few virus particles and the host cells are killed, but no infective particles or virus material is formed as indicated by plaque count, one-step growth curve, or protein or desoxyribonucleic determinations, the cells neither lyse nor release ribonucleic acid into the medium. 2. The "killing" effect of S. muscae phage is separate from its lytic property. 3. The release of ribonucleic acid into the medium is not simply due to the killing of the cell by the virus, and ribonucleic acid is never found in the medium unless virus material is synthesized. 4. Infected cells of S. muscae synthesizing virus release ribonucleic acid into the medium before cellular lysis begins and before any virus is liberated. 5. The higher the phage yield the more ribonucleic acid is released into the medium before any virus is released. 6. Phage may be released from one strain of Staphylococcus muscae without cellular lysis, although bacterial lysis begins shortly after the virus is released. In another strain, infected under similar conditions, virus liberation occurs simultaneously with cellular lysis. 7. The viruses liberated from both bacterial strains appear to be the same in so far as they cannot be distinguished by serological tests, have the same plaque type and plaque size, and need the same amino acids added to the medium in order to grow. Furthermore, the virus liberated from one strain can infect and multiply in the other strain and vice versa. 8. It is suggested that virus synthesis, in S. muscae cells infected with one or a few phage particles, leads to a disturbance of the normal cellular metabolism, resulting in lysis of the host cell. PMID:14898025

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

    PubMed

    Salmond, George P C; Fineran, Peter C

    2015-12-01

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

  9. Exploring the risks of phage application in the environment

    PubMed Central

    Meaden, Sean; Koskella, Britt

    2013-01-01

    Interest in using bacteriophages to control the growth and spread of bacterial pathogens is being revived in the wake of widespread antibiotic resistance. However, little is known about the ecological effects that high concentrations of phages in the environment might have on natural microbial communities. We review the current evidence suggesting phage-mediated environmental perturbation, with a focus on agricultural examples, and describe the potential implications for human health and agriculture. Specifically, we examine the known and potential consequences of phage application in certain agricultural practices, discuss the risks of evolved bacterial resistance to phages, and question whether the future of phage therapy will emulate that of antibiotic treatment in terms of widespread resistance. Finally, we propose some basic precautions that could preclude such phenomena and highlight existing methods for tracking bacterial resistance to phage therapeutic agents. PMID:24348468

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

    PubMed

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

    2017-02-01

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

  11. Phage therapy: delivering on the promise.

    PubMed

    Harper, D R; Anderson, J; Enright, M C

    2011-07-01

    Bacteriophages are viruses that infect and, in many cases, destroy their bacterial targets. Within a few years of their initial discovery they were being investigated as therapeutic agents for infectious disease, an approach known as phage therapy. However, the nature of these exquisitely specific agents was not understood and much early use was both uninformed and unsuccessful. As a result they were replaced by chemical antibiotics once these became available. Although work on phage therapy continued (and continues) in Eastern Europe, this was not conducted to a standard allowing it to support clinical uses in areas regulated by the European Medicines Agency or the US FDA. To develop phage therapy for these areas requires work carried out in accordance with the requirements of these agencies, and, driven by the current crisis of antibiotic resistance, such clinical trials are now under way. The first Phase I clinical trial of safety was reported in 2005, and the results of the first Phase II clinical trial of efficacy of a bacteriophage therapeutic was published in 2009. While the delivery of these relatively large and complex agents to the site of disease can be more challenging than for conventional, small-molecule antibiotics, bacteriophages are then able to multiply locally even from an extremely low (picogram range) initial dose. This multiplication where and only where they are needed underlies the potential for bacteriophage therapeutics to become a much needed and powerful weapon against bacterial disease.

  12. Diversity of phage integrases in Enterobacteriaceae: development of markers for environmental analysis of temperate phages.

    PubMed

    Balding, Claire; Bromley, Stephen A; Pickup, Roger W; Saunders, Jon R

    2005-10-01

    Viruses are the most abundant biological entities in aquatic systems. Temperate bacteriophages have enormous influences on microbial diversity, genetic exchange and bacterial population dynamics. However, development of molecular tools for their detection in the environment has been problematic. The integrase gene is used here as a molecular marker to analyse the diversity of temperate bacteriophages in a population of freshwater bacteria. Interrogation of the GenBank database revealed 32 non-cryptic enteric phage integrase sequences, leading to the development of a suite of 11 degenerate primer sets specific to the extant sequences elucidated. Application of these primer sets to enterobacterial isolates recovered from a freshwater pond and the temperate phages induced from them revealed a number of diverse integrase genes, including novel integrase-like sequences not represented in the databases. This highlights the potential of utilizing the integrase gene family as a marker for phage diversity.

  13. Probing Tumor Microenvironment with In Vivo Phage Display

    DTIC Science & Technology

    2013-07-01

    peptides may result in an efficient probe for breast tumor imaging and therapy . 15. SUBJECT TERMS Carcinoma-associated fibroblast; phage display...In Vivo Phage Display PRINCIPAL INVESTIGATOR: Dr. Erkki Ruoslahti CONTRACTING ORGANIZATION: Sanford Burnham Medical Research Institute...COVERED 01 July 2012 – 30 June 2013 4. TITLE AND SUBTITLE Probing Tumor Microenvironment with In Vivo Phage Display 5a. CONTRACT NUMBER W81XWH

  14. Probing Tumor Microenvironment with in Vivo Phage Display

    DTIC Science & Technology

    2013-07-01

    Vivo Phage Display PRINCIPAL INVESTIGATOR: Kazuki N. Sugahara, M.D., Ph.D...TITLE AND SUBTITLE 5a. CONTRACT NUMBER Probing Tumor Microenvironment with In Vivo Phage Display 5b. GRANT NUMBER W81XWH-12-1-0174 5c...cells and the matrix. The goal of our group is to make technical improvements in our phage display system, and find peptides that target carcinoma

  15. Evolutionary Rationale for Phages as Complements of Antibiotics.

    PubMed

    Torres-Barceló, Clara; Hochberg, Michael E

    2016-04-01

    Antibiotic-resistant bacterial infections are a major concern to public health. Phage therapy has been proposed as a promising alternative to antibiotics, but an increasing number of studies suggest that both of these antimicrobial agents in combination are more effective in controlling pathogenic bacteria than either alone. We advocate the use of phages in combination with antibiotics and present the evolutionary basis for our claim. In addition, we identify compelling challenges for the realistic application of phage-antibiotic combined therapy.

  16. Complete genome sequence of the giant Pseudomonas phage Lu11.

    PubMed

    Adriaenssens, E M; Mattheus, W; Cornelissen, A; Shaburova, O; Krylov, V N; Kropinski, A M; Lavigne, R

    2012-06-01

    The complete genome sequence of the giant Pseudomonas phage Lu11 was determined, comparing 454 and Sanger sequencing. The double-stranded DNA (dsDNA) genome is 280,538 bp long and encodes 391 open reading frames (ORFs) and no tRNAs. The closest relative is Ralstonia phage ϕRSL1, encoding 40 similar proteins. As such, Lu11 can be considered phylogenetically unique within the Myoviridae and indicates the diversity of the giant phages within this family.

  17. Using Phage Lytic Enzymes to Destroy Pathogenic and BW Bacteria

    DTIC Science & Technology

    2005-07-14

    B. anthracis than the gamma-phage since it will not recognize the occasional B. cereus strain yielding a false positive reaction. This phage is a...bacteriolytic agent that can rapidly and specifically detect and kill Bacillus anthracis. Nature. 418: 884– 889. Nelson, D., Schuch, R., S. Zhu, D...Djurkovic and V.A. Fischetti. 2003. The phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia and sepsis. Infect. Immun.71:6199

  18. Quality and safety requirements for sustainable phage therapy products.

    PubMed

    Pirnay, Jean-Paul; Blasdel, Bob G; Bretaudeau, Laurent; Buckling, Angus; Chanishvili, Nina; Clark, Jason R; Corte-Real, Sofia; Debarbieux, Laurent; Dublanchet, Alain; De Vos, Daniel; Gabard, Jérôme; Garcia, Miguel; Goderdzishvili, Marina; Górski, Andrzej; Hardcastle, John; Huys, Isabelle; Kutter, Elizabeth; Lavigne, Rob; Merabishvili, Maia; Olchawa, Ewa; Parikka, Kaarle J; Patey, Olivier; Pouilot, Flavie; Resch, Gregory; Rohde, Christine; Scheres, Jacques; Skurnik, Mikael; Vaneechoutte, Mario; Van Parys, Luc; Verbeken, Gilbert; Zizi, Martin; Van den Eede, Guy

    2015-07-01

    The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge. To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allows a timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies. This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.

  19. Revisiting phage therapy: new applications for old resources.

    PubMed

    Nobrega, Franklin L; Costa, Ana Rita; Kluskens, Leon D; Azeredo, Joana

    2015-04-01

    The success of phage therapy is dependent on the development of strategies able to overcome the limitations of bacteriophages as therapeutic agents, the creation of an adequate regulatory framework, the implementation of safety protocols, and acceptance by the general public. Many approaches have been proposed to circumvent phages' intrinsic limitations but none have proved to be completely satisfactory. In this review we present the major hurdles of phage therapy and the solutions proposed to circumvent them. A thorough discussion of the advantages and drawbacks of these solutions is provided and special attention is given to the genetic modification of phages as an achievable strategy to shape bacteriophages to exhibit desirable biological properties.

  20. Phage display as a promising approach for vaccine development.

    PubMed

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

    2016-09-29

    Bacteriophages are specific antagonists to bacterial hosts. These viral entities have attracted growing interest as optimal vaccine delivery vehicles. Phages are well-matched for vaccine design due to being highly stable under harsh environmental conditions, simple and inexpensive large scale production, and potent adjuvant capacities. Phage vaccines have efficient immunostimulatory effects and present a high safety profile because these viruses have made a constant relationship with the mammalian body during a long-standing evolutionary period. The birth of phage display technology has been a turning point in the development of phage-based vaccines. Phage display vaccines are made by expressing multiple copies of an antigen on the surface of immunogenic phage particles, thereby eliciting a powerful and effective immune response. Also, the ability to produce combinatorial peptide libraries with a highly diverse pool of randomized ligands has transformed phage display into a straightforward, versatile and high throughput screening methodology for the identification of potential vaccine candidates against different diseases in particular microbial infections. These libraries can be conveniently screened through an affinity selection-based strategy called biopanning against a wide variety of targets for the selection of mimotopes with high antigenicity and immunogenicity. Also, they can be panned against the antiserum of convalescent individuals to recognize novel peptidomimetics of pathogen-related epitopes. Phage display has represented enormous promise for finding new strategies of vaccine discovery and production and current breakthroughs promise a brilliant future for the development of different phage-based vaccine platforms.

  1. Ligand-directed profiling of organelles with internalizing phage libraries

    PubMed Central

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

    2015-01-01

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

  2. Bacteriophages and phage-derived proteins--application approaches.

    PubMed

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara

    2015-01-01

    Currently, the bacterial resistance, especially to most commonly used antibiotics has proved to be a severe therapeutic problem. Nosocomial and community-acquired infections are usually caused by multidrug resistant strains. Therefore, we are forced to develop an alternative or supportive treatment for successful cure of life-threatening infections. The idea of using natural bacterial pathogens such as bacteriophages is already well known. Many papers have been published proving the high antibacterial efficacy of lytic phages tested in animal models as well as in the clinic. Researchers have also investigated the application of non-lytic phages and temperate phages, with promising results. Moreover, the development of molecular biology and novel generation methods of sequencing has opened up new possibilities in the design of engineered phages and recombinant phage-derived proteins. Encouraging performances were noted especially for phage enzymes involved in the first step of viral infection responsible for bacterial envelope degradation, named depolymerases. There are at least five major groups of such enzymes - peptidoglycan hydrolases, endosialidases, endorhamnosidases, alginate lyases and hyaluronate lyases - that have application potential. There is also much interest in proteins encoded by lysis cassette genes (holins, endolysins, spanins) responsible for progeny release during the phage lytic cycle. In this review, we discuss several issues of phage and phage-derived protein application approaches in therapy, diagnostics and biotechnology in general.

  3. The E. Coli Response To A Phage Perturbation

    NASA Astrophysics Data System (ADS)

    Chapman-McQuiston, Emily; Wu, Xiao-Lun

    2007-03-01

    Bacteria have evolved a variety of defenses against extreme environmental pressure. While a majority of the population dies during times of stress, a portion of the population continues to survive due to the cell's phenotypic state. We study the response of the bacterial system to attack by a particular virus called lambda phage. During times of phage attack bacteria continue to create and lose receptors making the bacteria more or less sensitive to the applied phage concentration. We use experiment and modeling to study how the creation and loss of receptors affects the response and recovery of the bacterial population due to an applied phage pressure.

  4. Ligand-directed profiling of organelles with internalizing phage libraries.

    PubMed

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

    2015-02-02

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

  5. Isolation and Characterization of Phages Infecting Bacillus subtilis

    PubMed Central

    Krasowska, Anna; Biegalska, Anna; Augustyniak, Daria; Łoś, Marcin; Richert, Malwina; Łukaszewicz, Marcin

    2015-01-01

    Bacteriophages have been suggested as an alternative approach to reduce the amount of pathogens in various applications. Bacteriophages of various specificity and virulence were isolated as a means of controlling food-borne pathogens. We studied the interaction of bacteriophages with Bacillus species, which are very often persistent in industrial applications such as food production due to their antibiotic resistance and spore formation. A comparative study using electron microscopy, PFGE, and SDS-PAGE as well as determination of host range, pH and temperature resistance, adsorption rate, latent time, and phage burst size was performed on three phages of the Myoviridae family and one phage of the Siphoviridae family which infected Bacillus subtilis strains. The phages are morphologically different and characterized by icosahedral heads and contractile (SIOΦ, SUBω, and SPOσ phages) or noncontractile (ARπ phage) tails. The genomes of SIOΦ and SUBω are composed of 154 kb. The capsid of SIOΦ is composed of four proteins. Bacteriophages SPOσ and ARπ have genome sizes of 25 kbp and 40 kbp, respectively. Both phages as well as SUBω phage have 14 proteins in their capsids. Phages SIOΦ and SPOσ are resistant to high temperatures and to the acid (4.0) and alkaline (9.0 and 10.0) pH. PMID:26273592

  6. Salmonella phages and prophages: genomics, taxonomy, and applied aspects.

    PubMed

    Switt, Andrea I Moreno; Sulakvelidze, Alexander; Wiedmann, Martin; Kropinski, Andrew M; Wishart, David S; Poppe, Cornelis; Liang, Yongjie

    2015-01-01

    Since this book was originally published in 2007 there has been a significant increase in the number of Salmonella bacteriophages, particularly lytic virus, and Salmonella strains which have been fully sequenced. In addition, new insights into phage taxonomy have resulted in new phage genera, some of which have been recognized by the International Committee of Taxonomy of Viruses (ICTV). The properties of each of these genera are discussed, along with the role of phage as agents of genetic exchange, as therapeutic agents, and their involvement in phage typing.

  7. Bacteriophages and Phage-Derived Proteins – Application Approaches

    PubMed Central

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara

    2015-01-01

    Currently, the bacterial resistance, especially to most commonly used antibiotics has proved to be a severe therapeutic problem. Nosocomial and community-acquired infections are usually caused by multidrug resistant strains. Therefore, we are forced to develop an alternative or supportive treatment for successful cure of life-threatening infections. The idea of using natural bacterial pathogens such as bacteriophages is already well known. Many papers have been published proving the high antibacterial efficacy of lytic phages tested in animal models as well as in the clinic. Researchers have also investigated the application of non-lytic phages and temperate phages, with promising results. Moreover, the development of molecular biology and novel generation methods of sequencing has opened up new possibilities in the design of engineered phages and recombinant phage-derived proteins. Encouraging performances were noted especially for phage enzymes involved in the first step of viral infection responsible for bacterial envelope degradation, named depolymerases. There are at least five major groups of such enzymes – peptidoglycan hydrolases, endosialidases, endorhamnosidases, alginate lyases and hyaluronate lyases – that have application potential. There is also much interest in proteins encoded by lysis cassette genes (holins, endolysins, spanins) responsible for progeny release during the phage lytic cycle. In this review, we discuss several issues of phage and phage-derived protein application approaches in therapy, diagnostics and biotechnology in general. PMID:25666799

  8. 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

  9. Learning from bacteriophages - advantages and limitations of phage and phage-encoded protein applications.

    PubMed

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob

    2012-12-01

    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.

  10. Determination of the attP and attB sites of phage CD27 from Clostridium difficile NCTC 12727.

    PubMed

    Williams, Rachel; Meader, Emma; Mayer, Melinda; Narbad, Arjan; Roberts, Adam P; Mullany, Peter

    2013-09-01

    The attP region of the Clostridium difficile phage CD27 was identified, located immediately downstream of the putative recombinase. The phage could integrate into two specific sites (attB) in the C. difficile genome, one of which was in an open reading frame encoding a putative ATPase of an ABC transporter and the other in an open reading frame encoding a putative ATPase of the flagella protein export apparatus. The prophage was capable of excision and formation of a circular molecule and phages were spontaneously released at a low frequency during growth. Infection and lysogeny of a C. difficile strain previously shown to be sensitive to CD27 were demonstrated, leading to a reduction in toxin production. Finally, a putative repressor was identified which is likely to be involved in maintaining lysogeny in these strains.

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

    PubMed

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

    2012-01-01

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

  12. Genome Characteristics of a Novel Phage from Bacillus thuringiensis Showing High Similarity with Phage from Bacillus cereus

    PubMed Central

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

    2012-01-01

    Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the “late” region, the “lysogeny-lysis” region and the “early” region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor. PMID:22649540

  13. Identification of a new member of the phage shock protein response in Escherichia coli, the phage shock protein G (PspG).

    PubMed

    Lloyd, Louise J; Jones, Susan E; Jovanovic, Goran; Gyaneshwar, Prasad; Rolfe, Matthew D; Thompson, Arthur; Hinton, Jay C; Buck, Martin

    2004-12-31

    The phage shock protein operon (pspABCDE) of Escherichia coli is strongly up-regulated in response to overexpression of the filamentous phage secretin protein IV (pIV) and by many other stress conditions including defects in protein export. PspA has an established role in maintenance of the proton-motive force of the cell under stress conditions. Here we present evidence for a new member of the phage shock response in E. coli. Using transcriptional profiling, we show that the synthesis of pIV in E. coli leads to a highly restricted response limited to the up-regulation of the psp operon genes and yjbO. The psp operon and yjbO are also up-regulated in response to pIV in Salmonella enterica serovar Typhimurium. yjbO is a highly conserved gene found exclusively in bacteria that contain a psp operon but is physically unlinked to the psp operon. yjbO encodes a putative inner membrane protein that is co-controlled with the psp operon genes and is predicted to be an effector of the psp response in E. coli. We present evidence that yjbO expression is driven by sigma(54)-RNA polymerase, activated by PspF and integration host factor, and negatively regulated by PspA. PspF specifically regulates only members of the PspF regulon: pspABCDE and yjbO. We found that increased expression of YjbO results in decreased motility of bacteria. Because yjbO is co-conserved and co-regulated with the psp operon and is a member of the phage shock protein F regulon, we propose that yjbO be renamed pspG.

  14. Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks

    PubMed Central

    Bonilla, Natasha; Rojas, Maria Isabel; Netto Flores Cruz, Giuliano; Hung, Shr-Hau; Rohwer, Forest

    2016-01-01

    A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 1010–11 PFU·ml−1), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations and improve the molecular characterizations of phage. The method consists of five major parts, including phage propagation, phage clean up by 0.22 μm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of > 100 mL, the PoT protocol generated a clean, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays. PMID:27547567

  15. Characterization of Five Podoviridae Phages Infecting Citrobacter freundii.

    PubMed

    Hamdi, Sana; Rousseau, Geneviève M; Labrie, Simon J; Kourda, Rim S; Tremblay, Denise M; Moineau, Sylvain; Slama, Karim B

    2016-01-01

    Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus.

  16. Characterization of Five Podoviridae Phages Infecting Citrobacter freundii

    PubMed Central

    Hamdi, Sana; Rousseau, Geneviève M.; Labrie, Simon J.; Kourda, Rim S.; Tremblay, Denise M.; Moineau, Sylvain; Slama, Karim B.

    2016-01-01

    Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus. PMID:27446058

  17. Cryptic transposable phages of Pseudomonas aeruginosa

    SciTech Connect

    Krylov, V.N.; Mit`kina, L.N.; Pleteneva, E.A.; Aleshin, V.V.

    1995-11-01

    Frequencies of nucleotide sequences homologous to phage transposons (PT) of two species, D3112 and B3, were assessed in genomes of natural Pseudomonas aeruginosa strains by the dot-blot hybridization method. These strains were incapable of liberating viable phages on a lawn of the PA01 standard indicator strain of P. aeruginosa. It was shown that the homologies detected belong to two groups, high and intermediate, with respect to homology level. Homology patterns were classified as high when they provided signals comparable to those for hybridization in a positive control; patterns were classified as intermediate when the hybridization level was higher than the background level, but lower than in the positive control. Homologous PT sequences were designated as cryptic PT. Intact cryptic PT prophages were shown to exist in genomes of particular natural strains manifesting a higher level of hybridization. However, the growth of these phages was limited by the restriction system of strain PA01. It is possible to isolate strains maintaining the growth of some cryptic PT. These strains differed from P. aeruginosa with respect to the specificity of the restriction and modification system. Nevertheless, in most cases, the attempt to identify a novel host capable of maintaining growth of a cryptic PT failed. Natural strains often carry cryptic PT related to both known PT species, D3112 and B3. The frequency of cryptic PT is extremely high, reaching 30% in strains with a high level of homology only and up to 50% in all strains exhibiting homology. This high PT frequency is assumed to be associated with the considerable variation of P. aeruginosa. 15 refs., 1 fig., 2 tabs.

  18. Genome Sequences of Six Paenibacillus larvae Siphoviridae Phages.

    PubMed

    Carson, Susan; Bruff, Emily; DeFoor, William; Dums, Jacob; Groth, Adam; Hatfield, Taylor; Iyer, Aruna; Joshi, Kalyani; McAdams, Sarah; Miles, Devon; Miller, Delanie; Oufkir, Abdoullah; Raynor, Brinkley; Riley, Sara; Roland, Shelby; Rozier, Horace; Talley, Sarah; Miller, Eric S

    2015-06-18

    Six sequenced and annotated genomes of Paenibacillus larvae phages isolated from the combs of American foulbrood-diseased beehives are 37 to 45 kbp and have approximately 42% G+C content and 60 to 74 protein-coding genes. Phage Lily is most divergent from Diva, Rani, Redbud, Shelly, and Sitara.

  19. Genome Sequences of Six Paenibacillus larvae Siphoviridae Phages

    PubMed Central

    Carson, Susan; Bruff, Emily; DeFoor, William; Dums, Jacob; Groth, Adam; Hatfield, Taylor; Iyer, Aruna; Joshi, Kalyani; McAdams, Sarah; Miles, Devon; Miller, Delanie; Oufkir, Abdoullah; Raynor, Brinkley; Riley, Sara; Roland, Shelby; Rozier, Horace; Talley, Sarah

    2015-01-01

    Six sequenced and annotated genomes of Paenibacillus larvae phages isolated from the combs of American foulbrood-diseased beehives are 37 to 45 kbp and have approximately 42% G+C content and 60 to 74 protein-coding genes. Phage Lily is most divergent from Diva, Rani, Redbud, Shelly, and Sitara. PMID:26089405

  20. Computational models of populations of bacteria and lytic phage.

    PubMed

    Krysiak-Baltyn, Konrad; Martin, Gregory J O; Stickland, Anthony D; Scales, Peter J; Gras, Sally L

    2016-11-01

    The use of phages to control and reduce numbers of unwanted bacteria can be traced back to the early 1900s, when phages were explored as a tool to treat infections before the wide scale use of antibiotics. Recently, phage therapy has received renewed interest as a method to treat multiresistant bacteria. Phages are also widely used in the food industry to prevent the growth of certain bacteria in foods, and are currently being explored as a tool for use in bioremediation and wastewater treatment. Despite the large body of biological research on phages, relatively little attention has been given to computational modeling of the population dynamics of phage and bacterial interactions. The earliest model was described by Campbell in the 1960s. Subsequent modifications to this model include partial or complete resistance, multiple phage binding sites, and spatial heterogeneity. This review provides a general introduction to modeling of the population dynamics of bacteria and phage. The review introduces the basic model and relevant concepts and evaluates more complex variations of the basic model published to date, including a model of disease epidemics caused by infectious bacteria. Finally, the shortcomings and potential ways to improve the models are discussed.

  1. Chromosomal DNA deletion confers phage resistance to Pseudomonas aeruginosa.

    PubMed

    Le, Shuai; Yao, Xinyue; Lu, Shuguang; Tan, Yinling; Rao, Xiancai; Li, Ming; Jin, Xiaolin; Wang, Jing; Zhao, Yan; Wu, Nicholas C; Lux, Renate; He, Xuesong; Shi, Wenyuan; Hu, Fuquan

    2014-04-28

    Bacteria develop a broad range of phage resistance mechanisms, such as prevention of phage adsorption and CRISPR/Cas system, to survive phage predation. In this study, Pseudomonas aeruginosa PA1 strain was infected with lytic phage PaP1, and phage-resistant mutants were selected. A high percentage (~30%) of these mutants displayed red pigmentation phenotype (Red mutant). Through comparative genomic analysis, one Red mutant PA1r was found to have a 219.6 kb genomic fragment deletion, which contains two key genes hmgA and galU related to the observed phenotypes. Deletion of hmgA resulted in the accumulation of a red compound homogentisic acid; while A galU mutant is devoid of O-antigen, which is required for phage adsorption. Intriguingly, while the loss of galU conferred phage resistance, it significantly attenuated PA1r in a mouse infection experiment. Our study revealed a novel phage resistance mechanism via chromosomal DNA deletion in P. aeruginosa.

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

    PubMed Central

    Gillis, Annika; Mahillon, Jacques

    2014-01-01

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

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

    PubMed

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

    2017-02-01

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

  4. Filamentous Phages As a Model System in Soft Matter Physics

    PubMed Central

    Dogic, Zvonimir

    2016-01-01

    Filamentous phages have unique physical properties, such as uniform particle lengths, that are not found in other model systems of rod-like colloidal particles. Consequently, suspensions of such phages provided powerful model systems that have advanced our understanding of soft matter physics in general and liquid crystals in particular. We described some of these advances. In particular we briefly summarize how suspensions of filamentous phages have provided valuable insight into the field of colloidal liquid crystals. We also describe recent experiments on filamentous phages that have elucidated a robust pathway for assembly of 2D membrane-like materials. Finally, we outline unique structural properties of filamentous phages that have so far remained largely unexplored yet have the potential to further advance soft matter physics and material science. PMID:27446051

  5. Strategies for Vaccine Design Using Phage Display-Derived Peptides.

    PubMed

    Goulart, Luiz R; Santos, Paula de S

    2016-01-01

    Development of peptide vaccines through the phage display technology is a powerful strategy that relies on short peptides expressed in the phage capsid surface to induce highly targeted immune responses. Phage display-derived immunogenic peptides can be used directly as a phage-fused peptide reagent or as a synthetic peptide with specific modifications, according to target molecule and disease pathogen/parasite. Peptides' selection (mimotopes) can be performed against monoclonal or polyclonal antibodies to disclose determinant regions (epitopes) that can induce a neutralizing response. Validations of mimotopes are performed in vitro and in vivo, based on cell culture and animal models, to demonstrate its immunogenic potential for final vaccine formulations with an appropriate adjuvant. Here we present specific methods for the discovery of novel immunogenic peptides based on phage display.

  6. Chemical posttranslational modification of phage-displayed peptides.

    PubMed

    Ng, Simon; Tjhung, Katrina F; Paschal, Beth M; Noren, Christopher J; Derda, Ratmir

    2015-01-01

    Phage-displayed peptide library has fueled the discovery of novel ligands for diverse targets. A new type of phage libraries that displays not only linear and disulfide-constrained cyclic peptides but moieties that cannot be encoded genetically or incorporated easily by bacterial genetic machinery has emerged recently. Chemical posttranslational modification of phage library is one of the simplest approaches to encode nonnatural moieties. It confers the library with new functionality and makes it possible to select and evolve molecules with properties not found in the peptides, for instance, glycopeptides recognized by carbohydrate-binding protein and peptides with photoswitching capability. To this end, we describe the newly emerging techniques to chemically modify the phage library and quantify the efficiency of the reaction with a biotin-capture assay. Finally, we provide the methods to construct N-terminal Ser peptide library that allows site-selective modification of phage.

  7. Filamentous Phages As a Model System in Soft Matter Physics.

    PubMed

    Dogic, Zvonimir

    2016-01-01

    Filamentous phages have unique physical properties, such as uniform particle lengths, that are not found in other model systems of rod-like colloidal particles. Consequently, suspensions of such phages provided powerful model systems that have advanced our understanding of soft matter physics in general and liquid crystals in particular. We described some of these advances. In particular we briefly summarize how suspensions of filamentous phages have provided valuable insight into the field of colloidal liquid crystals. We also describe recent experiments on filamentous phages that have elucidated a robust pathway for assembly of 2D membrane-like materials. Finally, we outline unique structural properties of filamentous phages that have so far remained largely unexplored yet have the potential to further advance soft matter physics and material science.

  8. Phages in the global fruit and vegetable industry.

    PubMed

    Żaczek, M; Weber-Dąbrowska, B; Górski, A

    2015-03-01

    From recent articles, we have learned that phages can constitute a promising alternative in the food industry to eliminate bacterial pathogens from seedlings in greenhouse and field environments, as well as from fresh-cut food products. The fruit and vegetable industry requires quite a different approach than the meat or dairy industry. Several factors can inhibit efficacy of phage treatment such as plant watering or washing ready-to-eat products (water may dilute therapeutic doses), UV irradiation or extensive spreading of phytopathogens by wind, insects or even humans. Spontaneously occurring anomalous weather conditions in different parts of the world also may have an enormous impact on phage persistence in cultivations and on yields. Despite that, some phage preparations are commercially available and, without doubt, are much safer than chemical treatments. Along with increasing worldwide fruit and vegetable consumption, plant diseases and human foodborne illnesses are becoming a serious economic problem, resulting in a focus on optimization of phage treatment.

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

    PubMed Central

    2014-01-01

    Background In prior work, a phage engineered with a biofilm-degrading enzyme (dispersin B) cleared artificial, short-term biofilms more fully than the phage lacking the enzyme. An unresolved question is whether the transgene will be lost or maintained during phage growth – its loss would limit the utility of the engineering. Broadly supported evolutionary theory suggests that transgenes will be lost through a ‘tragedy of the commons’ mechanism unless the ecology of growth in biofilms meets specific requirements. We test that theory here. Results Functional properties of the transgenic phage were identified. Consistent with the previous study, the dispersin phage was superior to unmodified phage at clearing short term biofilms grown in broth, shown here to be an effect attributable to free enzyme. However, the dispersin phage was only marginally better than control phages on short term biofilms in minimal media and was no better than control phages in clearing long term biofilms. There was little empirical support for the tragedy of the commons framework despite a strong theoretical foundation for its supposed relevance. The framework requires that the transgene imposes an intrinsic cost, yet the transgene was intrinsically neutral or beneficial when expressed from one part of the phage genome. Expressed from a different part of the genome, the transgene did behave as if intrinsically costly, but its maintenance did not benefit from spatially structured growth per se – violating the tragedy framework. Conclusions Overall, the transgene was beneficial under many conditions, but no insight to its maintenance was attributable to the established evolutionary framework. The failure likely resides in system details that would be used to parameterize the models. Our study cautions against naive applications of evolutionary theory to synthetic biology, even qualitatively. PMID:25126112

  10. Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays

    PubMed Central

    Holguín, Angela V.; Rangel, Guillermo; Clavijo, Viviana; Prada, Catalina; Mantilla, Marcela; Gomez, María Catalina; Kutter, Elizabeth; Taylor, Corinda; Fineran, Peter C.; Barrios, Andrés Fernando González; Vives, Martha J.

    2015-01-01

    Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization. PMID:26274971

  11. The XXIIIrd Phage/Virus Assembly Meeting

    PubMed Central

    Serwer, Philip

    2014-01-01

    The XXIIIrd Phage/Virus Assembly (PVA) meeting returned to its birthplace in Lake Arrowhead, CA on September 8–13, 2013 (Fig. 1). The original meeting occurred in 1968, organized by Bob Edgar (Caltech, Pasadena, CA USA), Fred Eiserling (University of California, Los Angeles, Los Angeles, CA USA) and Bill Wood (Caltech, Pasadena, CA USA). The organizers of the 2013 meeting were Bill Gelbart (University of California, Los Angeles, Los Angeles, CA USA) and Jack Johnson (Scripps Research Institute, La Jolla, CA USA). This meeting specializes in an egalitarian format. Students are distinguished from senior faculty primarily by the signs of age. With the exception of historically based introductory talks, all talks were allotted the same time and freedom. This tradition began when the meeting was phage-only and has been continued now that all viruses are included. Many were the animated conversations about basic questions. New and international participants were present, a sign that the field has significant attraction, as it should, based on details below. The meeting was also characterized by a sense of humor and generally good times, a chance to both enjoy the science and forget the funding malaise to which many participants are exposed. I will present some of the meeting content, without attempting to be comprehensive. PMID:24498537

  12. Multivalent pIX phage display selects for distinct and improved antibody properties

    PubMed Central

    Høydahl, Lene S.; Nilssen, Nicolay R.; Gunnarsen, Kristin S.; Pré, M. Fleur du; Iversen, Rasmus; Roos, Norbert; Chen, Xi; Michaelsen, Terje E.; Sollid, Ludvig M.; Sandlie, Inger; Løset, Geir Å.

    2016-01-01

    Phage display screening readily allows for the identification of a multitude of antibody specificities, but to identify optimal lead candidates remains a challenge. Here, we direct the antibody-capsid fusion away from the signal sequence-dependent secretory SEC pathway in E. coli by utilizing the intrinsic signal sequence-independent property of pIX to obtain virion integration. This approach was combined with the use of an engineered helper phage known to improve antibody pIX display and retrieval. By direct comparison with pIII display, we demonstrate that antibody display using this pIX system translates into substantially improved retrieval of desired specificities with favorable biophysical properties in de novo selection. We show that the effect was due to less E. coli host toxicity during phage propagation conferred by the lack of a signal sequence. This pIX combinatorial display platform provides a generic alternative route for obtaining good binders with high stability and may thus find broad applicability. PMID:27966617

  13. A Broadly Implementable Research Course in Phage Discovery and Genomics for First-Year Undergraduate Students

    PubMed Central

    Jordan, Tuajuanda C.; Burnett, Sandra H.; Carson, Susan; Caruso, Steven M.; Clase, Kari; DeJong, Randall J.; Dennehy, John J.; Denver, Dee R.; Dunbar, David; Elgin, Sarah C. R.; Findley, Ann M.; Gissendanner, Chris R.; Golebiewska, Urszula P.; Guild, Nancy; Hartzog, Grant A.; Grillo, Wendy H.; Hollowell, Gail P.; Hughes, Lee E.; Johnson, Allison; King, Rodney A.; Lewis, Lynn O.; Li, Wei; Rosenzweig, Frank; Rubin, Michael R.; Saha, Margaret S.; Sandoz, James; Shaffer, Christopher D.; Taylor, Barbara; Temple, Louise; Vazquez, Edwin; Ware, Vassie C.; Barker, Lucia P.; Bradley, Kevin W.; Jacobs-Sera, Deborah; Pope, Welkin H.; Russell, Daniel A.; Cresawn, Steven G.; Lopatto, David; Bailey, Cheryl P.; Hatfull, Graham F.

    2014-01-01

    ABSTRACT Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students’ interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training. PMID:24496795

  14. Pseudomonas aeruginosa population structure revisited under environmental focus: impact of water quality and phage pressure.

    PubMed

    Selezska, Katherina; Kazmierczak, Marlon; Müsken, Mathias; Garbe, Julia; Schobert, Max; Häussler, Susanne; Wiehlmann, Lutz; Rohde, Christine; Sikorski, Johannes

    2012-08-01

    Pseudomonas aeruginosa attracts research attention as a common opportunistic nosocomial pathogen causing severe health problems in humans. Nevertheless, its primary habitat is the natural environment. Here, we relate the genetic diversity of 381 environmental isolates from rivers in northern Germany to ecological factors such as river system, season of sampling and different levels of water quality. From representatives of 99 environmental clones, also in comparison with 91 clinical isolates, we determined motility phenotypes, virulence factors, biofilm formation, serotype and the resistance to seven environmental P.aeruginosa phages. The integration of genetic, ecological and phenotypic data showed (i) the presence of several extended clonal complexes (ecc) which are non-uniformly distributed across different water qualities, and (ii) a correlation of the hosts' serotype composition with susceptibility towards distinct groups of environmental phages. For at least one ecc (eccB), we assumed the ecophysiological differences on environmental water adaptation and phage resistance to be so distinct as to reinforce an environmentally driven cladogenic split from the remainder of P.aeruginosa. In summary, we conclude that the majority of the microevolutionary population dynamics of P.aeruginosa were shaped by the natural environment and not by the clinical habitat.

  15. Multivalent pIX phage display selects for distinct and improved antibody properties.

    PubMed

    Høydahl, Lene S; Nilssen, Nicolay R; Gunnarsen, Kristin S; Pré, M Fleur du; Iversen, Rasmus; Roos, Norbert; Chen, Xi; Michaelsen, Terje E; Sollid, Ludvig M; Sandlie, Inger; Løset, Geir Å

    2016-12-14

    Phage display screening readily allows for the identification of a multitude of antibody specificities, but to identify optimal lead candidates remains a challenge. Here, we direct the antibody-capsid fusion away from the signal sequence-dependent secretory SEC pathway in E. coli by utilizing the intrinsic signal sequence-independent property of pIX to obtain virion integration. This approach was combined with the use of an engineered helper phage known to improve antibody pIX display and retrieval. By direct comparison with pIII display, we demonstrate that antibody display using this pIX system translates into substantially improved retrieval of desired specificities with favorable biophysical properties in de novo selection. We show that the effect was due to less E. coli host toxicity during phage propagation conferred by the lack of a signal sequence. This pIX combinatorial display platform provides a generic alternative route for obtaining good binders with high stability and may thus find broad applicability.

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

    PubMed

    Speck, Peter; Smithyman, Anthony

    2016-02-01

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

  17. Phage-bacteria infection networks: From nestedness to modularity

    NASA Astrophysics Data System (ADS)

    Flores, Cesar O.; Valverde, Sergi; Weitz, Joshua S.

    2013-03-01

    Bacteriophages (viruses that infect bacteria) are the most abundant biological life-forms on Earth. However, very little is known regarding the structure of phage-bacteria infections. In a recent study we re-evaluated 38 prior studies and demonstrated that phage-bacteria infection networks tend to be statistically nested in small scale communities (Flores et al 2011). Nestedness is consistent with a hierarchy of infection and resistance within phages and bacteria, respectively. However, we predicted that at large scales, phage-bacteria infection networks should be typified by a modular structure. We evaluate and confirm this hypothesis using the most extensive study of phage-bacteria infections (Moebus and Nattkemper 1981). In this study, cross-infections were evaluated between 215 marine phages and 286 marine bacteria. We develop a novel multi-scale network analysis and find that the Moebus and Nattkemper (1981) study, is highly modular (at the whole network scale), yet also exhibits nestedness and modularity at the within-module scale. We examine the role of geography in driving these modular patterns and find evidence that phage-bacteria interactions can exhibit strong similarity despite large distances between sites. CFG acknowledges the support of CONACyT Foundation. JSW holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund and acknowledges the support of the James S. McDonnell Foundation

  18. Plasmid carriage can limit bacteria-phage coevolution.

    PubMed

    Harrison, Ellie; Truman, Julie; Wright, Rosanna; Spiers, Andrew J; Paterson, Steve; Brockhurst, Michael A

    2015-08-01

    Coevolution with bacteriophages is a major selective force shaping bacterial populations and communities. A variety of both environmental and genetic factors has been shown to influence the mode and tempo of bacteria-phage coevolution. Here, we test the effects that carriage of a large conjugative plasmid, pQBR103, had on antagonistic coevolution between the bacterium Pseudomonas fluorescens and its phage, SBW25ϕ2. Plasmid carriage limited bacteria-phage coevolution; bacteria evolved lower phage-resistance and phages evolved lower infectivity in plasmid-carrying compared with plasmid-free populations. These differences were not explained by effects of plasmid carriage on the costs of phage resistance mutations. Surprisingly, in the presence of phages, plasmid carriage resulted in the evolution of high frequencies of mucoid bacterial colonies. Mucoidy can provide weak partial resistance against SBW25ϕ2, which may have limited selection for qualitative resistance mutations in our experiments. Taken together, our results suggest that plasmids can have evolutionary consequences for bacteria that go beyond the direct phenotypic effects of their accessory gene cargo.

  19. Evolution of Lactococcus lactis phages within a cheese factory.

    PubMed

    Rousseau, Geneviève M; Moineau, Sylvain

    2009-08-01

    We have sequenced the double-stranded DNA genomes of six lactococcal phages (SL4, CB13, CB14, CB19, CB20, and GR7) from the 936 group that were isolated over a 9-year period from whey samples obtained from a Canadian cheese factory. These six phages infected the same two industrial Lactococcus lactis strains out of 30 tested. The CB14 and GR7 genomes were found to be 100% identical even though they were isolated 14 months apart, indicating that a phage can survive in a cheese plant for more than a year. The other four genomes were related but notably different. The length of the genomes varied from 28,144 to 32,182 bp, and they coded for 51 to 55 open reading frames. All five genomes possessed a 3' overhang cos site that was 11 nucleotides long. Several structural proteins were also identified by nano-high-performance liquid chromatography-tandem mass spectrometry, confirming bioinformatic analyses. Comparative analyses suggested that the most recently isolated phages (CB19 and CB20) were derived, in part, from older phage isolates (CB13 and CB14/GR7). The organization of the five distinct genomes was similar to the previously sequenced lactococcal phage genomes of the 936 group, and from these sequences, a core genome was determined for lactococcal phages of the 936 group.

  20. Coevolution of CRISPR bacteria and phage in 2 dimensions

    NASA Astrophysics Data System (ADS)

    Han, Pu; Deem, Michael

    2014-03-01

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

  1. Biofilm control with natural and genetically-modified phages.

    PubMed

    Motlagh, Amir Mohaghegh; Bhattacharjee, Ananda Shankar; Goel, Ramesh

    2016-04-01

    Bacteriophages, as the most dominant and diverse entities in the universe, have the potential to be one of the most promising therapeutic agents. The emergence of multidrug-resistant bacteria and the antibiotic crisis in the last few decades have resulted in a renewed interest in phage therapy. Furthermore, bacteriophages, with the capacity to rapidly infect and overcome bacterial resistance, have demonstrated a sustainable approach against bacterial pathogens-particularly in biofilm. Biofilm, as complex microbial communities located at interphases embedded in a matrix of bacterial extracellular polysaccharide substances (EPS), is involved in health issues such as infections associated with the use of biomaterials and chronic infections by multidrug resistant bacteria, as well as industrial issues such as biofilm formation on stainless steel surfaces in food industry and membrane biofouling in water and wastewater treatment processes. In this paper, the most recent studies on the potential of phage therapy using natural and genetically-modified lytic phages and their associated enzymes in fighting biofilm development in various fields including engineering, industry, and medical applications are reviewed. Phage-mediated prevention approaches as an indirect phage therapy strategy are also explored in this review. In addition, the limitations of these approaches and suggestions to overcome these constraints are discussed to enhance the efficiency of phage therapy process. Finally, future perspectives and directions for further research towards a better understanding of phage therapy to control biofilm are recommended.

  2. Changes in Environmental Conditions Modify Infection Kinetics of Dairy Phages.

    PubMed

    Zaburlin, Delfina; Quiberoni, Andrea; Mercanti, Diego

    2017-04-08

    Latent period, burst time, and burst size, kinetic parameters of phage infection characteristic of a given phage/host system, have been measured for a wide variety of lactic acid bacteria. However, most studies to date were conducted in optimal growth conditions of host bacteria and did not consider variations due to changes in external factors. In this work, we determined the effect of temperature, pH, and starvation on kinetic parameters of phages infecting Lactobacillus paracasei, Lactobacillus plantarum, and Leuconostoc mesenteroides. For kinetics assessment, one-step growth curves were carried out in MRS broth at optimal conditions (control), lower temperature, pH 6.0 and 5.0 (MRS6 and MRS5, respectively), or in medium lacking carbon (MRSN) or nitrogen (MRSC) sources. Phage infection was progressively impaired as environmental conditions were modified from optimal. At lower temperature or pH, infection was delayed, as perceived by longer latent and burst times. Burst size, however, was lower, equal or higher than for controls, but this effect was highly dependent on the particular phage-host system studied. Phage infection was strongly inhibited in MRSC, but only mildly impaired in MRSN. Nevertheless, growth of all the bacterial strains tested was severely compromised by starvation, without significant differences between MRSC and MRSN, indicating that nitrogen compounds are specifically required for a successful phage infection, beyond their influence on bacterial growth.

  3. The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family

    PubMed Central

    Shen, Xiaona; Zhang, Jingyun; Xu, Jialiang; Du, Pengcheng; Pang, Bo; Li, Jie; Kan, Biao

    2016-01-01

    Bacteriophage 919TP is a temperate phage of Vibrio cholerae serogroup O1 El Tor and is used as a subtyping phage in the phage-biotyping scheme in cholera surveillance in China. In this study, sequencing of the 919TP genome showed that it belonged to the Vibrio phage K139 family. The mechanisms conferring resistance to 919TP infection of El Tor strains were explored to help understand the subtyping basis of phage 919TP and mutations related to 919TP resistance. Among the test strains resistant to phage 919TP, most contained the temperate 919TP phage genome, which facilitated superinfection exclusion to 919TP. Our data suggested that this immunity to Vibrio phage 919TP occurred after absorption of the phage onto the bacteria. Other strains contained LPS receptor synthesis gene mutations that disable adsorption of phage 919TP. Several strains resistant to 919TP infection possessed unknown resistance mechanisms, since they did not contain LPS receptor mutations or temperate K139 phage genome. Further research is required to elucidate the phage infection steps involved in the resistance of these strains to phage infection. PMID:27242744

  4. Phage approved in food, why not as a therapeutic?

    PubMed

    Sarhan, Wessam A; Azzazy, Hassan M E

    2015-01-01

    Bacterial resistance is not only restricted to human infections but is also a major problem in food. With the marked decrease in produced antimicrobials, the world is now reassessing bacteriophages. In 2006, ListShield™ received the US FDA approval for using phage in food. Nevertheless, regulatory approval of phage-based therapeutics is still facing many challenges. This review highlights the use of bacteriophages as biocontrol agents in the food industry. It also focuses on the challenges still facing the regulatory approval of phage-based therapeutics and the proposed approaches to overcome such challenges.

  5. Gene transfer agents: phage-like elements of genetic exchange

    PubMed Central

    Lang, Andrew S.; Zhaxybayeva, Olga; Beatty, J. Thomas

    2013-01-01

    Horizontal gene transfer is important in the evolution of bacterial and archaeal genomes. An interesting genetic exchange process is carried out by diverse phage-like gene transfer agents (GTAs) that are found in a wide range of prokaryotes. Although GTAs resemble phages, they lack the hallmark capabilities that define typical phages, and they package random pieces of the producing cell’s genome. In this Review, we discuss the defining characteristics of the GTAs that have been identified to date, along with potential functions for these agents and the possible evolutionary forces that act on the genes involved in their production. PMID:22683880

  6. The factors affecting effectiveness of treatment in phages therapy

    PubMed Central

    Ly-Chatain, Mai Huong

    2014-01-01

    In recent years, the use of lytic bacteriophages as antimicrobial agents controlling pathogenic bacteria has appeared as a promising new alternative strategy in the face of growing antibiotic resistance which has caused problems in many fields including medicine, veterinary medicine, and aquaculture. The use of bacteriophages has numerous advantages over traditional antimicrobials. The effectiveness of phage applications in fighting against pathogenic bacteria depends on several factors such as the bacteriophages/target bacteria ratio, the mode and moment of treatment, environmental conditions (pH, temperature...), the neutralization of phage and accessibility to target bacteria, amongst others. This report presents these factors and the challenges involved in developing phage therapy applications. PMID:24600439

  7. [Advances in the treatment of wound bacterial infection with phage].

    PubMed

    Cui, Zelong

    2015-10-01

    The treatment of wound bacterial infection is an extremely difficult problem in clinic, especially in patients with large wounds which are infected by multidrug resistant, pan-resistant or omni-resistant bacteria. In recent years, with a grim prospect of antibiotic resistance, phage therapy is re-valued by researchers after being ignored for nearly half a century. Phage therapy has made great achievements in prevention and control of bacterial infection of open wounds. This review is mainly focused on the latest research progress of phage therapy in wound bacterial infection.

  8. Review: phage therapy: a modern tool to control bacterial infections.

    PubMed

    Qadir, Muhammad Imran

    2015-01-01

    The evolution of antibiotic-resistant in bacteria has aggravated curiosity in development of alternative therapy to conventional drugs. One of the emerging drugs that can be used alternative to antibiotics is bacteriophage therapy. The use of living phages in the cure of lethal infectious life threatening diseases caused by Gram positive and Gram negative bacteria has been reported. Another development in the field of bacteriophage therapy is the use of genetically modified and non replicating phages in the treatment of bacterial infection. Genetically engineered bacteriophages can be used as adjuvant along with antibiotic therapy. Phages encoded with lysosomal enzymes are also effectual in the treatment of infectious diseases.

  9. Phage display library screening for identification of interacting protein partners.

    PubMed

    Addepalli, Balasubrahmanyam; Rao, Suryadevara; Hunt, Arthur G

    2015-01-01

    Phage display is a versatile high-throughput screening method employed to understand and improve the chemical biology, be it production of human monoclonal antibodies or identification of interacting protein partners. A majority of cell proteins operate in a concerted fashion either by stable or transient interactions. Such interactions can be mediated by recognition of small amino acid sequence motifs on the protein surface. Phage display can play a crucial role in identification of such motifs. This report describes the use of phage display for the identification of high affinity sequence motifs that could be responsible for interactions with a target (bait) protein.

  10. Introduction of Pseudomonas aeruginosa mutator phage D3112 into Alcaligenes eutrophus strain CH34.

    PubMed

    Krylov, V; Merlin, C; Toussaint, A

    1995-01-01

    We have investigated the possibility of growing mutator phages from Pseudomonas aeruginosa on various isolates of Alcaligenes eutrophus. Although none out of 10 A. eutrophus strains were susceptible to infection with any of the phages tested, phage D3112 could be readily transferred in our model strain CH34 by means of an RP4::D3112 plasmid. CH34/RP4::D3112 lysogens were stable and produced phages. However, neither mitomycin C nor UV treatment increased the phage yield.

  11. Core Lipopolysaccharide-Specific Phage SSU5 as an Auxiliary Component of a Phage Cocktail for Salmonella Biocontrol

    PubMed Central

    Kim, Minsik; Kim, Sujin; Park, Bookyung

    2014-01-01

    Salmonella spp. are among the major food-borne pathogens that cause mild diarrhea to severe bacteremia. The use of bacteriophages to control various food-borne pathogens, including Salmonella, has emerged as a promising alternative to traditional chemotherapy. We isolated the Siphoviridae family phage SSU5, which can infect only rough strains of Salmonella. The blocking of SSU5 adsorption by periodate treatment of host Salmonella cells and spotting and adsorption assays with mutants that contain various truncations in their lipopolysaccharide (LPS) cores revealed that the outer core region of the LPS is a receptor of SSU5. SSU5 could infect O-antigen (O-Ag)-deficient Salmonella mutants that developed by challenging of O-Ag-specific phages, and consequently, it delayed the emergence of the phage-resistant Salmonella population in broth culture when treated together with phages using O-Ag as a receptor. Therefore, these results suggested that phage SSU5 would be a promising auxiliary component of a phage cocktail to control rough strains of Salmonella enterica serovar Typhimurium, which might emerge as resistant mutants upon infection by phages using O-Ag as a receptor. PMID:24271179

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

    PubMed

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

    2014-01-01

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

  13. Amplification and Purification of T4-Like Escherichia coli Phages for Phage Therapy: from Laboratory to Pilot Scale

    PubMed Central

    Bourdin, Gilles; Schmitt, Bertrand; Marvin Guy, Laure; Germond, Jacques-Edouard; Zuber, Sophie; Michot, Lise; Reuteler, Gloria

    2014-01-01

    We investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 109 to 1010 PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-μm membrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10 mM magnesium ions, phages showed no loss of titer over 1 month at 30°C. PMID:24362424

  14. Phage-based nanomaterials for biomedical applications.

    PubMed

    Farr, Rebecca; Choi, Dong Shin; Lee, Seung-Wuk

    2014-04-01

    Recent advances in nanotechnology enable us to manipulate and produce materials with molecular level control. In the newly emerging field of bionanomedicine, it is essential to precisely control the physical, chemical and biological properties of materials. Among other biological building blocks, viruses are a promising nanomaterial that can be functionalized with great precision. Since the production of viral particles is directed by the genetic information encapsulated in their protein shells, the viral particles create precisely defined sizes and shapes. In addition, the composition and surface properties of the particles can be controlled through genetic engineering and chemical modification. In this manuscript, we review the advances of virus-based nanomaterials for biomedical applications in three different areas: phage therapy, drug delivery and tissue engineering. By exploiting and manipulating the original functions of viruses, viral particles hold great possibilities in these biomedical applications to improve human health.

  15. Interactions between phage-shock proteins in Escherichia coli.

    PubMed

    Adams, Hendrik; Teertstra, Wieke; Demmers, Jeroen; Boesten, Rolf; Tommassen, Jan

    2003-02-01

    Expression of the pspABCDE operon of Escherichia coli is induced upon infection by filamentous phage and by many other stress conditions, including defects in protein export. Expression of the operon requires the alternative sigma factor sigma54 and the transcriptional activator PspF. In addition, PspA plays a negative regulatory role, and the integral-membrane proteins PspB and PspC play a positive one. In this study, we investigated whether the suggested protein-protein interactions implicated in this complex regulatory network can indeed be demonstrated. Antisera were raised against PspB, PspC, and PspD, which revealed, in Western blotting experiments, that PspC forms stable sodium dodecyl sulfate-resistant dimers and that the hypothetical pspD gene is indeed expressed in vivo. Fractionation experiments showed that PspD localizes as a peripherally bound inner membrane protein. Cross-linking studies with intact cells revealed specific interactions of PspA with PspB and PspC, but not with PspD. Furthermore, affinity-chromatography suggested that PspB could bind PspA only in the presence of PspC. These data indicate that regulation of the psp operon is mediated via protein-protein interactions.

  16. Proposed Ancestors of Phage Nucleic Acid Packaging Motors (and Cells)

    PubMed Central

    Serwer, Philip

    2011-01-01

    I present a hypothesis that begins with the proposal that abiotic ancestors of phage RNA and DNA packaging systems (and cells) include mobile shells with an internal, molecule-transporting cavity. The foundations of this hypothesis include the conjecture that current nucleic acid packaging systems have imprints from abiotic ancestors. The abiotic shells (1) initially imbibe and later also bind and transport organic molecules, thereby providing a means for producing molecular interactions that are links in the chain of events that produces ancestors to the first molecules that are both information carrying and enzymatically active, and (2) are subsequently scaffolds on which proteins assemble to form ancestors common to both shells of viral capsids and cell membranes. Emergence of cells occurs via aggregation and merger of shells and internal contents. The hypothesis continues by using proposed imprints of abiotic and biotic ancestors to deduce an ancestral thermal ratchet-based DNA packaging motor that subsequently evolves to integrate a DNA packaging ATPase that provides a power stroke. PMID:21994778

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

    DOEpatents

    Bradbury, Andrew M.

    2012-07-24

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

  18. Metagenomic recovery of phage genomes of uncultured freshwater actinobacteria.

    PubMed

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

    2017-01-01

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

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

    PubMed Central

    Tan, Wen Siang; Ho, Kok Lian

    2014-01-01

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

  20. Recognition of Salmonella typhimurium by immobilized phage P22 monolayers

    NASA Astrophysics Data System (ADS)

    Handa, Hitesh; Gurczynski, Stephen; Jackson, Matthew P.; Auner, Gregory; Walker, Jeremy; Mao, Guangzhao

    2008-04-01

    Phages are promising alternatives to antibodies as the biorecognition element in a variety of biosensing applications. In this study, a monolayer of bacteriophage P22 whose tailspike proteins specifically recognize Salmonella serotypes was covalently bound to glass substrates through a bifunctional cross linker 3-aminopropyltrimethoxysilane. The specific binding of Salmonella typhimurium to the phage monolayer was studied by enzyme-linked immunosorbent assay and atomic force microscopy. Escherichia coli and a Gram-positive bacterium Listeria monocytogenes were also studied as control bacteria. The P22 particles show strong binding affinity to S. typhimurium. In addition, the dried P22 monolayer maintained 50% binding capacity to S. typhimurium after a one-week storage time. This is a promising method to prepare phage monolayer coatings on surface plasmon resonance and acoustic biosensor substrates in order to utilize the nascent phage display technology.

  1. Deep sequencing analysis of phage libraries using Illumina platform.

    PubMed

    Matochko, Wadim L; Chu, Kiki; Jin, Bingjie; Lee, Sam W; Whitesides, George M; Derda, Ratmir

    2012-09-01

    This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10(7) reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5'-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2×10(7) reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6bp), one complimentary region (12bp) and a variable region (36bp). We applied this sequencing to a model library of 10(6) unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve

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

    PubMed

    Sørensen, Martine C Holst; Gencay, Yilmaz Emre; Birk, Tina; Baldvinsson, Signe Berg; Jäckel, Claudia; Hammerl, Jens A; Vegge, Christina S; Neve, Horst; Brøndsted, Lone

    2015-01-01

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

  3. Properties of Klebsiella phage P13 and associated exopolysaccharide depolymerase

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Li, Guiyang; Mo, Zhaolan; Chai, Zihan; Shang, Anqi; Mou, Haijin

    2013-11-01

    The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide (EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL-1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL-1 in 60 min. Accordingly, the enzyme activity of depolymerase approached the maximum 60 min after infection. Treatment at 70°C for 30 min inactivated all the phage, but retained over 90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60°C and pH 6.5. Transmission electron microscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a doublestrand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation including thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.

  4. Phage-Coupled Piezoelectric Biodetector for Salmonella Typhimurium

    DTIC Science & Technology

    2005-08-01

    confirm phage specificity and selectivity. The phage was up to 22,000 times more specific for S. iv DISTRBUTIOM! ST•TAT7r T A Approved for Public...unit with holder and crystals attached. (5) AFC unit. (6) DFC unit. (7) Data processing unit. (8) External control keyboard for film control mechanics...immobilized to the surface of a sensor by physical adsorption; cf. Fig. 5.37. Magnification, x 3000 ; bar = 5 9m

  5. Phage therapy against Enterococcus faecalis in dental root canals

    PubMed Central

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

    2016-01-01

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

  6. The Legacy of 20th Century Phage Research.

    PubMed

    Campbell, Allan M

    2010-09-01

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

  7. The genome of the Lactobacillus sanfranciscensis temperate phage EV3

    PubMed Central

    2013-01-01

    Background Bacteriophages infection modulates microbial consortia and transduction is one of the most important mechanism involved in the bacterial evolution. However, phage contamination brings food fermentations to a halt causing economic setbacks. The number of phage genome sequences of lactic acid bacteria especially of lactobacilli is still limited. We analysed the genome of a temperate phage active on Lactobacillus sanfranciscensis, the predominant strain in type I sourdough fermentations. Results Sequencing of the DNA of EV3 phage revealed a genome of 34,834 bp and a G + C content of 36.45%. Of the 43 open reading frames (ORFs) identified, all but eight shared homology with other phages of lactobacilli. A similar genomic organization and mosaic pattern of identities align EV3 with the closely related Lactobacillus vaginalis ATCC 49540 prophage. Four unknown ORFs that had no homologies in the databases or predicted functions were identified. Notably, EV3 encodes a putative dextranase. Conclusions EV3 is the first L. sanfranciscensis phage that has been completely sequenced so far. PMID:24308641

  8. Phage phenomics: Physiological approaches to characterize novel viral proteins

    ScienceCinema

    Sanchez, Savannah E. [San Diego State Univ., San Diego, CA (United States); Cuevas, Daniel A. [San Diego State Univ., San Diego, CA (United States); Rostron, Jason E. [San Diego State Univ., San Diego, CA (United States); Liang, Tiffany Y. [San Diego State Univ., San Diego, CA (United States); Pivaroff, Cullen G. [San Diego State Univ., San Diego, CA (United States); Haynes, Matthew R. [San Diego State Univ., San Diego, CA (United States); Nulton, Jim [San Diego State Univ., San Diego, CA (United States); Felts, Ben [San Diego State Univ., San Diego, CA (United States); Bailey, Barbara A. [San Diego State Univ., San Diego, CA (United States); Salamon, Peter [San Diego State Univ., San Diego, CA (United States); Edwards, Robert A. [San Diego State Univ., San Diego, CA (United States); Argonne National Lab. (ANL), Argonne, IL (United States); Burgin, Alex B. [Broad Institute, Cambridge, MA (United States); Segall, Anca M. [San Diego State Univ., San Diego, CA (United States); Rohwer, Forest [San Diego State Univ., San Diego, CA (United States)

    2016-07-12

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

  9. Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery

    PubMed Central

    Yata, Teerapong; Lee, Koon-Yang; Dharakul, Tararaj; Songsivilai, Sirirurg; Bismarck, Alexander; Mintz, Paul J; Hajitou, Amin

    2014-01-01

    Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage), viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage. PMID:25118171

  10. Phage phenomics: Physiological approaches to characterize novel viral proteins

    SciTech Connect

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; Liang, Tiffany Y.; Pivaroff, Cullen G.; Haynes, Matthew R.; Nulton, Jim; Felts, Ben; Bailey, Barbara A.; Salamon, Peter; Edwards, Robert A.; Burgin, Alex B.; Segall, Anca M.; Rohwer, Forest

    2015-06-11

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

  11. Phage phenomics: Physiological approaches to characterize novel viral proteins

    DOE PAGES

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; ...

    2015-06-11

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysismore » by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.« less

  12. Phage Display of a Biologically Active Bacillus thuringiensis Toxin

    PubMed Central

    Kasman, Laura M.; Lukowiak, Andrew A.; Garczynski, Stephen F.; McNall, Rebecca J.; Youngman, Phil; Adang, Michael J.

    1998-01-01

    Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning. PMID:9687463

  13. RECOMBINATIONS OF MUTANT PHAGES OF BACILLUS MEGATHERIUM 899A

    PubMed Central

    Murphy, James S.

    1953-01-01

    A group of mutant phages stemming from the virus of B. megatherium 899a (lysogenic), growing on a sensitive B. megatherium strain (KM), have been studied with respect to their recombination reactions. All these mutants and many of their recombinations can be recognized by a characteristic plaque morphology. A similar group of phages have been isolated directly from a culture of B. megatherium 899a in this laboratory. Previous work has shown that when two different plaque mutant phages both infect essentially all the bacteria in a culture, a characteristic per cent of recombinants is produced. This percentage depends on the two recombinants used, each pair having its own value. Hershey and coworkers (2–5) have demonstrated with coli-phage T2, that the percentages of recombination found can be handled mathematically and that they demonstrate the existence of a relationship between the mutations entirely comparable to crossover percentages as used in gene locus maps in genetics. This has been found to hold true for the phages studied in the present work. Only one "linkage group" has been detected and all the mutants studied showed low percentages of recombination (0.8 to 7.6). B. megatherium 899a phage and some of its mutants have been examined with an electron microscope and no differences have been detected between the different mutant strains. PMID:13109115

  14. Phage therapy against Enterococcus faecalis in dental root canals.

    PubMed

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

    2016-01-01

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

  15. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa.

    PubMed

    Chan, Benjamin K; Sistrom, Mark; Wertz, John E; Kortright, Kaitlyn E; Narayan, Deepak; Turner, Paul E

    2016-05-26

    Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections.

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

    PubMed

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

    2015-05-01

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

  17. Influence of Stress Factors Related to Cheese-Making Process and to STEC Detection Procedure on the Induction of Stx Phages from STEC O26:H11

    PubMed Central

    Bonanno, Ludivine; Delubac, Benjamin; Michel, Valérie; Auvray, Frédéric

    2017-01-01

    Shiga toxin-producing Escherichia coli (STEC) are responsible for human infections, ranging from mild watery diarrhea to hemorrhagic colitis (CH) that may be complicated by hemolytic uremic syndrome (HUS). The main STEC virulence factor is Shiga toxin encoded by the stx gene, located in the genome of a bacteriophage integrated into the bacterial chromosome. The serotype O26:H11 is the second HUS-causing serotype worldwide (after O157:H7), and the first found in dairy products such as raw-milk cheeses. A small number of HUS cases identified each year in France are caused by serotype O26:H11. Stx phage induction is known to result in STEC lysis and release of new Stx phages particles. This phenomenon could negatively impact STEC screening in foods based on stx gene detection by PCR. Here, we evaluated the influence of physicochemical parameters related to cheese-making process on the induction rate of Stx phages from STEC O26:H11, including H2O2, NaCl, lactic acid and temperature. In addition, selective agents from the analytical STEC enrichment and detection procedure (XP CEN ISO/TS 13136) were tested, including novobiocin, acrifavin, cefixim-tellurite, and bile salts. An impact of H2O2 and NaCl on Stx phage induction was observed. Production of Stx phages was also observed during a real cheese-making process. By contrast, no significant effect could be demonstrated for the chemical agents of the STEC detection procedure when tested separately, except for acriflavin and novobiocin which reduced Stx1 phage production in some cases. In conclusion, these results suggest that the cheese-making process might trigger the production of Stx phages, potentially interfering with the analysis of STEC in food. PMID:28316592

  18. Evolutionary consequences of intra-patient phage predation on microbial populations.

    PubMed

    Seed, Kimberley D; Yen, Minmin; Shapiro, B Jesse; Hilaire, Isabelle J; Charles, Richelle C; Teng, Jessica E; Ivers, Louise C; Boncy, Jacques; Harris, Jason B; Camilli, Andrew

    2014-08-26

    The impact of phage predation on bacterial pathogens in the context of human disease is not currently appreciated. Here, we show that predatory interactions of a phage with an important environmentally transmitted pathogen, Vibrio cholerae, can modulate the evolutionary trajectory of this pathogen during the natural course of infection within individual patients. We analyzed geographically and temporally disparate cholera patient stool samples from Haiti and Bangladesh and found that phage predation can drive the genomic diversity of intra-patient V. cholerae populations. Intra-patient phage-sensitive and phage-resistant isolates were isogenic except for mutations conferring phage resistance, and moreover, phage-resistant V. cholerae populations were composed of a heterogeneous mix of many unique mutants. We also observed that phage predation can significantly alter the virulence potential of V. cholerae shed from cholera patients. We provide the first molecular evidence for predatory phage shaping microbial community structure during the natural course of infection in humans.

  19. Diversity of Streptococcus thermophilus phages in a large-production cheese factory in Argentina.

    PubMed

    Quiberoni, A; Tremblay, D; Ackermann, H-W; Moineau, S; Reinheimer, J A

    2006-10-01

    Phage infections still represent a serious risk to the dairy industry, in which Streptococcus thermophilus is used in starter cultures for the manufacture of yogurt and cheese. The goal of the present study was to analyze the biodiversity of the virulent S. thermophilus phage population in one Argentinean cheese plant. Ten distinct S. thermophilus phages were isolated from cheese whey samples collected in a 2-mo survey. They were then characterized by their morphology, host range, and restriction patterns. These phages were also classified within the 2 main groups of S. thermophilus phages (cos- and pac-type) using a newly adapted multiplex PCR method. Six phages were classified as cos-type phages, whereas the 4 others belonged to the pac-type group. This study illustrates the phage diversity that can be found in one factory that rotates several cultures of S. thermophilus. Limiting the number of starter cultures is likely to reduce phage biodiversity within a fermentation facility.

  20. Baseplate assembly of phage Mu: Defining the conserved core components of contractile-tailed phages and related bacterial systems

    PubMed Central

    Büttner, Carina R.; Wu, Yingzhou; Maxwell, Karen L.; Davidson, Alan R.

    2016-01-01

    Contractile phage tails are powerful cell puncturing nanomachines that have been co-opted by bacteria for self-defense against both bacteria and eukaryotic cells. The tail of phage T4 has long served as the paradigm for understanding contractile tail-like systems despite its greater complexity compared with other contractile-tailed phages. Here, we present a detailed investigation of the assembly of a “simple” contractile-tailed phage baseplate, that of Escherichia coli phage Mu. By coexpressing various combinations of putative Mu baseplate proteins, we defined the required components of this baseplate and delineated its assembly pathway. We show that the Mu baseplate is constructed through the independent assembly of wedges that are organized around a central hub complex. The Mu wedges are comprised of only three protein subunits rather than the seven found in the equivalent structure in T4. Through extensive bioinformatic analyses, we found that homologs of the essential components of the Mu baseplate can be identified in the majority of contractile-tailed phages and prophages. No T4-like prophages were identified. The conserved simple baseplate components were also found in contractile tail-derived bacterial apparatuses, such as type VI secretion systems, Photorhabdus virulence cassettes, and R-type tailocins. Our work highlights the evolutionary connections and similarities in the biochemical behavior of phage Mu wedge components and the TssF and TssG proteins of the type VI secretion system. In addition, we demonstrate the importance of the Mu baseplate as a model system for understanding bacterial phage tail-derived systems. PMID:27555589

  1. Baseplate assembly of phage Mu: Defining the conserved core components of contractile-tailed phages and related bacterial systems.

    PubMed

    Büttner, Carina R; Wu, Yingzhou; Maxwell, Karen L; Davidson, Alan R

    2016-09-06

    Contractile phage tails are powerful cell puncturing nanomachines that have been co-opted by bacteria for self-defense against both bacteria and eukaryotic cells. The tail of phage T4 has long served as the paradigm for understanding contractile tail-like systems despite its greater complexity compared with other contractile-tailed phages. Here, we present a detailed investigation of the assembly of a "simple" contractile-tailed phage baseplate, that of Escherichia coli phage Mu. By coexpressing various combinations of putative Mu baseplate proteins, we defined the required components of this baseplate and delineated its assembly pathway. We show that the Mu baseplate is constructed through the independent assembly of wedges that are organized around a central hub complex. The Mu wedges are comprised of only three protein subunits rather than the seven found in the equivalent structure in T4. Through extensive bioinformatic analyses, we found that homologs of the essential components of the Mu baseplate can be identified in the majority of contractile-tailed phages and prophages. No T4-like prophages were identified. The conserved simple baseplate components were also found in contractile tail-derived bacterial apparatuses, such as type VI secretion systems, Photorhabdus virulence cassettes, and R-type tailocins. Our work highlights the evolutionary connections and similarities in the biochemical behavior of phage Mu wedge components and the TssF and TssG proteins of the type VI secretion system. In addition, we demonstrate the importance of the Mu baseplate as a model system for understanding bacterial phage tail-derived systems.

  2. Effects of surface functionalization on the surface phage coverage and the subsequent performance of phage-immobilized magnetoelastic biosensors.

    PubMed

    Horikawa, Shin; Bedi, Deepa; Li, Suiqiong; Shen, Wen; Huang, Shichu; Chen, I-Hsuan; Chai, Yating; Auad, Maria L; Bozack, Michael J; Barbaree, James M; Petrenko, Valery A; Chin, Bryan A

    2011-01-15

    One of the important applications for which phage-immobilized magnetoelastic (ME) biosensors are being developed is the wireless, on-site detection of pathogenic bacteria for food safety and bio-security. Until now, such biosensors have been constructed by immobilizing a landscape phage probe on gold-coated ME resonators via physical adsorption. Although the physical adsorption method is simple, the immobilization stability and surface coverage of phage probes on differently functionalized sensor surfaces need to be evaluated as a potential way to enhance the detection capabilities of the biosensors. As a model study, a filamentous fd-tet phage that specifically binds streptavidin was adsorbed on either bare or surface-functionalized gold-coated ME resonators. The surface functionalization was performed through the formation of three self-assembled monolayers with a different terminator, based on the sulfur-gold chemistry: AC (activated carboxy-terminated), ALD (aldehyde-terminated), and MT (methyl-terminated). The results, obtained by atomic force microscopy, showed that surface functionalization has a large effect on the surface phage coverage (46.8%, 49.4%, 4.2%, and 5.2% for bare, AC-, ALD-, and MT-functionalized resonators, respectively). In addition, a direct correlation of the observed surface phage coverage with the quantity of subsequently captured streptavidin-coated microbeads was found by scanning electron microscopy and by resonance frequency measurements of the biosensors. The differences in surface phage coverage on the differently functionalized surfaces may then be used to pattern the phage probe layer onto desired parts of the sensor surface to enhance the detection capabilities of ME biosensors.

  3. Isolation of phages for phage therapy: a comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy.

    PubMed

    Khan Mirzaei, Mohammadali; Nilsson, Anders S

    2015-01-01

    Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.

  4. Phage P4 DNA replication in vitro.

    PubMed Central

    Díaz Orejas, R; Ziegelin, G; Lurz, R; Lanka, E

    1994-01-01

    Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 alpha protein [Krevolin and Calendar (1985), J. Mol. Biol. 182, 507-517]. Using a modified in vitro replication assay, we have further characterized this process. Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product. Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo. Replication proceeds from this origin bidirectionally, resulting in theta-type molecules. In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates. The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process. The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase III holoenzyme. Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase. Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate. Images PMID:8029013

  5. 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.

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

    PubMed

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

    2014-01-01

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

  7. Bacteriophage exploitation of bacterial biofilms: phage preference for less mature targets?

    PubMed

    Abedon, Stephen T

    2016-02-01

    Robust evidence is somewhat lacking for biofilm susceptibility to bacteriophages in nature, contrasting often substantial laboratory biofilm vulnerability to phages. To help bridge this divide, I review a two-part scenario for 'heterogeneous' phage interaction even with phage-permissive single-species biofilms. First, through various mechanisms, those bacteria which are both more newly formed and located at biofilm surfaces may be particularly vulnerable to phage adsorption, rather than biofilm matrix being homogeneously resistant to phage penetration. Second, though phage infection of older, less metabolically active bacteria may still be virion productive, nevertheless the majority of phage population growth in association with biofilm bacteria could involve infection particularly of those bacteria which are more metabolically active and thereby better able to support larger phage bursts, versus clonally related biofilm bacteria equivalently supporting phage production. To the extent that biofilms are physiologically or structurally heterogeneous, with phages exploiting particularly relatively newly divided biofilm-surface bacteria, then even effective phage predation of natural biofilms could result in less than complete overall biofilm clearance. Phage tendencies toward only partial exploitation of even single-species biofilms could be consistent with observations that chronic bacterial infections in the clinic can require more aggressive or extensive phage therapy to eradicate.

  8. Proteomic Analysis of a Novel Bacillus Jumbo Phage Revealing Glycoside Hydrolase As Structural Component

    PubMed Central

    Yuan, Yihui; Gao, Meiying

    2016-01-01

    Tailed phages with genomes of larger than 200 kbp are classified as Jumbo phages and exhibited extremely high uncharted diversity. The genomic annotation of Jumbo phage is often disappointing because most of the predicted proteins, including structural proteins, failed to make good hits to the sequences in the databases. In this study, 23 proteins of a novel Bacillus Jumbo phage, vB_BpuM_BpSp, were identified as phage structural proteins by the structural proteome analysis, including 14 proteins of unknown function, 5 proteins with predicted function as structural proteins, a glycoside hydrolase, a Holliday junction resolvase, a RNA-polymerase β-subunit, and a host-coding portal protein, which might be hijacked from the host strain during phage virion assembly. The glycoside hydrolase (Gp255) was identified as phage virion component and was found to interact with the phage baseplate protein. Gp255 shows specific lytic activity against the phage host strain GR8 and has high temperature tolerance. In situ peptidoglycan-hydrolyzing activities analysis revealed that the expressed Gp255 and phage structural proteome exhibited glycoside hydrolysis activity against the tested GR8 cell extracts. This study identified the first functional individual structural glycoside hydrolase in phage virion. The presence of activated glycoside hydrolase in phage virions might facilitate the injection of the phage genome during infection by forming pores on the bacterial cell wall. PMID:27242758

  9. Impact of a single phage and a phage cocktail application in broilers on reduction of Campylobacter jejuni and development of resistance.

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  11. Ricin Detection Using Phage Displayed Single Domain Antibodies

    PubMed Central

    Goldman, Ellen R.; Liu, Jinny L.; Bernstein, Rachael D.; Swain, Marla D.; Mitchell, Stanley Q.; Anderson, George P.

    2009-01-01

    Phage-displayed single domain antibodies (sdAb) were compared to monomeric solubly expressed sdAb and llama polyclonal antibodies for the detection of ricin. SdAb are comprised of the variable domain derived from camelid heavy chain only antibodies (HcAb). Although HcAb lack variable light chains, they as well as their derivative sdAb are able to bind antigens with high affinity. The small size of sdAb (∼16 kDa), while advantageous in many respects, limits the number of labels that can be incorporated. The ability to incorporate multiple labels is a beneficial attribute for reporter elements. Opportunely, sdAb are often selected using phage display methodology. Using sdAb displayed on bacteriophage M13 as the reporter element gives the potential for incorporating a very high number of labels. We have demonstrated the use of both sdAb and phage- displayed sdAb for the detection of ricin using both enzyme linked immunosorbent assays (ELISAs) and Luminex fluid array assays. The phage-displayed sdAb led to five to ten fold better detection of ricin in both the ELISA and Luminex assays, resulting in limits of detection of 1 ng/mL and 64 pg/mL respectively. The phage-displayed sdAb were also dramatically more effective for the visualization of binding to target in nitrocellulose dot blot assays, a method frequently used for epitope mapping. PMID:22389616

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

    PubMed

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

    2015-08-01

    The compositional stability of the complex Gouda cheese starter culture Ur is thought to be influenced by diversity in phage resistance of highly related strains that co-exist together with bacteriophages. To analyze the role of bacteriophages in maintaining culture diversity at the level of genetic lineages, simple blends of Lactococcus lactis strains were made and subsequently propagated for 152 generations in the absence and presence of selected bacteriophages. We first screened 102 single-colony isolates (strains) from the complex cheese starter for resistance to bacteriophages isolated from this starter. The collection of isolates represents all lactococcal genetic lineages present in the culture. Large differences were found in bacteriophage resistance among strains belonging to the same genetic lineage and among strains from different lineages. The blends of strains were designed such that 3 genetic lineages were represented by strains with different levels of phage resistance. The relative abundance of the lineages in blends with phages was not stable throughout propagation, leading to continuous changes in composition up to 152 generations. The individual resistance of strains to phage predation was confirmed as one of the factors influencing starter culture diversity. Furthermore, loss of proteolytic activity of initially proteolytic strains was found. Reconstituted blends with only 4 strains with a variable degree of phage resistance showed complex behavior during prolonged propagation.

  13. Phage display selection and evaluation of cancer drug targets.

    PubMed

    Romanov, Victor I

    2003-04-01

    Techniques for the construction of phage display libraries of combinatorial proteins have dramatically improved. This has allowed researchers to expand the applications to the field of cancer biology. The most direct use of protein phage-displayed libraries is the selection of ligands for individual proteins. This includes identification of peptide ligands for receptor signaling molecules: integrins, cytokine and growth factor receptors. Selected peptides may be used as competitors for natural ligands and for the mapping of binding epitopes. This approach has been exploited for delineation of intracellular signal transduction pathways and for the selection of enzyme substrates and inhibitors. Recently, more complicated biological systems were used as targets for biopanning. This includes combination of soluble proteins, cellular surfaces and even the vasculature of whole organs. cDNA expression libraries in phage-based vectors have been recently introduced. The use of phage as a vector for targeted gene therapy is also considered. These and other applications of phage display for cancer research will be reviewed.

  14. Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage in Streptococcus thermophilus.

    PubMed

    Wei, Yunzhou; Chesne, Megan T; Terns, Rebecca M; Terns, Michael P

    2015-02-18

    CRISPR-Cas systems are RNA-based immune systems that protect prokaryotes from invaders such as phages and plasmids. In adaptation, the initial phase of the immune response, short foreign DNA fragments are captured and integrated into host CRISPR loci to provide heritable defense against encountered foreign nucleic acids. Each CRISPR contains a ∼100-500 bp leader element that typically includes a transcription promoter, followed by an array of captured ∼35 bp sequences (spacers) sandwiched between copies of an identical ∼35 bp direct repeat sequence. New spacers are added immediately downstream of the leader. Here, we have analyzed adaptation to phage infection in Streptococcus thermophilus at the CRISPR1 locus to identify cis-acting elements essential for the process. We show that the leader and a single repeat of the CRISPR locus are sufficient for adaptation in this system. Moreover, we identified a leader sequence element capable of stimulating adaptation at a dormant repeat. We found that sequences within 10 bp of the site of integration, in both the leader and repeat of the CRISPR, are required for the process. Our results indicate that information at the CRISPR leader-repeat junction is critical for adaptation in this Type II-A system and likely other CRISPR-Cas systems.

  15. Bacteriophages of Pseudomonas aeruginosa: long-term prospects for use in phage therapy.

    PubMed

    Krylov, Victor N

    2014-01-01

    Bacteria Pseudomonas aeruginosa, being opportunistic pathogens, are the major cause of nosocomial infections and, in some cases, the primary cause of death. They are virtually untreatable with currently known antibiotics. Phage therapy is considered as one of the possible approaches to the treatment of P. aeruginosa infections. Difficulties in the implementation of phage therapy in medical practice are related, for example, to the insufficient number and diversity of virulent phages that are active against P. aeruginosa. Results of interaction of therapeutic phages with bacteria in different conditions and environments are studied insufficiently. A little is known about possible interactions of therapeutic phages with resident prophages and plasmids in clinical strains in the foci of infections. This chapter highlights the different approaches to solving these problems and possible ways to expand the diversity of therapeutic P. aeruginosa phages and organizational arrangements (as banks of phages) to ensure long-term use of phages in the treatment of P. aeruginosa infections.

  16. 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.

  17. Phages Bearing Affinity Peptides to Bovine Rotavirus Differentiate the Virus from Other Viruses

    PubMed Central

    Wang, Xin; Li, Guangxing; Ren, Yudong; Ren, Xiaofeng

    2011-01-01

    The aim of this study was to identify potential ligands and develop a novel diagnostic test to pathogenic bovine rotavirus (BRV) using phage display technology. The viruses were used as an immobilized target followed by incubation with a 12-mer phage display random peptide library. After five rounds of biopanning, phages had a specific binding activity to BRV were isolated. DNA sequencing indicated that phage displayed peptides HVHPPLRPHSDK, HATNHLPTPHNR or YPTHHAHTTPVR were potential ligands to BRV. Using the specific peptide-expressing phages, we developed a phage-based ELISA to differentiate BRV from other viruses. Compared with quantitative real-time PCR (qPCR), the phage-mediated ELISA was more suitable for the capture of BRV and the detection limitation of this approach was 0.1 µg/ml of samples. The high sensitivity, specificity and low cross-reactivity for the phage-based ELISA were confirmed in receiver operating characteristics (ROC) analysis. PMID:22163050

  18. 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

  19. 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.

  20. Characterization of phage receptors in Streptococcus thermophilus using purified cell walls obtained by a simple protocol.

    PubMed

    Quiberoni, A; Stiefel, J I; Reinheimer, J A

    2000-12-01

    A simple protocol was designed and applied to obtain Streptococcus thermophilus purified cell walls. To identify the structures involved in phage adsorption, the cell walls of two Strep. thermophilus strains were treated with sodium dodecyl sulphate and proteinase K. These treatments did not reduce the adsorption of phages CYM and 0BJ to the cell walls of Strep. thermophilus YSD10 and Strep. thermophilus BJ15, respectively. However, phage binding was reduced when the cell envelopes were treated with mutanolysin or trichloroacetic acid 5%, suggesting that the phage receptor component is part of the peptidoglycan or a polymer closely linked to it. The ability of several saccharides to inactivate both phages was also assayed. These phage inhibition experiments suggested that the phage CYM adsorbed to a component involving glucosamine and rhamnose, while glucosamine and ribose interfered with the adsorption of phage 0BJ.

  1. Recent findings about the Yersinia enterocolitica phage shock protein response.

    PubMed

    Yamaguchi, Saori; Darwin, Andrew J

    2012-02-01

    The phage shock protein (Psp) system is a conserved extracytoplasmic stress response in bacteria that is essential for virulence of the human pathogen Yersinia enterocolitica. This article summarizes some recent findings about Y. enterocolitica Psp system function. Increased psp gene expression requires the transcription factor PspF, but under non-inducing conditions PspF is inhibited by an interaction with another protein, PspA, in the cytoplasm. A Psp-inducing stimulus causes PspA to relocate to the cytoplasmic membrane, freeing PspF to induce psp gene expression. This PspA relocation requires the integral cytoplasmic membrane proteins, PspB and PspC, which might sense an inducing trigger and sequester PspA by direct interaction. The subsequent induction of psp gene expression increases the PspA concentration, which also allows it to contact the membrane directly, perhaps for its physiological function. Mutational analysis of the PspB and PspC proteins has revealed that they both positively and negatively regulate psp gene expression and has also identified PspC domains associated with each function. We also compare the contrasting physiological roles of the Psp system in the virulence of Y. enterocolitica and Salmonella enterica sv. Typhimurium (S. Typhimurium). In S. Typhimurium, PspA maintains the proton motive force, which provides the energy needed to drive ion importers required for survival within macrophages. In contrast, in the extracellular pathogen Y. enterocolitica, PspB and PspC, but not PspA, are the Psp components needed for virulence. PspBC protect Y. enterocolitica from damage caused by the secretin component of its type 3 secretion system, an essential virulence factor.

  2. Nano-magnetic immunosensor based on staphylococcus protein a and the amplification effect of HRP-conjugated phage antibody.

    PubMed

    Mu, Xihui; Tong, Zhaoyang; Huang, Qibin; Liu, Bing; Liu, Zhiwei; Hao, Lanqun; Zhang, Jinping; Gao, Chuan; Wang, Fenwei

    2015-02-09

    In this research, super-paramagnetic Fe3O4 nanoparticles (magnetic particles) were coated with Staphylococcus protein A (SPA) and coupled with polyclonal antibody (pcAb) to construct magnetic capturing probes, and HRP-conjugated phage antibody was then used as specific detecting probe to design a labeled immunosensor for trace detection of Staphylococcus aureus enterotoxin B (SEB). The linear detection range of the sensor was 0.008~125 µg/L, the regression equation was Y = 0.487X + 1.2 (R = 0.996, N = 15, p < 0.0001), the limit of detection (LOD) was 0.008 µg/L, and the limit of quantification (LOQ) was 0.008 µg/L. HRP-conjugated phage antibody, SPA and magnetic particles can enhance the sensitivity 4-fold, 3-fold and 2.6-fold higher, respectively. Compared with conventional double-antibody sandwich ELISA, the detection sensitivity of the sensor was 31-fold higher resulting from the integrated amplifying effect. The immunosensor integrates the unique advantages of SPA-oriented antibody as magnetic capturing probe, HRP-conjugated phage antibody as detecting probe, magnetic separation immunoassay technique, and several other advanced techniques, so it achieves high sensitivity, specificity and interference-resistance. It is proven to be well suited for analysis of trace SEB in various environmental samples with high recovery rate and reproducibility.

  3. Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly

    PubMed Central

    Benítez Quintana, Andrea Denisse; Bosch, Martine A.; Coll De Peña, Adriana; Aguilera, Elizabeth; Coulibaly, Assitan; Wu, Weimin; Osier, Michael V.; Hudson, André O.; Weintraub, Susan T.; Black, Lindsay W.

    2016-01-01

    ABSTRACT Giant tailed bacterial viruses, or phages, such as Pseudomonas aeruginosa phage ϕKZ, have long genomes packaged into large, atypical virions. Many aspects of ϕKZ and related phage biology are poorly understood, mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240-kb SPN3US genome shares a core set of 91 genes with ϕKZ and related phages, ∼61 of which are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of 18 mutants enabled assignment of 13 genes as essential, information which could not have been determined by sequence-based searches for 11 genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 provided new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241, which encodes an RNA polymerase β subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a “missing” β′ subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host interactions, and evolution. IMPORTANCE In recent years, there has been a paradigm shift in virology with the realization that extremely large viruses infecting prokaryotes (giant phages) can be found in many environments. A group of phages related to the prototype giant phage ϕKZ are of great interest due to their virions being among the most complex of prokaryotic viruses and their potential for

  4. Primary Adsorption Site of Phage PBS1: the Flagellum of Bacillus subtilis

    PubMed Central

    Raimondo, Linda M.; Lundh, Nancy P.; Martinez, Rafael J.

    1968-01-01

    The adsorption of Bacillus subtilis phage PBS1 was studied, and it was demonstrated that the primary adsorption site for this phage is the flagellum of B. subtilis. The capacity of flagella to function for motility may be lost without the loss of their capacity to adsorb the phage and permit infection. Deoxyribonucleic acid injection by the phage is inhibited by cyanide, suggesting the requirement for cellular energy in the infection process. Images PMID:4986906

  5. Phage typing of Salmonella enteritidis from different sources in Brazil.

    PubMed

    Nunes, Iolanda A; Helmuth, Reiner; Schroeter, Andreus; Mead, Geoffrey C; Santos, Manoel A A; Solari, Claude A; Silva, Oyama R; Ferreira, Antonio J Piantino

    2003-02-01

    The occurrence of Salmonella Enteritidis (SE) phage types (PTs) in samples collected from healthy and diseased chickens, in outbreaks of human gastroenteritis related to the consumption of egg products, in samples of poultry meat, in pipped embryos of broiler chickens, in meat meal, in poultry-rearing environments, and in many foods (cheese, mayonnaise, cake, and bacon) is described for strains isolated from 1995 to 1997 in Brazil. SE strains were isolated, and the most common PT was found to be PT 4, followed by PTs 7, 21, 35, 6, 4a, 8, 30, 6a, 5a, 1, and 1b. Fourteen strains were classified as react-but-do-not-conform strains, and one strain was not typeable. The results of this study demonstrate that PT 4 has a wider distribution among the sources studied than do any other SE phage types and is the most important phage type in human salmonellosis.

  6. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    NASA Astrophysics Data System (ADS)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  7. Phage cluster relationships identified through single gene analysis

    PubMed Central

    2013-01-01

    Background Phylogenetic comparison of bacteriophages requires whole genome approaches such as dotplot analysis, genome pairwise maps, and gene content analysis. Currently mycobacteriophages, a highly studied phage group, are categorized into related clusters based on the comparative analysis of whole genome sequences. With the recent explosion of phage isolation, a simple method for phage cluster prediction would facilitate analysis of crude or complex samples without whole genome isolation and sequencing. The hypothesis of this study was that mycobacteriophage-cluster prediction is possible using comparison of a single, ubiquitous, semi-conserved gene. Tape Measure Protein (TMP) was selected to test the hypothesis because it is typically the longest gene in mycobacteriophage genomes and because regions within the TMP gene are conserved. Results A single gene, TMP, identified the known Mycobacteriophage clusters and subclusters using a Gepard dotplot comparison or a phylogenetic tree constructed from global alignment and maximum likelihood comparisons. Gepard analysis of 247 mycobacteriophage TMP sequences appropriately recovered 98.8% of the subcluster assignments that were made by whole-genome comparison. Subcluster-specific primers within TMP allow for PCR determination of the mycobacteriophage subcluster from DNA samples. Using the single-gene comparison approach for siphovirus coliphages, phage groupings by TMP comparison reflected relationships observed in a whole genome dotplot comparison and confirm the potential utility of this approach to another widely studied group of phages. Conclusions TMP sequence comparison and PCR results support the hypothesis that a single gene can be used for distinguishing phage cluster and subcluster assignments. TMP single-gene analysis can quickly and accurately aid in mycobacteriophage classification. PMID:23777341

  8. Isolation and characterization of phage-host systems from the Baltic Sea ice.

    PubMed

    Luhtanen, Anne-Mari; Eronen-Rasimus, Eeva; Kaartokallio, Hermanni; Rintala, Janne-Markus; Autio, Riitta; Roine, Elina

    2014-01-01

    In search for sea ice bacteria and their phages from the Baltic Sea ice, two ice samples were collected from land-fast ice in a south-west Finland coastal site in February and March 2011. Bacteria were isolated from the melted sea ice samples and phages were screened from the same samples for 43 purified isolates. Plaque-producing phages were found for 15 bacterial isolates at 3 °C. Ten phage isolates were successfully plaque purified and eight of them were chosen for particle purification to analyze their morphology and structural proteins. Phage 1/32 infecting an isolate affiliated to phylum Bacteroidetes (Flavobacterium sp.) is a siphovirus and six phages infecting isolates affiliated to γ-Proteobacteria (Shewanella sp.) hosts were myoviruses. Cross titrations between the hosts showed that all studied phages are host specific. Phage solutions, host growth and phage infection were tested in different temperatures revealing phage temperature tolerance up to 45 °C, whereas phage infection was in most of the cases retarded above 15 °C. This study is the first to report isolation and cultivation of ice bacteria and cold-active phages from the Baltic Sea ice.

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

    SciTech Connect

    Aziz, Ramy K.; Dwivedi, Bhakti; Akhter, Sajia; Breitbart, Mya; Edwards, Robert A.

    2015-05-08

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

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

    DOE PAGES

    Aziz, Ramy K.; Dwivedi, Bhakti; Akhter, Sajia; ...

    2015-05-08

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

  11. Characterization of a ViI-like phage specific to Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phage vB_EcoM_CBA120 (CBA120) isolated against Escherichia coli O157:H7 from a cattle feedlot is morphologically very similar to the classic phage ViI of Salmonella enterica serovar Typhi. Until recently, little was known genetically or physiologically about the ViI-like phages, and non targeting E...

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

    PubMed

    Aziz, Ramy K; Dwivedi, Bhakti; Akhter, Sajia; Breitbart, Mya; Edwards, Robert A

    2015-01-01

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

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

    PubMed Central

    Aziz, Ramy K.; Dwivedi, Bhakti; Akhter, Sajia; Breitbart, Mya; Edwards, Robert A.

    2015-01-01

    Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set of publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. We propose adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution. PMID:26005436

  14. Phage mutations in response to CRISPR diversification in a bacterial population.

    PubMed

    Sun, Christine L; Barrangou, Rodolphe; Thomas, Brian C; Horvath, Philippe; Fremaux, Christophe; Banfield, Jillian F

    2013-02-01

    Interactions between bacteria and their coexisting phage populations impact evolution and can strongly influence biogeochemical processes in natural ecosystems. Periodically, mutation or migration results in exposure of a host to a phage to which it has no immunity; alternatively, a phage may be exposed to a host it cannot infect. To explore the processes by which coexisting, co-evolving hosts and phage populations establish, we cultured Streptococcus thermophilus DGCC7710 with phage 2972 and tracked CRISPR (clustered regularly interspaced short palindromic repeats) diversification and host-phage co-evolution in a population derived from a colony that acquired initial CRISPR-encoded immunity. After 1 week of co-culturing, the coexisting host-phage populations were metagenomically characterized using 454 FLX Titanium sequencing. The evolved genomes were compared with reference genomes to identify newly incorporated spacers in S. thermophilus DGCC7710 and recently acquired single-nucleotide polymorphisms (SNPs) in phage 2972. Following phage exposure, acquisition of immune elements (spacers) led to a genetically diverse population with multiple subdominant strain lineages. Phage mutations that circumvented three early immunization events were localized in the proto-spacer adjacent motif (PAM) or near the PAM end of the proto-spacer, suggesting a strong selective advantage for the phage that mutated in this region. The sequential fixation or near fixation of these single mutations indicates selection events so severe that single phage genotypes ultimately gave rise to all surviving lineages and potentially carried traits unrelated to immunity to fixation.

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

    PubMed Central

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

    2016-01-01

    Gordonia phages BaxterFox, Kita, Nymphadora, and Yeezy are newly characterized phages of Gordonia terrae, isolated from soil samples in Pittsburgh, Pennsylvania. These phages have genome lengths between 50,346 and 53,717 bp, and encode on average 84 predicted proteins. All have G+C content of 66.6%. PMID:27516501

  16. Selective posttranslational modification of phage-displayed polypeptides

    SciTech Connect

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

    2013-11-19

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

  17. Selective posttranslational modification of phage-displayed polypeptides

    SciTech Connect

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

    2013-02-05

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

  18. Phage display in the study of infectious diseases.

    PubMed

    Mullen, Lisa M; Nair, Sean P; Ward, John M; Rycroft, Andrew N; Henderson, Brian

    2006-03-01

    Microbial infections are dependent on the panoply of interactions between pathogen and host and identifying the molecular basis of such interactions is necessary to understand and control infection. Phage display is a simple functional genomic methodology for screening and identifying protein-ligand interactions and is widely used in epitope mapping, antibody engineering and screening for receptor agonists or antagonists. Phage display is also used widely in various forms, including the use of fragment libraries of whole microbial genomes, to identify peptide-ligand and protein-ligand interactions that are of importance in infection. In particular, this technique has proved successful in identifying microbial adhesins that are vital for colonization.

  19. Electron microscopy of phages in serotypes of Actinobacillus actinomycetemcomitans.

    PubMed

    Olsen, I; Namork, E; Myhrvold, V

    1993-12-01

    Actinobacillus actinomycetemcomitans, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica and Pasteurella multocida strains were examined by transmission electron microscopy for the presence of bacteriophages. Phages were detected in serotype a (SUNY 75) and e (UOH 1705) and in the fresh clinical isolates UOH Q1243 and UOH Q1247 of A. actinomycetemcomitans. Phages were not found in serotype b, c and d strains of A. actinomycetemcomitans, in the fresh clinical isolate UOH Q1244 of this species or in old strains (including reference strains) of related species from the Actinobacillus-Haemophilus-Pasteurella group.

  20. Integration

    ERIC Educational Resources Information Center

    Kalyn, Brenda

    2006-01-01

    Integrated learning is an exciting adventure for both teachers and students. It is not uncommon to observe the integration of academic subjects such as math, science, and language arts. However, educators need to recognize that movement experiences in physical education also can be linked to academic curricula and, may even lead the…

  1. The filamentous phage XacF1 causes loss of virulence in Xanthomonas axonopodis pv. citri, the causative agent of citrus canker disease.

    PubMed

    Ahmad, Abdelmonim Ali; Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2014-01-01

    In this study, filamentous phage XacF1, which can infect Xanthomonas axonopodis pv. citri (Xac) strains, was isolated and characterized. Electron microscopy showed that XacF1 is a member of the family Inoviridae and is about 600 nm long. The genome of XacF1 is 7325 nucleotides in size, containing 13 predicted open reading frames (ORFs), some of which showed significant homology to Ff-like phage proteins such as ORF1 (pII), ORF2 (pV), ORF6 (pIII), and ORF8 (pVI). XacF1 showed a relatively wide host range, infecting seven out of 11 strains tested in this study. Frequently, XacF1 was found to be integrated into the genome of Xac strains. This integration occurred at the host dif site (attB) and was mediated by the host XerC/D recombination system. The attP sequence was identical to that of Xanthomonas phage Cf1c. Interestingly, infection by XacF1 phage caused several physiological changes to the bacterial host cells, including lower levels of extracellular polysaccharide production, reduced motility, slower growth rate, and a dramatic reduction in virulence. In particular, the reduction in virulence suggested possible utilization of XacF1 as a biological control agent against citrus canker disease.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  3. Transmission of phage by glassy-winged sharpshooters, a vector of Xylella fastidiosa

    PubMed Central

    Bhowmick, Tushar Suvra; Das, Mayukh; Heinz, Kevin M.; Krauter, Peter C.; Gonzalez, Carlos F.

    2016-01-01

    ABSTRACT Xylella fastidiosa subsp. fastidiosa (Xff) is the causal agent of Pierce's Disease (PD) of grapevines and is vectored by the glassy-winged sharpshooter (GWSS, Homalodisca vitripennis). Previously we have reported the development of a bacteriophage (phage) based biocontrol system for PD, but no information on insect transmission of phages has been reported. Here we communicate that laboratory reared GWSSs fed on cowpea plants (Vigna unguiculata subsp. unguiculata) harboring the virulent phage Paz were able to uptake of phage efficiently when the phage was present in high concentration, but were inefficient in transfer to plants. PMID:27738554

  4. Expression of Cre recombinase during transient phage infection permits efficient marker removal in Streptomyces

    PubMed Central

    Khodakaramian, Gholam; Lissenden, Sarah; Gust, Bertolt; Moir, Laura; Hoskisson, Paul A.; Chater, Keith F.; Smith, Margaret C. M.

    2006-01-01

    We report a system for the efficient removal of a marker flanked by two loxP sites in Streptomyces coelicolor, using a derivative of the temperate phage φC31 that expresses Cre recombinase during a transient infection. As the test case for this recombinant phage (called Cre-phage), we present the construction of an in-frame deletion of a gene, pglW, required for phage growth limitation or Pgl in S.coelicolor. Cre-phage was also used for marker deletion in other strains of S.coelicolor. PMID:16473843

  5. Genetic and Physical Structures of Salmonella-coli Phage Hybrids and Development of New Generalized Transducing Hybrid Phages for E. coli.

    DTIC Science & Technology

    1983-01-01

    34 Transducing Hybrid Phages for E . coli . Annual Progress Report -- Nobuto Yamamoto, Ph.D. "-"- January, 1983 ,’. °."Supported by . . U.S. ARMY MEDICAL...bacteriophage, Salmonella typhimurium, Hybrid bacteria, E . coli -S. typhimurium hybrid, Genetic homology, Genetic recombination, Antigenicity...and identify hp otrk number) E . coli -S. typhimurium provided excellent systems to isolate bacterio- phage hybrids between Salmonella phage P22 and E

  6. In defense of phage: viral suppressors of CRISPR-mediated adaptive immunity in bacteria.

    PubMed

    Wiedenheft, Blake

    2013-05-01

    Viruses that infect bacteria are the most abundant biological agents on the planet and bacteria have evolved diverse defense mechanisms to combat these genetic parasites. One of these bacterial defense systems relies on a repetitive locus, referred to as a CRISPR (clusters of regularly interspaced short palindromic repeats). Bacteria and archaea acquire resistance to invading viruses and plasmids by integrating short fragments of foreign nucleic acids at one end of the CRISPR locus. CRISPR loci are transcribed and the long primary CRISPR transcript is processed into a library of small RNAs that guide the immune system to invading nucleic acids, which are subsequently degraded by dedicated nucleases. However, the development of CRISPR-mediated immune systems has not eradicated phages, suggesting that viruses have evolved mechanisms to subvert CRISPR-mediated protection. Recently, Bondy-Denomy and colleagues discovered several phage-encoded anti-CRISPR proteins that offer new insight into the ongoing molecular arms race between viral parasites and the immune systems of their hosts.

  7. Phages and HIV-1: From Display to Interplay

    PubMed Central

    Delhalle, Sylvie; Schmit, Jean-Claude; Chevigné, Andy

    2012-01-01

    The complex hide-and-seek game between HIV-1 and the host immune system has impaired the development of an efficient vaccine. In addition, the high variability of the virus impedes the long-term control of viral replication by small antiviral drugs. For more than 20 years, phage display technology has been intensively used in the field of HIV-1 to explore the epitope landscape recognized by monoclonal and polyclonal HIV-1-specific antibodies, thereby providing precious data about immunodominant and neutralizing epitopes. In parallel, biopanning experiments with various combinatorial or antibody fragment libraries were conducted on viral targets as well as host receptors to identify HIV-1 inhibitors. Besides these applications, phage display technology has been applied to characterize the enzymatic specificity of the HIV-1 protease. Phage particles also represent valuable alternative carriers displaying various HIV-1 antigens to the immune system and eliciting antiviral responses. This review presents and summarizes the different studies conducted with regard to the nature of phage libraries, target display mode and biopanning procedures. PMID:22606007

  8. Genome Sequences of Streptomyces Phages Amela and Verse

    PubMed Central

    Layton, Sonya R.; Hemenway, Ryan M.; Munyoki, Christine M.; Barnes, Emory B.; Barnett, Sierra E.; Bond, Alec M.; Narvaez, Jessi M.; Sirisakd, Christie D.; Smith, Brandt R.; Swain, Justin; Syed, Orooj; Bowman, Charles A.; Russell, Daniel A.; Bhuiyan, Swapan; Donegan-Quick, Richard; Benjamin, Robert C.

    2016-01-01

    Amela and Verse are two Streptomyces phages isolated by enrichment on Streptomyces venezuelae (ATCC 10712) from two different soil samples. Amela has a genome length of 49,452, with 75 genes. Verse has a genome length of 49,483, with 75 genes. Both belong to the BD3 subcluster of Actinobacteriophage. PMID:26893416

  9. Prospective identification of parasitic sequences in phage display screens

    PubMed Central

    Matochko, Wadim L.; Cory Li, S.; Tang, Sindy K.Y.; Derda, Ratmir

    2014-01-01

    Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones. PMID:24217917

  10. The phage-driven microbial loop in petroleum bioremediation.

    PubMed

    Rosenberg, Eugene; Bittan-Banin, Gili; Sharon, Gil; Shon, Avital; Hershko, Galit; Levy, Itzik; Ron, Eliora Z

    2010-07-01

    During the drilling process and transport of crude oil, water mixes with the petroleum. At oil terminals, the water settles to the bottom of storage tanks. This drainage water is contaminated with emulsified oil and water-soluble hydrocarbons and must be treated before it can be released into the environment. In this study, we tested the efficiency of a continuous flow, two-stage bioreactor for treating drainage water from an Israeli oil terminal. The bioreactor removed all of the ammonia, 93% of the sulfide and converted 90% of the total organic carbon (TOC) into carbon dioxide. SYBR Gold staining indicated that reactor 1 contained 1.7 × 10(8) bacteria and 3.7 × 10(8) phages per millilitre, and reactor 2 contained 1.3 × 10(8) bacteria and 1.7 × 10(9) phages per millilitre. The unexpectedly high mineralization of TOC and high concentration of phage in reactor 2 support the concept of a phage-driven microbial loop in the bioremediation of the drainage water. In general, application of this concept in bioremediation of contaminated water has the potential to increase the efficiency of processes.

  11. CRISPR: new horizons in phage resistance and strain identification.

    PubMed

    Barrangou, Rodolphe; Horvath, Philippe

    2012-01-01

    Bacteria have been widely used as starter cultures in the food industry, notably for the fermentation of milk into dairy products such as cheese and yogurt. Lactic acid bacteria used in food manufacturing, such as lactobacilli, lactococci, streptococci, Leuconostoc, pediococci, and bifidobacteria, are selectively formulated based on functional characteristics that provide idiosyncratic flavor and texture attributes, as well as their ability to withstand processing and manufacturing conditions. Unfortunately, given frequent viral exposure in industrial environments, starter culture selection and development rely on defense systems that provide resistance against bacteriophage predation, including restriction-modification, abortive infection, and recently discovered CRISPRs (clustered regularly interspaced short palindromic repeats). CRISPRs, together with CRISPR-associated genes (cas), form the CRISPR/Cas immune system, which provides adaptive immunity against phages and invasive genetic elements. The immunization process is based on the incorporation of short DNA sequences from virulent phages into the CRISPR locus. Subsequently, CRISPR transcripts are processed into small interfering RNAs that guide a multifunctional protein complex to recognize and cleave matching foreign DNA. Hypervariable CRISPR loci provide insights into the phage and host population dynamics, and new avenues for enhanced phage resistance and genetic typing and tagging of industrial strains.

  12. Nitrous acid induced damage in T7 DNA and phage

    SciTech Connect

    Scearce, L.M.; Masker, W.E.

    1986-05-01

    The response of bacteriophage T7 to nitrous acid damage was investigated. The T7 system allows in vitro mimicry of most aspects of in vivo DNA metabolism. Nitrous acid is of special interest since it has been previously shown to induce deletions and point mutations as well as novel adducts in DNA. T7 phage was exposed to 56 mM nitrous acid at pH 4.6 in vivo, causing a time dependent 98% decrease in survival for each 10 min duration of exposure to nitrous acid. These studies were extended to include examination of pure T7 DNA exposed in vitro to nitrous acid conditions identical to those used in the in vivo survival studies. The treated DNA was dialyzed to remove the nitrous acid and the DNA was encapsulated into empty phage heads. These in vitro packaged phage showed a survival curve analogous to the in vivo system. There was no change in survival when either in vitro or in vivo exposed phage were grown on wild type E. coli or on E. coli strains deficient in DNA repair due to mutations in DNA polymerase I, exonuclease III or a uvrA mutation. Survival was not increased when nitrous acid treated T7 were grown on E. coli induced for SOS repair. In vitro replication of nitrous acid treated DNA showed a time dependent decrease in the total amount of DNA synthesized.

  13. IDENTIFICATION OF KNOWN PHAGE ENDOLYSIN GENES BY PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    S. aureus phage endolysins degrade staphylococcal peptidoglycan when released from within an infected cell. Some have been shown to also be effective when purified and exposed to the bacteria externally (lysis 'from without'). These have been proposed for use in antimicrobial applications, especia...

  14. Genome Sequences of Gordonia terrae Phages Benczkowski14 and Katyusha

    PubMed Central

    Benczkowski, Matthew S.; Green, Daryn E.; Hwang, Melina; Kennedy, Bryan; Kocak, Bradley; Kruczek, Ellen; Lin, Leon; Moretti, Matthew L.; Onelangsy, Faith L.; Mezghani, Nadia; Milliken, Katherine A.; Toner, Chelsea L.; Thompson, Paige K.; Ulbrich, Megan C.; Furbee, Emily C.; Grubb, Sarah R.; Warner, Marcie H.; Montgomery, Matthew T.; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah; Hatfull, Graham F.

    2016-01-01

    Bacteriophages Katyusha and Benczkowski14 are newly isolated phages that infect Gordonia terrae 3612. Both have siphoviral morphologies with isometric heads and long tails (500 nm). The genomes are 75,380 bp long and closely related, and the tape measure genes (9 kbp) are among the largest to be identified. PMID:27340062

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

    PubMed Central

    Dennehy, John J.

    2012-01-01

    The outcomes of host-parasite interactions depend on the coevolutionary forces acting upon them, but because every host-parasite relation is enmeshed in a web of biotic and abiotic interactions across a heterogeneous landscape, host-parasite coevolution has proven difficult to study. Simple laboratory phage-bacteria microcosms can ameliorate this difficulty by allowing controlled, well-replicated experiments with a limited number of interactors. Genetic, population, and life history data obtained from these studies permit a closer examination of the fundamental correlates of host-parasite coevolution. In this paper, I describe the results of phage-bacteria coevolutionary studies and their implications for the study of host-parasite coevolution. Recent experimental studies have confirmed phage-host coevolutionary dynamics in the laboratory and have shown that coevolution can increase parasite virulence, specialization, adaptation, and diversity. Genetically, coevolution frequently proceeds in a manner best described by the Gene for Gene model, typified by arms race dynamics, but certain contexts can result in Red Queen dynamics according to the Matching Alleles model. Although some features appear to apply only to phage-bacteria systems, other results are broadly generalizable and apply to all instances of antagonistic coevolution. With laboratory host-parasite coevolutionary studies, we can better understand the perplexing array of interactions that characterize organismal diversity in the wild. PMID:23213618

  16. Characterization of phages virulent for Sarothamnus scoparius bradyrhizobia.

    PubMed

    Małek, Wanda; Sajnaga, Ewa; Wdowiak-Wróbel, Sylwia; Studzińska, Bozena; Icka, Izabela Swie; Nosalewicz, Izabela; Słomka, Marta; Tatara, Agnieszka; Gawron, Antoni

    2005-10-01

    Four virulent phages--PhiDl, PhiTl, PhiCYT21, and PhiOS6, infective on Sarothamnus scoparius rhizobia--were isolated from the soil and characterized for morphology, host range, rate of adsorption to bacterial cells, and genome size. New phages were separated into two morphological families: Siphoviridae with long, noncontractile tails (PhiDl, PhiTl) and Myoviridae with long, contractile tails (PhiCYT21, PhiOS6). They were also classified into two groups by a host specificity. One of them included viruses (PhiDl and PhiTl) that lysed S. scoparius bradyrhizobia and Bradyrhizobium sp. (Lupinus) strain Dl, and the second one comprised phages (PhiCYT21 and PhiOS6) that parasitized only Scotch broom native microsymbionts. Phages specific for S. scoparius rhizobia were differentiated not only by morphology and host range but also by a genome size that was in the range from 47,583 to 60,098 b.p.

  17. Functional characterization of a novel lytic phage EcSw isolated from Sus scrofa domesticus and its potential for phage therapy.

    PubMed

    Easwaran, Maheswaran; Paudel, Sarita; De Zoysa, Mahanama; Shin, Hyun-Jin

    2015-06-01

    In this study, multi-drug resistant Escherichia coli Sw1 (E. coli Sw1) and active lytic phage EcSw was isolated from feces samples of Sus scrofa domesticus (piglet) suffering from diarrhea. Transmission electron microscopy (TEM) indicated that isolated EcSw belongs to the Myoviridae family with an icosahedral head (80 ± 4) and a long tail (180 ± 5 nm). The EcSw phage genome size was estimated to be approximately 75 Kb of double-stranded DNA (dsDNA). Phage dynamic studies show that the latent period and burst size of EcSw were approximately 20 min and 28 PFU per cell, respectively. Interestingly, the EcSw phage can tolerate a wide range of environmental conditions, such as temperature, pH and ions (Ca(2+) and Mg(2+)). Furthermore, genome sequence analysis revealed that the lytic genes of the EcSw phage are notably similar to those of enterobacteria phages. In addition, phage-antibiotic synergy has notable effects compared with the effects of phages or antibiotics alone. Inhibition of E. coli Sw1 and 0157:H7 strains showed that the limitations of host specificity and infectivity of EcSw. Even though, it has considerable potential for phage therapy for handling the problem of the emergence of multidrug resistant pathogens.

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

    PubMed

    Hobbs, Zack; Abedon, Stephen T

    2016-04-01

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

  19. Phage Biodiversity in Artisanal Cheese Wheys Reflects the Complexity of the Fermentation Process

    PubMed Central

    Mahony, Jennifer; Moscarelli, Angelo; Kelleher, Philip; Lugli, Gabriele A.; Ventura, Marco; Settanni, Luca; van Sinderen, Douwe

    2017-01-01

    Dairy fermentations constitute a perfect “breeding ground” for bacteriophages infecting starter cultures, particularly strains of Lactococcus lactis. In modern fermentations, these phages typically belong to one of three groups, i.e., the 936, P335, and c2 phage groups. Traditional production methods present fewer chemical and physical barriers to phage proliferation compared to modern production systems, while the starter cultures used are typically complex, variable, and undefined. In the current study, a variety of cheese whey, animal-derived rennet, and vat swab samples from artisanal cheeses produced in Sicily were analysed for the presence of lactococcal phages to assess phage diversity in such environments. The complete genomes of 18 representative phage isolates were sequenced, allowing the identification of 10 lactococcal 949 group phages, six P087 group phages, and two members of the 936 group phages. The genetic diversity of these isolates was examined using phylogenetic analysis as well as a focused analysis of the receptor binding proteins, which dictate specific interactions with the host-encoded receptor. Thermal treatments at 63 °C and 83 °C indicate that the 949 phages are particularly sensitive to thermal treatments, followed by the P087 and 936 isolates, which were shown to be much less sensitive to such treatments. This difference may explain the relatively low frequency of isolation of the so-called “rare” 949 and P087 group phages in modern fermentations. PMID:28300778

  20. Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine

    PubMed Central

    Nobrega, Franklin L.; Costa, Ana Rita; Santos, José F.; Siliakus, Melvin F.; van Lent, Jan W. M.; Kengen, Servé W. M.; Azeredo, Joana; Kluskens, Leon D.

    2016-01-01

    Orally administered phages to control zoonotic pathogens face important challenges, mainly related to the hostile conditions found in the gastrointestinal tract (GIT). These include temperature, salinity and primarily pH, which is exceptionally low in certain compartments. Phage survival under these conditions can be jeopardized and undermine treatment. Strategies like encapsulation have been attempted with relative success, but are typically complex and require several optimization steps. Here we report a simple and efficient alternative, consisting in the genetic engineering of phages to display lipids on their surfaces. Escherichia coli phage T7 was used as a model and the E. coli PhoE signal peptide was genetically fused to its major capsid protein (10 A), enabling phospholipid attachment to the phage capsid. The presence of phospholipids on the mutant phages was confirmed by High Performance Thin Layer Chromatography, Dynamic Light Scattering and phospholipase assays. The stability of phages was analysed in simulated GIT conditions, demonstrating improved stability of the mutant phages with survival rates 102–107 pfu.mL−1 higher than wild-type phages. Our work demonstrates that phage engineering can be a good strategy to improve phage tolerance to GIT conditions, having promising application for oral administration in veterinary medicine. PMID:27976713

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

    PubMed

    Laanto, Elina; Bamford, Jaana K H; Ravantti, Janne J; Sundberg, Lotta-Riina

    2015-01-01

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

  2. Heterogeneity in phage induction enables the survival of the lysogenic population.

    PubMed

    Imamovic, Lejla; Ballesté, Elisenda; Martínez-Castillo, Alexandre; García-Aljaro, Cristina; Muniesa, Maite

    2016-03-01

    Lysogeny by temperate phages provides novel functions for bacteria and shelter for phages. However, under conditions that activate the phage lytic cycle, the benefit of lysogeny becomes a paradox that poses a threat for bacterial population survival. Using Escherichia coli lysogens for Shiga toxin (Stx) phages as model, we demonstrate how lysogenic bacterial populations circumvent extinction after phage induction. A fraction of cells maintains lysogeny, allowing population survival, whereas the other fraction of cells lyse, increasing Stx production and spreading Stx phages. The uninduced cells were still lysogenic for the Stx phage and equally able to induce phages as the original cells, suggesting heterogeneity of the E. coli lysogenic population. The bacterial population can modulate phage induction under stress conditions by the stress regulator RpoS. Cells overexpressing RpoS reduce Stx phage induction and compete with and survive better than cells with baseline RpoS levels. Our observations suggest that population heterogeneity in phage induction could be widespread among other bacterial genera and we propose this is a mechanism positively selected to prevent the extinction of the lysogenic population that can be modulated by environmental conditions.

  3. Bioengineering bacteriophages to enhance the sensitivity of phage amplification-based paper fluidic detection of bacteria.

    PubMed

    Alcaine, S D; Law, K; Ho, S; Kinchla, A J; Sela, D A; Nugen, S R

    2016-08-15

    Bacteriophage (phage) amplification is an attractive method for the detection of bacteria due to a narrow phage-host specificity, short amplification times, and the phages' ability to differentiate between viable and non-viable bacterial cells. The next step in phage-based bacteria detection is leveraging bioengineered phages to create low-cost, rapid, and easy-to-use detection platforms such as lateral flow assays. Our work establishes the proof-of-concept for the use of bioengineered T7 phage strains to increase the sensitivity of phage amplification-based lateral flow assays. We have demonstrated a greater than 10-fold increase in sensitivity using a phage-based protein reporter, maltose-binding protein, over the detection of replicated T7 phage viron itself, and a greater then 100-fold increase in sensitivity using a phage-based enzymatic reporter, alkaline phosphatase. This increase in sensitivity enabled us to detect 10(3)CFU/mL of Escherichia coli in broth after 7h, and by adding a filter concentration step, the ability to detect a regulatory relevant E. coli concentration of 100CFU/100mL in inoculated river water after 9h, where the current standard requires days for results. The combination of the paper fluidic format with phage-based detection provides a platform for the development of novel diagnostics that are sensitive, rapid, and easy to use.

  4. Identification and characterisation of new Campylobacter group III phages of animal origin.

    PubMed

    Janež, Nika; Kokošin, Andreja; Zaletel, Eva; Vranac, Tanja; Kovač, Jasna; Vučković, Darinka; Smole Možina, Sonja; Curin Šerbec, Vladka; Zhang, Qijing; Accetto, Tomaž; Podgornik, Aleš; Peterka, Matjaž

    2014-10-01

    Campylobacter-specific bacteriophages (phages) are considered as an alternative intervention strategy to decrease the level of poultry contamination with Campylobacter, a leading cause of gastroenteritis worldwide. Eradication efficiency depends primarily on phage-host interaction mediated by phage tail-spike proteins and bacterial receptors. Here, this interaction was characterised using tail-spike gene sequence analysis, phage neutralisation by antiserum and host range analysis of newly isolated group III Campylobacter phages with 68 Campylobacter jejuni and Campylobacter coli strains. Three different groups of phages were obtained using antibody neutralisation assay, and they were further divided according to polymorphisms observed within tail fibre sequences and host range. Only moderate congruence was observed between these criteria with notable exception of two phages. The infection relied on capsule in all phages isolated, and flagella were found to influence phage propagation on agar plates, but not in broth. Their specificity was more C. jejuni oriented with tendency to lyse human isolates more efficiently. Additionally, natural resistance of C. jejuni to phages did not correlate with their antibiotic resistance patterns. These findings provide new insights into Campylobacter-phage interaction.

  5. Genome analysis of two virulent Streptococcus thermophilus phages isolated in Argentina.

    PubMed

    Guglielmotti, Daniela M; Deveau, Hélène; Binetti, Ana G; Reinheimer, Jorge A; Moineau, Sylvain; Quiberoni, Andrea

    2009-11-30

    Two Streptococcus thermophilus phages (ALQ13.2 and phiAbc2) were previously isolated from breakdowns of cheese manufacture in Argentina. Complete nucleotide sequence analysis indicated that both phages contained linear double-stranded DNA: 35,525 bp in length for the pac-type phage ALQ13.2 and 34,882 bp for the cos-type phage phiAbc2. Forty-four and 48 open reading frames (ORF) were identified for ALQ13.2 and phiAbc2, respectively. Comparative genomic analysis showed that these isolates shared many similarities with the eight previously studied cos- and pac-phages infecting different S. thermophilus strains. In particular, part of the phiAbc2 genome was highly similar to a region of phage 7201, which was thought to be unique to this latter phage. Protein analysis of the pac-phage ALQ13.2 using SDS polyacrylamide gel electrophoresis (SDS-PAGE) identified three major proteins and seven minor proteins. Parallel structural proteome analysis of phiAbc2 revealed seven protein bands, two of which were related to major structural proteins, as expected for a cos-type phage. Similarities to other S. thermophilus phages suggest that the streptococcal phage diversity is not extensive in worldwide dairy factories possibly because related high-performing bacterial strains are used in starter cultures.

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

    PubMed Central

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

    2017-01-01

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

  7. Development of a highly sensitive noncompetitive electrochemical immunosensor for the detection of atrazine by phage anti-immunocomplex assay.

    PubMed

    González-Techera, Andrés; Zon, María Alicia; Molina, Patricia Gabriela; Fernández, Héctor; González-Sapienza, Gualberto; Arévalo, Fernando Javier

    2015-02-15

    The development of immunosensors for the detection of small molecules is of great interest because of their simplicity, high sensitivity and extended analytical range. Due to their size, small compounds cannot be simultaneously recognized by two antibodies impeding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. In this work, we combine the advantages of magneto-electrochemical immunosensors with the improved sensitivity and direct proportional signal of noncompetitive immunoassays to develop a new Phage Anti-Immunocomplex Electrochemical Immunosensor (PhAIEI) for the detection of the herbicide atrazine. The noncompetitive assay is based on the use of recombinant M13 phage particles bearing a peptide that specifically recognizes the immunocomplex of atrazine with an anti-atrazine monoclonal antibody. The PhAIEI performed with a limit of detection (LOD) of 0.2 pg mL(-1), which is 200-fold better than the LOD obtained using the same antibody in an optimized conventional competitive ELISA, with a large increase in working range. The developed PhAIEI was successfully used to assay undiluted river water samples with no pretreatment and excellent recoveries. Apart from the first demonstration of the benefits of integrating phage anti-immunocomplex particles into electrochemical immunosensors, the extremely low and environmentally relevant detection limits of atrazine attained with the PhAIEIS may have direct applicability to fast and sensitive detection of this herbicide in the environment.

  8. Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.

    PubMed

    Rossant, Christine J; Carroll, Danielle; Huang, Ling; Elvin, John; Neal, Frances; Walker, Edward; Benschop, Joris J; Kim, Eldar E; Barry, Simon T; Vaughan, Tristan J

    2014-01-01

    Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.

  9. Architectonics of phage-liposome nanowebs as optimized photosensitizer vehicles for photodynamic cancer therapy

    PubMed Central

    Sreeram, Kalarical Janardhanan; Narayan, Shoba; Gopal, Abbineni; Hayhurst, Andrew; Mao, Chuanbin

    2010-01-01

    Filamentous M13 phage can be engineered to display cancer cell-targeting or tumor-homing peptides through phage display. It would be highly desirable if the tumor targeting phage can also carry anti-cancer drugs to deliver them to the cancer cells. We studied the evolution of structures of the complexes between anionic filamentous M13 phage and cationic serum-stable liposomes which encapsulate the monomeric photosensitizer, zinc naphthalocyanine. At specific phage-liposome ratios, multiple phage nanofibers and liposomes are interwoven into a “nanoweb”. The chemical and biological properties of the phage-liposome nanoweb were evaluated for possible application in drug delivery. This study highlights the ability of phageliposome nanowebs to serve as efficient carriers to transport photosensitizers to cancer cells. PMID:20807781

  10. Genome organization of temperate phage 11143 from emetic Bacillus cereus NCTC11143.

    PubMed

    Lee, Young-Duck; Park, Jong-Hyun

    2012-05-01

    A temperate phage was isolated from emetic Bacillus cereus NCTC 11143 by mitomycin C and characterized by transmission electron microscopy and DNA and protein analyses. Whole genome sequencing of Bacillus phage 11143 was performed by GS-FLX. The phage has a dsDNA genome of 39,077 bp and a 35% G+C content. Bioinformatic analysis of the phage genome revealed 49 putative ORFs involved in replication, morphogenesis, DNA packaging, lysogeny, and host lysis. Bacillus phage 11143 could be classified as a member of the Siphoviridae family by morphology and genome structure. Genomic comparisons at the DNA and protein levels revealed homologous genetic modules with patterns and morphogenesis proteins similar to those of other Bacillus phages. Thus, Bacillus phages might have a mosaic genetic relationship.

  11. A new small temperate DNA phage BcP15 isolated from Burkholderia cepacia DR11.

    PubMed

    Hens, D K; Ghosh, A N; Kumar, R

    2005-12-01

    A Burkholderia cepacia DR11 strain was isolated during the survey of microorganisms from coastal water of deltaic Sunderbans. This strain always released temperate phage BcP15 into culture supernatant. UV irradiation of the strain also induced phage induction. The phage titer was 2.3 x 10(8). New temperate phage BcP15 has unusual structure. It has a hexagonal head, 65 nm in diameter and a tail 200 nm long, attached with single thick wavy tail fiber (424-705 nm). Phage DNA is double stranded 11.9 kb long. Southern hybridization result indicated that the phage DNA was in lysogenic state into the B. cepacia DR11 genome. SDS-PAGE of phage protein showed two major bands of molecular weight 20 kDa and 40 kDa.

  12. The phage-related chromosomal islands of Gram-positive bacteria.

    PubMed

    Novick, Richard P; Christie, Gail E; Penadés, Jose R

    2010-08-01

    The phage-related chromosomal islands (PRCIs) were first identified in Staphylococcus aureus as highly mobile, superantigen-encoding genetic elements known as the S. aureus pathogenicity islands (SaPIs). These elements are characterized by a specific set of phage-related functions that enable them to use the phage reproduction cycle for their own transduction and inhibit phage reproduction in the process. SaPIs produce many phage-like infectious particles; their streptococcal counterparts have a role in gene regulation but may not be infectious. These elements therefore represent phage satellites or parasites, not defective phages. In this Review, we discuss the shared genetic content of PRCIs, their life cycle and their ability to be transferred across large phylogenetic distances.

  13. Typing of Panton-Valentine Leukocidin-Encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China

    PubMed Central

    Zhao, Huanqiang; Hu, Fupin; Jin, Shu; Xu, Xiaogang; Zou, Yuhan; Ding, Baixing; He, Chunyan; Gong, Fang; Liu, Qingzhong

    2016-01-01

    Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages. PMID:27536288

  14. 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

    2017-03-13

    The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeriamonocytogenes but is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance from 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 identity 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 extends

  15. Characterizing RecA-Independent Induction of Shiga toxin2-Encoding Phages by EDTA Treatment

    PubMed Central

    Imamovic, Lejla; Muniesa, Maite

    2012-01-01

    Background The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA. Methodology/Principal Findings The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA) were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg2+. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction. Conclusions/Significance Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and in the emergence of new pathogenic strains. PMID:22393404

  16. Role of the phi 11 phage genome in competence of Staphylococcus aureus.

    PubMed

    Sjöström, J E; Philipson, L

    1974-07-01

    Both phage ø11 and 83A, when present as prophage or when used as helper phage, induce competence for transfection and transformation to the same level in Staphylococcus aureus, strain 8325-4. Cells lysogenized with certain temperature-sensitive (ts) mutants of phage ø11 show competence at the nonpermissive temperature (41 C) without production of infectious phages. Phage ø11ts allele 31 can neither as a prophage nor as a helper phage develop competence under nonpermissive conditions. This mutant appears, therefore, to be mutated in the region of the phage genome controlling competence. The competence level for both transfection and transformation is increased by superinfecting strain 8325-4 (ø11) or 8325-4 (83A) at high multiplicities with phage ø11 with some of its mutants or with phage 83A. This superinfection enhancement appears to require protein synthesis but not deoxyribonucleic acid synthesis as judged from studies with inhibitors of macromolecular synthesis. Besides the phage particle, no extracellular or cell-bound factors so far detected can induce competence. The phage-induced product conferring competence is rapidly synthesized by strain 8325-4 (tsø11(31)) after shift to permissive conditions, but requires deoxyribonucleic acid and protein synthesis to be expressed. Recombination between the sus mutants of phage ø11 of Kretschmer and Egan and tsø11(31) indicate that competence is controlled by an early gene in the lytic cycle which may be expressed also in lysogenic cells. The phage product inducing competence appears to have a half-life of 10 to 15 min in the conditional lethal mutant at shift to nonpermissive temperature. Ultraviolet inactivation of phage ø11 infectivity occurs more rapidly than inactivation of competence induction. In fact, the number of transformants is increased at low doses of irradiation. Competence induction is, however, decreased at high does of ultraviolet irradiation.

  17. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

    PubMed Central

    Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

    2016-01-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 − 2.0 × 108 cells mL−1), a low limit of detection (79 cells mL−1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis. PMID:26908277

  18. Development of a High Throughput Assay for Indirectly Measuring Phage Growth Using the OmniLog (trademark) System

    DTIC Science & Technology

    2012-09-01

    and phage Giraffe exhibits a similar morphology (Fig. 1A). Phage BA39 (Fig. 1E) appears to belong to the Myoviridae family (icosahedral head and...see later) but this assignment is tentative. Monitoring the kinetics of bacterial growth using OmniLogTM system upon infection with Giraffe phage...spores with and without infection by Giraffe phage are shown in Figure 2A and B respectively. The growth of 7702 without phage infection followed a

  19. Dispersal and survival of Flavobacterium psychrophilum phages in vivo in rainbow trout and in vitro under laboratory conditions: implications for their use in phage therapy.

    PubMed

    Madsen, Lone; Bertelsen, Sif K; Dalsgaard, Inger; Middelboe, Mathias

    2013-08-01

    Attention has been drawn to phage therapy as an alternative approach for controlling pathogenic bacteria such as Flavobacterium psychrophilum in salmonid aquaculture, which can give rise to high mortalities, especially in rainbow trout fry. Recently, phages have been isolated with a broad host range and a strong lytic potential against pathogenic F. psychrophilum under experimental conditions. However, little is known about the fate of phages at environmental conditions. Here, we quantified the dispersal and fate of F. psychrophilum phages and hosts in rainbow trout fry after intraperitoneal injection. Both phages and bacteria were isolated from the fish organs for up to 10 days after injection, and coinjection with both bacteria and phages resulted in a longer persistence of the phage in the fish organs, than when the fish had been injected with the phages only. The occurrence of both phage and bacterium was most prevalent in the kidney and spleen, with only minor occurrence in the brain. The experiment showed that injected phages were rapidly spread in the internal organs of the fish, also in the absence of bacteria. Parallel examination of the regulation of bacteriophage infectivity in controlled laboratory experiments at various environmental conditions showed that pH had only minor effects on long-term (3 months) phage infectivity within a pH range of 4.5 to 7.5, whereas phage infectivity was immediately lost at pH 3. In the absence of host cells, phage infectivity decreased by a factor of 10,000 over 55 days in untreated pond water, while the sterilization and removal of particles caused a 100-fold increase in phage survival relative to the control. In addition, F. psychrophilum-specific phages maintained their infectivity for ∼2 months in glycerol at -80°C, whereas infectivity decreased by a factor 10 when kept in a buffer at 20°C. Only a very small degradation in infectivity was seen when bacteriophages were added and dried on fish feed pellets

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

    PubMed Central

    2012-01-01

    Background Phages (viruses that infect bacteria) have gained significant attention because of their abundance, diversity and important ecological roles. However, the lack of a universal gene shared by all phages presents a challenge for phage identification and characterization, especially in environmental samples where it is difficult to culture phage-host systems. Homologous conserved genes (or "signature genes") present in groups of closely-related phages can be used to explore phage diversity and define evolutionary relationships amongst these phages. Bioinformatic approaches are needed to identify candidate signature genes and design PCR primers to amplify those genes from environmental samples; however, there is currently no existing computational tool that biologists can use for this purpose. Results Here we present PhiSiGns, a web-based and standalone application that performs a pairwise comparison of each gene present in user-selected phage genomes, identifies signature genes, generates alignments of these genes, and designs potential PCR primer pairs. PhiSiGns is available at (http://www.phantome.org/phisigns/; http://phisigns.sourceforge.net/) with a link to the source code. Here we describe the specifications of PhiSiGns and demonstrate its application with a case study. Conclusions PhiSiGns provides phage biologists with a user-friendly tool to identify signature genes and design PCR primers to amplify related genes from uncultured phages in environmental samples. This bioinformatics tool will facilitate the development of novel signature genes for use as molecular markers in studies of phage diversity, phylogeny, and evolution. PMID:22385976

  1. Listeria phage and phage tail induction triggered by components of bacterial growth media (phosphate, LiCl, nalidixic acid, and acriflavine).

    PubMed

    Lemaître, Jean-Paul; Duroux, Amandine; Pimpie, Romain; Duez, Jean-Marie; Milat, Marie-Louise

    2015-03-01

    The detection of Listeria monocytogenes from food is currently carried out using a double enrichment. For the ISO methodology, this double enrichment is performed using half-Fraser and Fraser broths, in which the overgrowth of L. innocua can occur in samples where both species are present. In this study, we analyzed the induction of phages and phage tails of Listeria spp. in these media and in two brain heart infusion (BHI) broths (BHIM [bioMérieux] and BHIK [Biokar]) to identify putative effectors. It appears that Na2HPO4 at concentrations ranging from 1 to 40 g/liter with an initial pH of 7.5 can induce phage or phage tail production of Listeria spp., especially with 10 g/liter of Na2HPO4 and a pH of 7.5, conditions present in half-Fraser and Fraser broths. Exposure to LiCl in BHIM (18 to 21 g/liter) can also induce phage and phage tail release, but in half-Fraser and Fraser broths, the concentration of LiCl is much lower (3 g/liter). Low phage titers were induced by acriflavine and/or nalidixic acid. We also show that the production of phages and phage tails can occur in half-Fraser and Fraser broths. This study points out that induction of phages and phage tails could be triggered by compounds present in enrichment media. This could lead to a false-negative result for the detection of L. monocytogenes in food products.

  2. Salmonella phages isolated from dairy farms in Thailand show wider host range than a comparable set of phages isolated from U.S. dairy farms.

    PubMed

    Wongsuntornpoj, Sarach; Moreno Switt, Andrea I; Bergholz, Peter; Wiedmann, Martin; Chaturongakul, Soraya

    2014-08-06

    Salmonella is a zoonotic pathogen with globally distributed serovars as well as serovars predominantly found in certain regions; for example, serovar Weltevreden is rarely isolated in the U.S., but is common in Thailand. Relative to our understanding of Salmonella diversity, our understanding of the global diversity of Salmonella phages is limited. We hypothesized that the serovar diversity in a given environment and farming system will affect the Salmonella phage diversity associated with animal hosts. We thus isolated and characterized Salmonella phages from 15 small-scale dairy farms in Thailand and compared the host ranges of the 62 Salmonella phage isolates obtained with host range diversity for 129 phage isolates obtained from dairy farms in the U.S. The 62 phage isolates from Thailand represented genome sizes ranging from 40 to 200 kb and showed lysis of 6-25 of the 26 host strains tested (mean number of strain lysed=19). By comparison, phage isolates previously obtained in a survey of 15 U.S. dairy farms showed a narrow host range (lysis of 1-17; mean number of strains lysed=4); principal coordinate analysis also confirmed U.S. and Thai phages had distinct host lysis profiles. Our data indicate that dairy farms that differ in management practices and are located on different continents can yield phage isolates that differ in their host ranges, providing an avenue for isolation of phages with desirable host range characteristics for commercial applications. Farming systems characterized by coexistence of different animals may facilitate presence of Salmonella phages with wide host ranges.

  3. Oligopeptidase A is required for normal phage P22 development.

    PubMed Central

    Conlin, C A; Vimr, E R; Miller, C G

    1992-01-01

    The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carrying opdA mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations. Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host. This decrease resulted from a reduced efficiency of plating of particles from an opdA infection. In the absence of a functional opdA gene, most of the P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated. Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment. The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins. The opdA-independent mutations lead to mutant forms of gp7 which function without processing. Images PMID:1522065

  4. Evolution of Pseudomonas aeruginosa virulence as a result of phage predation.

    PubMed

    Hosseinidoust, Zeinab; van de Ven, Theo G M; Tufenkji, Nathalie

    2013-10-01

    The rapid increase in the emergence of antibiotic-resistant bacteria has attracted attention to bacteriophages for treating and preventing bacterial infections. Bacteriophages can drive the diversification of Pseudomonas aeruginosa, giving rise to phage-resistant variants with different phenotypes from their ancestral hosts. In this study, we sought to investigate the effect of phage resistance on cytotoxicity of host populations toward cultured mammalian cells. The library of phage-resistant P. aeruginosa PAO1 variants used was developed previously via experimental evolution of an isogenic host population using phages PP7 and E79. Our results presented herein indicate that the phage-resistant variants developed in a heterogeneous phage environment exhibit a greater ability to impede metabolic action of cultured human keratinocytes and have a greater tendency to cause membrane damage even though they cannot invade the cells in large numbers. They also show a heightened resistance to phagocytosis by model murine macrophages. Furthermore, all isolates produced higher levels of at least one of the secreted virulence factors, namely, total proteases, elastase, phospholipase C, and hemolysins. Reverse transcription-quantitative PCR (RT-qPCR) revealed upregulation in the transcription of a number of genes associated with virulence of P. aeruginosa for the phage-resistant variants. The results of this study indicate a significant change in the in vitro virulence of P. aeruginosa following phage predation and highlight the need for caution in the selection and design of phages and phage cocktails for therapeutic use.

  5. Phage Therapy in Bacterial Infections Treatment: One Hundred Years After the Discovery of Bacteriophages.

    PubMed

    Cisek, Agata Anna; Dąbrowska, Iwona; Gregorczyk, Karolina Paulina; Wyżewski, Zbigniew

    2017-02-01

    The therapeutic use of bacteriophages has seen a renewal of interest blossom in the last few years. This reversion is due to increased difficulties in the treatment of antibiotic-resistant strains of bacteria. Bacterial resistance to antibiotics, a serious problem in contemporary medicine, does not implicate resistance to phage lysis mechanisms. Lytic bacteriophages are able to kill antibiotic-resistant bacteria at the end of the phage infection cycle. Thus, the development of phage therapy is potentially a way to improve the treatment of bacterial infections. However, there are antibacterial phage therapy difficulties specified by broadening the knowledge of the phage nature and influence on the host. It has been shown during experiments that both innate and adaptive immunity are involved in the clearance of phages from the body. Immunological reactions against phages are related to the route of administration and may vary depending on the type of bacterial viruses. For that reason, it is very important to test the immunological response of every single phage, particularly if intravenous therapy is being considered. The lack of these data in previous years was one of the reasons for phage therapy abandonment despite its century-long study. Promising results of recent research led us to look forward to a phage therapy that can be applied on a larger scale and subsequently put it into practice.

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

    NASA Astrophysics Data System (ADS)

    Chapman, Emily; Wu, Xiao-Lun

    2006-03-01

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

  7. Genomic evolution of bacterial populations under coselection by antibiotics and phage.

    PubMed

    Cairns, Johannes; Frickel, Jens; Jalasvuori, Matti; Hiltunen, Teppo; Becks, Lutz

    2016-12-15

    Bacteria live in dynamic systems where selection pressures can alter rapidly, forcing adaptation to the prevailing conditions. In particular, bacteriophages and antibiotics of anthropogenic origin are major bacterial stressors in many environments. We previously observed that populations of the bacterium Pseudomonas fluorescens SBW25 exposed to the lytic bacteriophage SBW25Φ2 and a noninhibitive concentration of the antibiotic streptomycin (coselection) achieved higher levels of phage resistance compared to populations exposed to the phage alone. In addition, the phage became extinct under coselection while remaining present in the phage alone environment. Further, phenotypic tests indicated that these observations might be associated with increased mutation rate under coselection. In this study, we examined the genetic causes behind these phenotypes by whole-genome sequencing clones isolated from the end of the experiments. We were able to identify genetic factors likely responsible for streptomycin resistance, phage resistance and hypermutable (mutator) phenotypes. This constitutes genomic evidence in support of the observation that while the presence of phage did not affect antibiotic resistance, the presence of antibiotic affected phage resistance. We had previously hypothesized an association between mutators and elevated levels of phage resistance under coselection. However, our evidence regarding the mechanism was inconclusive, as although with phage mutators were only found under coselection, additional genomic evidence was lacking and phage resistance was also observed in nonmutators under coselection. More generally, our study provides novel insights into evolution between univariate and multivariate selection (here two stressors), as well as the potential role of hypermutability in natural communities.

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

    PubMed

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

    2014-08-01

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

  9. Safety assessment of Staphylococcus phages of the family Myoviridae based on complete genome sequences

    PubMed Central

    Cui, Zelin; Guo, Xiaokui; Dong, Ke; Zhang, Yan; Li, Qingtian; Zhu, Yongzhang; Zeng, Lingbing; Tang, Rong; Li, Li

    2017-01-01

    Staphylococcus phages of the Myoviridae family have a wide host range and potential applications in phage therapy. In this report, safety assessments of these phages were conducted based on their complete genome sequences. The complete genomes of Staphylococcus phages of the Myoviridae family were analyzed, and the Open Reading Frame (ORFs) were compared with a pool of virulence and antibiotic resistance genes using the BLAST algorithm. In addition, the lifestyle of the phages (virulent or temperate) was also confirmed using PHACTS. The results showed that all phages were lytic and did not contain resistance or virulence genes based on bioinformatic analyses, excluding the possibility that they could be vectors for the dissemination of these undesirable genes. These findings suggest that the phages are safe at the genome level. The SceD-like transglycosylase, which is a biomarker for vancomycin-intermediate strains, was widely distributed in the phage genomes. Approximately 70% of the ORFs encoded in the phage genomes have unknown functions; therefore, their roles in the antibiotic resistance and virulence of Staphylococcus aureus are still unknown and require consideration before use in phage therapy. PMID:28117392

  10. Single-stranded DNA phages: from early molecular biology tools to recent revolutions in environmental microbiology.

    PubMed

    Székely, Anna J; Breitbart, Mya

    2016-03-01

    Single-stranded DNA (ssDNA) phages are profoundly different from tailed phages in many aspects including the nature and size of their genome, virion size and morphology, mutation rate, involvement in horizontal gene transfer, infection dynamics and cell lysis mechanisms. Despite the importance of ssDNA phages as molecular biology tools and model systems, the environmental distribution and ecological roles of these phages have been largely unexplored. Viral metagenomics and other culture-independent viral diversity studies have recently challenged the perspective of tailed, double-stranded DNA (dsDNA) phages, dominance by demonstrating the prevalence of ssDNA phages in diverse habitats. However, the differences between ssDNA and dsDNA phages also substantially limit the efficacy of simultaneously assessing the abundance and diversity of these two phage groups. Here we provide an overview of the major differences between ssDNA and tailed dsDNA phages that may influence their effects on bacterial communities. Furthermore, through the analysis of 181 published metaviromes we demonstrate the environmental distribution of ssDNA phages and present an analysis of the methodological biases that distort their study through metagenomics.

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

    PubMed Central

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

    2011-01-01

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

  12. Inactivation of Escherichia coli phage by pulsed electric field treatment and analysis of inactivation mechanism

    NASA Astrophysics Data System (ADS)

    Tanino, Takanori; Yoshida, Tomoki; Sakai, Kazuki; Ohshima, Takayuki

    2013-03-01

    Inactivation of bacteriophage by pulsed electric field (PEF) treatment, one of the effective procedures for bacteria nonthermal inactivation, was studied. Model phage particles Escherichia coli bacteriophages M13mp18 and λ phage, were successfully inactivated by PEF treatment. The survival ratios of both bacteriophages decreased depending on the PEF treatment time when applied peak voltage was 5 or 7 kV, and the survival ratios after 12 min PEF treatment were 10-4 - 10-5. Electrophoresis analyses of biological molecules of inactivated λ phage detected no degradation of total protein and genomic DNA. These results suggested that the factor of phage inactivation by PEF treatment was not based on the degradation of protein or DNA, but on the destruction of phage particle structure. Sensitivity of E. coli phage to PEF treatment was compared with that of E. coli cell. Phage and MV1184 cell were treated with same condition PEF at 5 kV, respectively. After 12 min treatment, the survival ration of λ phage and MV1184 were 4.0 × 10-5 and 1.7 × 10-3, respectively. The survival ratio of phage was lower than that of MV1184. E. coli cell is more tolerant to inactivation with PEF treatment than coli phage.

  13. Phage sensitivity and prophage carriage in Staphylococcus aureus isolated from foods in Spain and New Zealand.

    PubMed

    Gutiérrez, Diana; Rodríguez-Rubio, Lorena; García, Pilar; Billington, Craig; Premarante, Aruni; Rodríguez, Ana; Martínez, Beatriz

    2016-08-02

    Bacteriophages (phages) are a promising tool for the biocontrol of pathogenic bacteria, including those contaminating food products and causing infectious diseases. However, the success of phage preparations is limited by the host ranges of their constituent phages. The phage resistance/sensitivity profile of eighty seven Staphylococcus aureus strains isolated in Spain and New Zealand from dairy, meat and seafood sources was determined for six phages (Φ11, K, ΦH5, ΦA72, CAPSa1 and CAPSa3). Most of the S. aureus strains were sensitive to phage K (Myoviridae) and CAPSa1 (Siphoviridae) regardless of their origin. There was a higher sensitivity of New Zealand S. aureus strains to phages isolated from both Spain (ΦH5 and ΦA72) and New Zealand (CAPSa1 and CAPSa3). Spanish phages had a higher infectivity on S. aureus strains of Spanish dairy origin, while Spanish strains isolated from other environments were more sensitive to New Zealand phages. Lysogeny was more prevalent in Spanish S. aureus compared to New Zealand strains. A multiplex PCR reaction, which detected ΦH5 and ΦA72 sequences, indicated a high prevalence of these prophages in Spanish S. aureus strains, but were infrequently detected in New Zealand strains. Overall, the correlation between phage resistance and lysogeny in S. aureus strains was found to be weak.

  14. Characterization of Pseudomonas aeruginosa Phage C11 and Identification of Host Genes Required for Virion Maturation

    PubMed Central

    Cui, Xiaoli; You, Jiajia; Sun, Li; Yang, Xiaojing; Zhang, Tian; Huang, Kechong; Pan, Xuewei; Zhang, Fenjiao; He, Yang; Yang, Hongjiang

    2016-01-01

    The underlying mechanisms of phage-host interactions largely remained to be elucidated. In this work, Pseudomonas aeruginosa phage C11 was first characterized as a Myoviridae virus having a linear dsDNA molecule of 94109 bp with 1173 bp identical terminal direct repeats (TDR). Then the mutants resistant to phage C11 were screened in a Tn5G transposon mutant library of P. aeruginosa PAK, including two mutants with decreased adsorption rates (DAR) and five mutants with wild-type adsorption rates (WAR). When the WAR mutants were incubated with phage C11, their growth rates were significantly inhibited; the replication of the phage genomic DNA was detected in all the WAR mutants with the real-time quantitative PCR analysis; and the synthesized phage genomic DNA was processed into monomers for packaging evidenced by the southern blot analysis. Moreover, with strain PAK as indicator, small quantities of phage C11 were synthesized in the WAR mutants. Taken together, these data suggested the identified genes of the WAR mutants are necessary for efficient synthesis of the infectious phage particles. Finally, the WAR mutants were detected sensitive to two other Pseudomonas phages closely related with C11, further implying the evolved diversity and complexity of the phage-host interactions in both sides. PMID:28000703

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

    PubMed

    Christiansen, Rói Hammershaimb; Madsen, Lone; Dalsgaard, Inger; Castillo, Daniel; Kalatzis, Panos G; Middelboe, Mathias

    2016-05-01

    The controlling effect of single and multiple phages on the density of Flavobacterium psychrophilum at different initial multiplicity of infection (MOI) was assessed in batch cultures to explore the potential for phage-based treatment of this important fish pathogen. A high initial phage concentration (MOI = 0.3-4) was crucial for efficient viral lysis, resulting in a 10(4)-10(5)-fold reduction of phage-sensitive cells (both single phages and phage cocktails), which was maintained throughout the incubation (>10 days). Following cell lysis, regrowth of phage-resistant strains was examined and resistant strains were isolated for further characterization. The application of a mathematical model allowed simulation of phage-host interactions and resistance development, confirming indications from strain isolations that phage-sensitive strains dominated the regrowing population (>99.8%) at low MOI and phage-resistant strains (>87.8%) dominated at high MOI. A cross-infectivity test covering 68 isolated strains and 22 phages resulted in 23 different host susceptibility patterns, with 20 of the isolates being resistant to all the applied phages. Eleven isolated strains with different susceptibility patterns had lower growth rates (0.093 to 0.31 h(-1)) than the host strain (0.33 h(-1)), while 10 of 14 examined strains had lost the ability to take up specific substrates as shown by BIOLOG profiles. Despite increased selection for phage resistance at high MOI, the results emphasize that high initial MOI is essential for fast and effective control of F. psychrophilum infection and suggest that the small populations of resistant clones had reduced competitive abilities relative to the sensitive ancestral strain.

  16. The population and evolutionary dynamics of phage and bacteria with CRISPR-mediated immunity.

    PubMed

    Levin, Bruce R; Moineau, Sylvain; Bushman, Mary; Barrangou, Rodolphe

    2013-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with associated genes (cas), form the CRISPR-cas adaptive immune system, which can provide resistance to viruses and plasmids in bacteria and archaea. Here, we use mathematical models, population dynamic experiments, and DNA sequence analyses to investigate the host-phage interactions in a model CRISPR-cas system, Streptococcus thermophilus DGCC7710 and its virulent phage 2972. At the molecular level, the bacteriophage-immune mutant bacteria (BIMs) and CRISPR-escape mutant phage (CEMs) obtained in this study are consistent with those anticipated from an iterative model of this adaptive immune system: resistance by the addition of novel spacers and phage evasion of resistance by mutation in matching sequences or flanking motifs. While CRISPR BIMs were readily isolated and CEMs generated at high rates (frequencies in excess of 10(-6)), our population studies indicate that there is more to the dynamics of phage-host interactions and the establishment of a BIM-CEM arms race than predicted from existing assumptions about phage infection and CRISPR-cas immunity. Among the unanticipated observations are: (i) the invasion of phage into populations of BIMs resistant by the acquisition of one (but not two) spacers, (ii) the survival of sensitive bacteria despite the presence of high densities of phage, and (iii) the maintenance of phage-limited communities due to the failure of even two-spacer BIMs to become established in populations with wild-type bacteria and phage. We attribute (i) to incomplete resistance of single-spacer BIMs. Based on the results of additional modeling and experiments, we postulate that (ii) and (iii) can be attributed to the phage infection-associated production of enzymes or other compounds that induce phenotypic phage resistance in sensitive bacteria and kill resistant BIMs. We present evidence in support of these hypotheses and discuss the implications of these results

  17. Methicillin-resistant Staphylococcus aureus phage plaque size enhancement using sublethal concentrations of antibiotics.

    PubMed

    Kaur, Sandeep; Harjai, Kusum; Chhibber, Sanjay

    2012-12-01

    Phage therapy presents an alternative approach against the emerging methicillin-resistant Staphylococcus aureus (MRSA) threat. Some of the problems encountered during isolation of MRSA phages include the high prevalence of enteric phages in natural sources, nonspecific absorption of viable phage, and the formation of pinpoint or tiny plaques. The phage isolated in this study, MR-5, also formed tiny plaques against its host S. aureus ATCC 43300 (MRSA), making its detection and enumeration difficult. An improved method of increasing the plaque size of MRSA phage by incorporating sublethal concentrations of three different classes of antibiotics (inhibitors of protein synthesis) in the classical double-layer agar (DLA) method was investigated. The β-lactam and quinolone antibiotics commonly employed in earlier studies for increasing the plaque size did not show any significant effect on the plaque size of isolated MR-5 phage. Linezolid (oxazolidinone class), tetracycline, and ketolide antibiotics brought significant enhancements (3 times the original size) in the plaque size of MR-5 phage. Prior treatment with these antibiotics resulted in significant reductions in the time of adsorption and the latent period of MR-5 phage. To rule out whether the action of linezolid (which brought the maximum increase in plaque size) was specific for a single phage only, its effect on the plaque size of seven other S. aureus-specific phages was also assessed. Significant enhancements in the plaque size of these phages were observed. These results indicate that this modification can therefore safely be incorporated in the traditional DLA overlay method to search for new MRSA-virulent phages.

  18. Genomics of staphylococcal Twort-like phages--potential therapeutics of the post-antibiotic era.

    PubMed

    Łobocka, Małgorzata; Hejnowicz, Monika S; Dąbrowski, Kamil; Gozdek, Agnieszka; Kosakowski, Jarosław; Witkowska, Magdalena; Ulatowska, Magdalena I; Weber-Dąbrowska, Beata; Kwiatek, Magdalena; Parasion, Sylwia; Gawor, Jan; Kosowska, Helena; Głowacka, Aleksandra

    2012-01-01

    Polyvalent bacteriophages of the genus Twort-like that infect clinically relevant Staphylococcus strains may be among the most promising phages with potential therapeutic applications. They are obligatorily lytic, infect the majority of Staphylococcus strains in clinical strain collections, propagate efficiently and do not transfer foreign DNA by transduction. Comparative genomic analysis of 11 S. aureus/S. epidermidis Twort-like phages, as presented in this chapter, emphasizes their strikingly high similarity and clear divergence from phage Twort of the same genus, which might have evolved in hosts of a different species group. Genetically, these phages form a relatively isolated group, which minimizes the risk of acquiring potentially harmful genes. The order of genes in core parts of their 127 to 140-kb genomes is conserved and resembles that found in related representatives of the Spounavirinae subfamily of myoviruses. Functions of certain conserved genes can be predicted based on their homology to prototypical genes of model spounavirus SPO1. Deletions in the genomes of certain phages mark genes that are dispensable for phage development. Nearly half of the genes of these phages have no known homologues. Unique genes are mostly located near termini of the virion DNA molecule and are expressed early in phage development as implied by analysis of their potential transcriptional signals. Thus, many of them are likely to play a role in host takeover. Single genes encode homologues of bacterial virulence-associated proteins. They were apparently acquired by a common ancestor of these phages by horizontal gene transfer but presumably evolved towards gaining functions that increase phage infectivity for bacteria or facilitate mature phage release. Major differences between the genomes of S. aureus/S. epidermidis Twort-like phages consist of single nucleotide polymorphisms and insertions/deletions of short stretches of nucleotides, single genes, or introns of group I

  19. Genetic and Physical Structure of Salmonella-coli Phage Hybrids and Development of New Generalized Transducing Hybrid Phages for E. Coli.

    DTIC Science & Technology

    1984-05-01

    Walter Reed Army Institute of Research began searching for E . coli -S. typhimurium recombinants in 1971. After isolating E . coli -S. typhimurium hybrids...which are common hosts for the various coli and Salmonella phages, we developed efficient selective methods of hybrids between Salmonella and E . coli phages

  20. Purification of molecular machines and nanomotors using phage-derived monoclonal antibody fragments.

    PubMed

    Esteban, Olga; Christ, Daniel; Stock, Daniela

    2013-01-01

    Molecular machines and nanomotors are sophisticated biological assemblies that convert potential energy stored either in transmembrane ion gradients or in ATP into kinetic energy. Studying these highly dynamic biological devices by X-ray crystallography is challenging, as they are difficult to produce, purify, and crystallize. Phage display technology allows us to put a handle on these molecules in the form of highly specific antibody fragments that can also stabilize conformations and allow versatile labelling for electron microscopy, immunohistochemistry, and biophysics experiments.Here, we describe a widely applicable protocol for selecting high-affinity monoclonal antibody fragments against a complex molecular machine, the A-type ATPase from T. thermophilus that allows fast and simple purification of this transmembrane rotary motor from its wild-type source. The approach can be readily extended to other integral membrane proteins and protein complexes as well as to soluble molecular machines and nanomotors.

  1. Negative selection and stringency modulation in phage-assisted constinuous evolution

    PubMed Central

    Carlson, Jacob C.; Badran, Ahmed H.; Guggiana-Nilo, Drago A.; Liu, David R.

    2014-01-01

    Phage-assisted continuous evolution (PACE) uses a modified filamentous bacteriophage life cycle to dramatically accelerate laboratory evolution experiments. In this work we expand the scope and capabilities of the PACE method with two key advances that enable the evolution of biomolecules with radically altered or highly specific new activities. First, we implemented small molecule-controlled modulation of selection stringency that enables otherwise inaccessible activities to be evolved directly from inactive starting libraries through a period of evolutionary drift. Second, we developed a general negative selection that enables continuous counter-selection against undesired activities. We integrated these developments to continuously evolve mutant T7 RNA polymerase enzymes with ∼10,000-fold altered, rather than merely broadened, substrate specificities during a single three-day PACE experiment. The evolved enzymes exhibit specificity for their target substrate that exceeds that of wild-type RNA polymerases for their cognate substrates, while maintaining wild-type-like levels of activity. PMID:24487694

  2. Analyses of the Distribution Patterns of Burkholderia pseudomallei and Associated Phages in Soil Samples in Thailand Suggest That Phage Presence Reduces the Frequency of Bacterial Isolation

    PubMed Central

    Withatanung, Patoo; Chantratita, Narisara; Muangsombut, Veerachat; Saiprom, Natnaree; Lertmemongkolchai, Ganjana; Klumpp, Jochen; Clokie, Martha R. J.; Galyov, Edouard E.

    2016-01-01

    Background Burkholderia pseudomallei is a soil saprophytic bacterium that causes melioidosis. The infection occurs through cutaneous inoculation, inhalation or ingestion. Bacteriophages (phages) in the same ecosystem may significantly impact the biology of this bacterium in the environment, and in their culturability in the laboratory. Methods/Principal Findings The soil samples were analysed for the presence of bacteria using culture methods, and for phages using plaque assays on B. pseudomallei strain 1106a lawns. Of the 86 soil samples collected from northeastern Thailand, B. pseudomallei was cultured from 23 (26.7%) samples; no phage capable of infecting B. pseudomallei was detected in these samples. In contrast, phages capable of infecting B. pseudomallei, but no bacteria, were present in 10 (11.6%) samples. B. pseudomallei and their phages were co-isolated from only 3 (3.5%) of soil samples. Since phage capable of infecting B. pseudomallei could not have appeared in the samples without the prior presence of bacteria, or exposure to bacteria nearby, our data suggest that all phage-positive/bacteria-negative samples have had B. pseudomallei in or in a close proximity to them. Taken together, these findings indicate that the presence of phages may influence the success of B. pseudomallei isolation. Transmission electron microscopy revealed that the isolated phages are podoviruses. The temperate phages residing in soil-isolated strains of B. pseudomallei that were resistant to the dominant soil borne phages could be induced by mitomycin C. These induced-temperate phages were closely related, but not identical, to the more dominant soil-isolated phage type. Conclusion/Significance The presence of podoviruses capable of infecting B. pseudomallei may affect the success of the pathogen isolation from the soil. The currently used culture-based methods of B. pseudomallei isolation appear to under-estimate the bacterial abundance. The detection of phage capable of

  3. Safety analysis of a Russian phage cocktail: from metagenomic analysis to oral application in healthy human subjects.

    PubMed

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

    2013-09-01

    Phage therapy has a long tradition in Eastern Europe, where preparations are comprised of complex phage cocktails whose compositions have not been described. We investigated the composition of a phage cocktail from the Russian pharmaceutical company Microgen targeting Escherichia coli/Proteus infections. Electron microscopy identified six phage types, with numerically T7-like phages dominating over T4-like phages. A metagenomic approach using taxonomical classification, reference mapping and de novo assembly identified 18 distinct phage types, including 7 genera of Podoviridae, 2 established and 2 proposed genera of Myoviridae, and 2 genera of Siphoviridae. De novo assembly yielded 7 contigs greater than 30 kb, including a 147-kb Myovirus genome and a 42-kb genome of a potentially new phage. Bioinformatic analysis did not reveal undesired genes and a small human volunteer trial did not associate adverse effects with oral phage exposure.

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

    PubMed

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

    2015-02-01

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

  5. Mobile DNA elements in T4 and related phages

    PubMed Central

    2010-01-01

    Mobile genetic elements are common inhabitants of virtually every genome where they can exert profound influences on genome structure and function in addition to promoting their own spread within and between genomes. Phage T4 and related phage have long served as a model system for understanding the molecular mechanisms by which a certain class of mobile DNA, homing endonucleases, promote their spread. Homing endonucleases are site-specific DNA endonucleases that initiate mobility by introducing double-strand breaks at defined positions in genomes lacking the endonuclease gene, stimulating repair and recombination pathways that mobilize the endonuclease coding region. In phage T4, homing endonucleases were first discovered as encoded within the self-splicing td, nrdB and nrdD introns of T4. Genomic data has revealed that homing endonucleases are extremely widespread in T-even-like phage, as evidenced by the astounding fact that ~11% of the T4 genome encodes homing endonuclease genes, with most of them located outside of self-splicing introns. Detailed studies of the mobile td intron and its encoded endonuclease, I-TevI, have laid the foundation for genetic, biochemical and structural aspects that regulate the mobility process, and more recently have provided insights into regulation of homing endonuclease function. Here, we summarize the current state of knowledge regarding T4-encoded homing endonucleases, with particular emphasis on the td/I-TevI model system. We also discuss recent progress in the biology of free-standing endonucleases, and present areas of future research for this fascinating class of mobile genetic elements. PMID:21029434

  6. Phage display as a technology delivering on the promise of peptide drug discovery.

    PubMed

    Hamzeh-Mivehroud, Maryam; Alizadeh, Ali Akbar; Morris, Michael B; Church, W Bret; Dastmalchi, Siavoush

    2013-12-01

    Phage display represents an important approach in the development pipeline for producing peptides and peptidomimetics therapeutics. Using randomly generated DNA sequences and molecular biology techniques, large diverse peptide libraries can be displayed on the phage surface. The phage library can be incubated with a target of interest and the phage which bind can be isolated and sequenced to reveal the displayed peptides' primary structure. In this review, we focus on the 'mechanics' of the phage display process, whilst highlighting many diverse and subtle ways it has been used to further the drug-development process, including the potential for the phage particle itself to be used as a drug carrier targeted to a particular pathogen or cell type in the body.

  7. Isolation and characterization of marine psychrophilic phage-host systems from Arctic sea ice.

    PubMed

    Borriss, Michael; Helmke, Elisabeth; Hanschke, Renate; Schweder, Thomas

    2003-10-01

    Phage-host systems from extreme cold environments have rarely been surveyed. This study is concerned with the isolation and characterization of three different phage-host systems from Arctic sea ice and melt pond samples collected north-west of Svalbard (Arctic). On the basis of 16S rDNA sequences, the three bacterial phage hosts exhibited the greatest similarity to the species Shewanella frigidimarina (96.0%), Flavobacterium hibernum (94.0%), and Colwellia psychrerythraea (98.4%), respectively. The host bacteria are psychrophilic with good growth at 0 degrees C, resulting in a rapid formation of visible colonies at this temperature. The phages showed an even more pronounced adaptation to cold temperatures than the bacteria, with growth maxima below 14 degrees C and good plaque formation at 0 degrees C. Transmission electron microscopy (TEM) examinations revealed that the bacteriophages belonged to the tailed, double-stranded DNA phage families Siphoviridae and Myoviridae. All three phages were host-specific.

  8. Lactococcal 949 Group Phages Recognize a Carbohydrate Receptor on the Host Cell Surface

    PubMed Central

    Mahony, Jennifer; Randazzo, Walter; Neve, Horst; Settanni, Luca

    2015-01-01

    Lactococcal bacteriophages represent one of the leading causes of dairy fermentation failure and product inconsistencies. A new member of the lactococcal 949 phage group, named WRP3, was isolated from cheese whey from a Sicilian factory in 2011. The genome sequence of this phage was determined, and it constitutes the largest lactococcal phage genome currently known, at 130,008 bp. Detailed bioinformatic analysis of the genomic region encoding the presumed initiator complex and baseplate of WRP3 has aided in the functional assignment of several open reading frames (ORFs), particularly that for the receptor binding protein required for host recognition. Furthermore, we demonstrate that the 949 phages target cell wall phospho-polysaccharides as their receptors, accounting for the specificity of the interactions of these phages with their lactococcal hosts. Such information may ultimately aid in the identification of strains/strain blends that do not present the necessary saccharidic target for infection by these problematic phages. PMID:25746988

  9. Genomic sequence of temperate phage TEM126 isolated from wild type S. aureus.

    PubMed

    Lee, Young-Duck; Chang, Hyo-Ihl; Park, Jong-Hyun

    2011-04-01

    Bacteriophage TEM126, a newly isolated temperate phage from a mitomycin-C-induced lysate of wild-type Staphylococcus aureus isolated from food, has an isometric head, a noncontractile tail, and a double-stranded DNA genome with a length of 33,540 bp and a G+C content of 33.94%. Bioinformatics analysis of the phage genome revealed 44 putative open reading frames (ORFs). Predicted protein products of the ORFs were determined and described. Temperate phage TEM126 can be classified as a member of the family Siphoviridae by morphology and genome structure. Temperate phage TEM126 showed 84% similarity with Staphylococcus phage phiNM1. To our knowledge, this is the first report of genomic sequencing and characterization of temperate phage TEM126 from a wild-type S. aureus isolated from foods in Korea.

  10. Targeting human embryonic stem cells with quantum dot-conjugated phages.

    PubMed

    Zhao, Wenxiu; Jin, Lei; Yuan, Hang; Tan, Zhiyang; Zhou, Changhua; Li, Lin Song; Ma, Lan

    2013-11-05

    Targeting embryonic stem cells (ESCs) is important for ESC labeling, drug delivery and cell fate control. In this study, we identified twenty-two phage clones that bind specifically to the hESC cell line X-01, which was derived from human blastocysts of Chinese origin. One phage (H178), which displays the sequence VGGEAWSSPTDL, showed higher binding affinity to hESCs than to a monkey ES cell line (RS366.4) and two mouse ES cell lines (R1 and E14). Using quantum dots (QDs) conjugated to the H178 phage, we demonstrate that the phage can specifically bind to hESCs in vitro. Our results suggest a possible interaction between the selected peptide and the stem cell extracellular matrix (ECM). The selection method described here allows rapid and efficient screening of unique phage clones and targeting cells. The phages displaying peptides identified by this study have potential applications for cargo delivery and receptor studies.

  11. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity.

    PubMed

    Pope, Welkin H; Bowman, Charles A; Russell, Daniel A; Jacobs-Sera, Deborah; Asai, David J; Cresawn, Steven G; Jacobs, William R; Hendrix, Roger W; Lawrence, Jeffrey G; Hatfull, Graham F

    2015-04-28

    The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery.

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

    SciTech Connect

    Stern, Adi; Sorek, Rotem

    2010-10-26

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

  13. Bacteriophage-encoded bacterial virulence factors and phage-pathogenicity island interactions.

    PubMed

    Boyd, E Fidelma

    2012-01-01

    The role of bacteriophages as natural vectors for some of the most potent bacterial toxins is well recognized and includes classical type I membrane-acting superantigens, type II pore-forming lysins, and type III exotoxins, such as diphtheria and botulinum toxins. Among Gram-negative pathogens, a novel class of bacterial virulence factors called effector proteins (EPs) are phage encoded among pathovars of Escherichia coli, Shigella spp., and Salmonella enterica. This chapter gives an overview of the different types of virulence factors encoded within phage genomes based on their role in bacterial pathogenesis. It also discusses phage-pathogenicity island interactions uncovered from studies of phage-encoded EPs. A detailed examination of the filamentous phage CTXφ that encodes cholera toxin is given as the sole example to date of a single-stranded DNA phage that encodes a bacterial toxin.

  14. Listeria phage A511, a model for the contractile tail machineries of SPO1-related bacteriophages.

    PubMed

    Habann, Matthias; Leiman, Petr G; Vandersteegen, Katrien; Van den Bossche, An; Lavigne, Rob; Shneider, Mikhail M; Bielmann, Regula; Eugster, Marcel R; Loessner, Martin J; Klumpp, Jochen

    2014-04-01

    Recognition of the bacterial host and attachment to its surface are two critical steps in phage infection. Here we report the identification of Gp108 as the host receptor-binding protein of the broad host-range, virulent Listeria phage A511. The ligands for Gp108 were found to be N-acetylglucosamine and rhamnose substituents of the wall teichoic acids of the bacterial cell wall. Transmission electron microscopy and immunogold-labelling allowed us to create a model of the A511 baseplate in which Gp108 forms emanating short tail fibres. Data obtained for related phages, such as Staphylococcus phages ISP and Twort, demonstrate the evolutionary conservation of baseplate components and receptor-binding proteins within the Spounavirinae subfamily, and contractile tail machineries in general. Our data reveal key elements in the infection process of large phages infecting Gram-positive bacteria and generate insights into the complex adsorption process of phage A511 to its bacterial host.

  15. 60Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage.

    PubMed

    Yamamoto, Tatsuo; Kojio, Seiichi; Taneike, Ikue; Nakagawa, Saori; Iwakura, Nobuhiro; Wakisaka-Saito, Noriko

    2003-05-16

    Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by (60)Co irradiation at 1 x l0(3) gray (1 kGy) or higher. However, a low dose of irradiation (0.1-0.3 kGy) markedly induced Stx phage from STEC. Stx production was observed in parallel to the phage induction. Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing. Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage. The findings suggest that (1). although (60)Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2). irradiation differentially inactivates some activities of Stx.

  16. Cell fate decisions emerge as phages cooperate or compete inside their host

    PubMed Central

    Trinh, Jimmy T.; Székely, Tamás; Shao, Qiuyan; Balázsi, Gábor; Zeng, Lanying

    2017-01-01

    The system of the bacterium Escherichia coli and its virus, bacteriophage lambda, is paradigmatic for gene regulation in cell-fate development, yet insight about its mechanisms and complexities are limited due to insufficient resolution of study. Here we develop a 4-colour fluorescence reporter system at the single-virus level, combined with computational models to unravel both the interactions between phages and how individual phages determine cellular fates. We find that phages cooperate during lysogenization, compete among each other during lysis, and that confusion between the two pathways occasionally occurs. Additionally, we observe that phage DNAs have fluctuating cellular arrival times and vie for resources to replicate, enabling the interplay during different developmental paths, where each phage genome may make an individual decision. These varied strategies could separate the selection for replication-optimizing beneficial mutations during lysis from sequence diversification during lysogeny, allowing rapid adaptation of phage populations for various environments. PMID:28165024

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

    PubMed

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

    2016-01-19

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

  18. Phage therapy treatment of the coral pathogen Vibrio coralliilyticus

    PubMed Central

    Cohen, Yossi; Joseph Pollock, F; Rosenberg, Eugene; Bourne, David G

    2013-01-01

    Vibrio coralliilyticus is an important coral pathogen demonstrated to cause disease outbreaks worldwide. This study investigated the feasibility of applying bacteriophage therapy to treat the coral pathogen V. coralliilyticus. A specific bacteriophage for V. coralliilyticus strain P1 (LMG23696), referred to here as bacteriophage YC, was isolated from the seawater above corals at Nelly Bay, Magnetic Island, central Great Barrier Reef (GBR), the same location where the bacterium was first isolated. Bacteriophage YC was shown to be a lytic phage belonging to the Myoviridae family, with a rapid replication rate, high burst size, and high affinity to its host. By infecting its host bacterium, bacteriophage YC was able to prevent bacterial-induced photosystem inhibition in pure cultures of Symbiodinium, the photosymbiont partner of coral and a target for virulence factors produced by the bacterial pathogen. Phage therapy experiments using coral juveniles in microtiter plates as a model system revealed that bacteriophage YC was able to prevent V. coralliilyticus-induced photoinactivation and tissue lysis. These results demonstrate that bacteriophage YC has the potential to treat coral disease outbreaks caused by the bacterial pathogen V. coralliilyticus, making it a good candidate for phage therapy treatment of coral disease. PMID:23239510

  19. Probing ADAMTS13 substrate specificity using phage display.

    PubMed

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

    2015-01-01

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

  20. Aptamer-phage reporters for ultrasensitive lateral flow assays

    PubMed Central

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

    2015-01-01

    We introduce the modification of bacteriophage particles with aptamers for the use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin-biotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ~100 times lower than those of previously reported IgE assays. PMID:26456715

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

    PubMed

    Szymczak, Paula; Janzen, Thomas; Neves, Ana Rute; Kot, Witold; Hansen, Lars H; Lametsch, René; Neve, Horst; Franz, Charles M A P; Vogensen, Finn K

    2017-03-01

    Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed.IMPORTANCEStreptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.

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

    PubMed Central

    Szymczak, Paula; Neves, Ana Rute; Kot, Witold; Hansen, Lars H.; Lametsch, René; Neve, Horst; Franz, Charles M. A. P.

    2016-01-01

    ABSTRACT Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed. IMPORTANCE Streptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations. PMID:28039135

  3. Salmonella typhimurium phage type 141 infections in Sheffield during 1984 and 1985: association with hens' eggs.

    PubMed Central

    Chapman, P. A.; Rhodes, P.; Rylands, W.

    1988-01-01

    Food poisoning due to Salmonella typhimurium phage type 141 was unusual in the Sheffield area before 1984. The sudden increase in incidence of this phage type during 1984 and 1985, and its causative role in several small outbreaks in this period have been investigated. Epidemiological and laboratory investigations suggested that hens' eggs were the most likely source of S. typhimurium phage type 141. PMID:3042440

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

    DTIC Science & Technology

    2016-09-01

    isolates. However, phage assays generally require that suspect colonies be sub- cultured onto a fresh agar plate to generate a dense lawn against...consume valuable time. Researchers have shown that temporal changes in the dielectric permittivity of bacterial micro- cultures differ for chemically or...to detect ɣ phage-induced stress in susceptible B. anthracis micro- cultures and thereby reduce both the time and biomass required to perform phage

  5. Identification of gliadin-binding peptides by phage display

    PubMed Central

    2011-01-01

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

  6. Efficient identification of phosphatidylserine-binding proteins by ORF phage display

    SciTech Connect

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

    2009-08-14

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

  7. Phage display for the generation of antibodies for proteome research, diagnostics and therapy.

    PubMed

    Schirrmann, Thomas; Meyer, Torsten; Schütte, Mark; Frenzel, André; Hust, Michael

    2011-01-10

    Twenty years after its development, antibody phage display using filamentous bacteriophage represents the most successful in vitro antibody selection technology. Initially, its development was encouraged by the unique possibility of directly generating recombinant human antibodies for therapy. Today, antibody phage display has been developed as a robust technology offering great potential for automation. Generation of monospecific binders provides a valuable tool for proteome research, leading to highly enhanced throughput and reduced costs. This review presents the phage display technology, application areas of antibodies in research, diagnostics and therapy and the use of antibody phage display for these applications.

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

    PubMed

    Mattila, Sari; Ruotsalainen, Pilvi; Jalasvuori, Matti

    2015-01-01

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

  9. Synergy and Order Effects of Antibiotics and Phages in Killing Pseudomonas aeruginosa Biofilms

    PubMed Central

    Chaudhry, Waqas Nasir; Concepción-Acevedo, Jeniffer; Park, Taehyun; Andleeb, Saadia; Bull, James J.

    2017-01-01

    In contrast to planktonic cells, bacteria imbedded biofilms are notoriously refractory to treatment by antibiotics or bacteriophage (phage) used alone. Given that the mechanisms of killing differ profoundly between drugs and phages, an obvious question is whether killing is improved by combining antibiotic and phage therapy. However, this question has only recently begun to be explored. Here, in vitro biofilm populations of Pseudomonas aeruginosa PA14 were treated singly and with combinations of two phages and bactericidal antibiotics of five classes. By themselves, phages and drugs commonly had only modest effects in killing the bacteria. However some phage-drug combinations reduced bacterial densities to well below that of the best single treatment; in some cases, bacterial densities were reduced even below the level expected if both agents killed independently of each other (synergy). Furthermore, there was a profound order effect in some cases: treatment with phages before drugs achieved maximum killing. Combined treatment was particularly effective in killing in Pseudomonas biofilms grown on layers of cultured epithelial cells. Phages were also capable of limiting the extent to which minority populations of bacteria resistant to the treating antibiotic ascend. The potential of combined antibiotic and phage treatment of biofilm infections is discussed as a realistic way to evaluate and establish the use of bacteriophage for the treatment of humans. PMID:28076361

  10. Conservation of phage reference materials and water samples containing bacteriophages of enteric bacteria.

    PubMed

    Mendez, J; Jofre, J; Lucena, F; Contreras, N; Mooijman, K; Araujo, R

    2002-12-01

    The survival was determined in different conservation conditions of: somatic coliphages, F-specific RNA bacteriophages and phages infecting Bacteroides fragilis proposed as model micro-organisms for water quality control. Titres of phages of all groups either in pure culture phage suspensions or in naturally occurring phage suspensions were stable at (-70+/-10) degrees C and at (-20+/-5) degrees C when protected with glycerol. Moreover, phage analysis of stored suspensions demonstrated that their numbers were homogeneous, both between vials and within vials, and consequently they can be used as reference materials. Furthermore, changes in the storage temperature of the vials cause unpredictable changes in the numbers of bacteriophages. Consequently, phage reference materials and samples containing a quantitative number of phages must be maintained and dispatched at a constant temperature. Consequently, the results indicate that bacteriophages should be packed in dry ice during transport and storage. Finally, the number of phages in water samples stored at (5+/-3) degrees C in the dark does not decrease significantly during the first 72 h of storage. In addition, phage concentrates from natural samples obtained by adsorption-elution to cellulose nitrate filters and mixed with 10% glycerol were stable at least for 2 months at (-70+/-10) degrees C and at (-20+/-5) degrees C.

  11. Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages.

    PubMed

    Chen, John; Carpena, Nuria; Quiles-Puchalt, Nuria; Ram, Geeta; Novick, Richard P; Penadés, José R

    2015-05-01

    Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.

  12. Coexistence of phage and bacteria on the boundary of self-organized refuges

    PubMed Central

    Heilmann, Silja; Sneppen, Kim; Krishna, Sandeep

    2012-01-01

    Bacteriophage are voracious predators of bacteria and a major determinant in shaping bacterial life strategies. Many phage species are virulent, meaning that infection leads to certain death of the host and immediate release of a large batch of phage progeny. Despite this apparent voraciousness, bacteria have stably coexisted with virulent phages for eons. Here, using individual-based stochastic spatial models, we study the conditions for achieving coexistence on the edge between two habitats, one of which is a bacterial refuge with conditions hostile to phage whereas the other is phage friendly. We show how bacterial density-dependent, or quorum-sensing, mechanisms such as the formation of biofilm can produce such refuges and edges in a self-organized manner. Coexistence on these edges exhibits the following properties, all of which are observed in real phage–bacteria ecosystems but difficult to achieve together in nonspatial ecosystem models: (i) highly efficient virulent phage with relatively long lifetimes, high infection rates and large burst sizes; (ii) large, stable, and high-density populations of phage and bacteria; (iii) a fast turnover of both phage and bacteria; and (iv) stability over evolutionary timescales despite imbalances in the rates of phage vs. bacterial evolution. PMID:22807479

  13. Anti-CRISPR Proteins: Counterattack of Phages on Bacterial Defense (CRISPR/Cas) System.

    PubMed

    Chaudhary, Kulbhushan; Chattopadhyay, Anirudha; Pratap, Dharmendra

    2017-03-01

    Since the dawn of life there is a never ending strife between bacteria and phages. Both are perpetually changing their strategies to take over each other. CRISPR/Cas is the most widespread defense system used by bacteria against mobile genetic elements (MGEs) such as phages, cojugative palsmids, transoposons and pathogenicity islands. This system utilizes small guide RNA molecules to protect against phages infection and invasion by MGEs. Phages circumvent to these antiviral barriers by point mutation in PAM (protospacer-adjacent motif) sequence, genome rearrangements and by using anti-CRISPR proteins. This article is protected by copyright. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  16. Going viral: next-generation sequencing applied to phage populations in the human gut.

    PubMed

    Reyes, Alejandro; Semenkovich, Nicholas P; Whiteson, Katrine; Rohwer, Forest; Gordon, Jeffrey I

    2012-09-01

    Over the past decade, researchers have begun to characterize viral diversity using metagenomic methods. These studies have shown that viruses, the majority of which infect bacteria, are probably the most genetically diverse components of the biosphere. Here, we briefly review the incipient rise of a phage biology renaissance, which has been catalysed by advances in next-generation sequencing. We explore how work characterizing phage diversity and lifestyles in the human gut is changing our view of ourselves as supra-organisms. Finally, we discuss how a renewed appreciation of phage dynamics may yield new applications for phage therapies designed to manipulate the structure and functions of our gut microbiomes.

  17. The targeted recognition of Lactococcus lactis phages to their polysaccharide receptors.

    PubMed

    McCabe, Orla; Spinelli, Silvia; Farenc, Carine; Labbé, Myriam; Tremblay, Denise; Blangy, Stéphanie; Oscarson, Stefan; Moineau, Sylvain; Cambillau, Christian

    2015-05-01

    Each phage infects a limited number of bacterial strains through highly specific interactions of the receptor-binding protein (RBP) at the tip of phage tail and the receptor at the bacterial surface. Lactococcus lactis is covered with a thin polysaccharide pellicle (hexasaccharide repeating units), which is used by a subgroup of phages as a receptor. Using L. lactis and phage 1358 as a model, we investigated the interaction between the phage RBP and the pellicle hexasaccharide of the host strain. A core trisaccharide (TriS), derived from the pellicle hexasaccharide repeating unit, was chemically synthesised, and the crystal structure of the RBP/TriS complex was determined. This provided unprecedented structural details of RBP/receptor site-specific binding. The complete hexasaccharide repeating unit was modelled and found to aptly fit the extended binding site. The specificity observed in in vivo phage adhesion assays could be interpreted in view of the reported structure. Therefore, by combining synthetic carbohydrate chemistry, X-ray crystallography and phage plaquing assays, we suggest that phage adsorption results from distinct recognition of the RBP towards the core TriS or the remaining residues of the hexasacchride receptor. This study provides a novel insight into the adsorption process of phages targeting saccharides as their receptors.

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

    PubMed Central

    Mattila, Sari; Ruotsalainen, Pilvi; Jalasvuori, Matti

    2015-01-01

    Bacteriophages are bacterial viruses, capable of killing even multi-drug resistant bacterial cells. For this reason, therapeutic use of phages is considered as a possible alternative to conventional antibiotics. However, phages are very host specific in comparison to wide-spectrum antibiotics and thus preparation of phage-cocktails beforehand against pathogens can be difficult. In this study, we evaluate whether it may be possible to isolate phages on-demand from environmental reservoir. We attempted to enrich infectious bacteriophages from sewage against nosocomial drug-resistant bacterial strains of different medically important species in order to evaluate the probability of discovering novel therapeutic phages. Stability and host-range were determined for the acquired phages. Our results suggest that on-demand isolation of phages is possible against Pseudomonas aeruginosa, Salmonella and extended spectrum beta-lactamase Escherichia coli and Klebsiella pneumoniae. The probability of finding suitable phages was less than 40% against vancomycin resistant Enterococcus and Acinetobacter baumannii strains. Furthermore, isolation of new phages against methicillin resistant Staphylococcus aureus strains was found to be very difficult. PMID:26617601

  19. Staphylococcal pathogenicity island interference with helper phage reproduction is a paradigm of molecular parasitism.

    PubMed

    Ram, Geeta; Chen, John; Kumar, Krishan; Ross, Hope F; Ubeda, Carles; Damle, Priyadarshan K; Lane, Kristin D; Penadés, José R; Christie, Gail E; Novick, Richard P

    2012-10-02

    Staphylococcal pathogenicity islands (SaPIs) carry superantigen and resistance genes and are extremely widespread in Staphylococcus aureus and in other Gram-positive bacteria. SaPIs represent a major source of intrageneric horizontal gene transfer and a stealth conduit for intergeneric gene transfer; they are phage satellites that exploit the life cycle of their temperate helper phages with elegant precision to enable their rapid replication and promiscuous spread. SaPIs also interfere with helper phage reproduction, blocking plaque formation, sharply reducing burst size and enhancing the survival of host cells following phage infection. Here, we show that SaPIs use several different strategies for phage interference, presumably the result of convergent evolution. One strategy, not described previously in the bacteriophage microcosm, involves a SaPI-encoded protein that directly and specifically interferes with phage DNA packaging by blocking the phage terminase small subunit. Another strategy involves interference with phage reproduction by diversion of the vast majority of virion proteins to the formation of SaPI-specific small infectious particles. Several SaPIs use both of these strategies, and at least one uses neither but possesses a third. Our studies illuminate a key feature of the evolutionary strategy of these mobile genetic elements, in addition to their carriage of important genes-interference with helper phage reproduction, which could ensure their transferability and long-term persistence.

  20. Romulus and Remus, two phage isolates representing a distinct clade within the Twortlikevirus genus, display suitable properties for phage therapy applications.

    PubMed

    Vandersteegen, Katrien; Kropinski, Andrew M; Nash, John H E; Noben, Jean-Paul; Hermans, Katleen; Lavigne, Rob

    2013-03-01

    The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.

  1. A minireview on the in vitro and in vivo experiments with anti-Escherichia coli O157:H7 phages as potential biocontrol and phage therapy agents.

    PubMed

    Sabouri, Salehe; Sepehrizadeh, Zargham; Amirpour-Rostami, Sahar; Skurnik, Mikael

    2017-02-21

    Phage therapy is an old method of combating bacterial pathogens that has recently been taken into consideration due to the alarming spread of antibiotic resistance. Escherichia coli O157:H7 is a foodborne pathogen that causes hemorrhagic colitis and life-threatening Hemolytic Uremic Syndrome (HUS). There are several studies on isolation of specific phages against E. coli O157:H7 and more than 60 specific phages have been published so far. Although in vitro experiments have been successful in elimination or reduction of E. coli O157:H7numbers, in vivo experiments have not been as promising. This may be due to escape of bacteria to locations where phages have difficulties to enter or due to the adverse conditions in the gastrointestinal tract that affect phage viability and proliferation. To get around the latter obstacle, an alternative phage delivery method such as polymer microencapsulation should be tried. While the present time results are not very encouraging the work should be continued as more efficient phage treatment regimens might be found in future.

  2. Genome sequence and global gene expression of Q54, a new phage species linking the 936 and c2 phage species of Lactococcus lactis.

    PubMed

    Fortier, Louis-Charles; Bransi, Ali; Moineau, Sylvain

    2006-09-01

    The lytic lactococcal phage Q54 was previously isolated from a failed sour cream production. Its complete genomic sequence (26,537 bp) is reported here, and the analysis indicated that it represents a new Lactococcus lactis phage species. A striking feature of phage Q54 is the low level of similarity of its proteome (47 open reading frames) with proteins in databases. A global gene expression study confirmed the presence of two early gene modules in Q54. The unusual configuration of these modules, combined with results of comparative analysis with other lactococcal phage genomes, suggests that one of these modules was acquired through recombination events between c2- and 936-like phages. Proteolytic cleavage and cross-linking of the major capsid protein were demonstrated through structural protein analyses. A programmed translational frameshift between the major tail protein (MTP) and the receptor-binding protein (RBP) was also discovered. A "shifty stop" signal followed by putative secondary structures is likely involved in frameshifting. To our knowledge, this is only the second report of translational frameshifting (+1) in double-stranded DNA bacteriophages and the first case of translational coupling between an MTP and an RBP. Thus, phage Q54 represents a fascinating member of a new species with unusual characteristics that brings new insights into lactococcal phage evolution.

  3. Romulus and Remus, Two Phage Isolates Representing a Distinct Clade within the Twortlikevirus Genus, Display Suitable Properties for Phage Therapy Applications

    PubMed Central

    Vandersteegen, Katrien; Kropinski, Andrew M.; Nash, John H. E.; Noben, Jean-Paul; Hermans, Katleen

    2013-01-01

    The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment. PMID:23302893

  4. Reduction of Campylobacter jejuni in Broiler Chicken by Successive Application of Group II and Group III Phages

    PubMed Central

    Hammerl, Jens A.; Jäckel, Claudia; Alter, Thomas; Janzcyk, Pawel; Stingl, Kerstin; Knüver, Marie Theres; Hertwig, Stefan

    2014-01-01

    Background Bacteriophage treatment is a promising tool to reduce Campylobacter in chickens. Several studies have been published where group II or group III phages were successfully applied. However, these two groups of phages are different regarding their host ranges and host cell receptors. Therefore, a concerted activity of group II and group III phages might enhance the efficacy of a treatment and decrease the number of resistant bacteria. Results In this study we have compared the lytic properties of some group II and group III phages and analysed the suitability of various phages for a reduction of C. jejuni in broiler chickens. We show that group II and group III phages exhibit different kinetics of infection. Two group III and one group II phage were selected for animal experiments and administered in different combinations to three groups of chickens, each containing ten birds. While group III phage CP14 alone reduced Campylobacter counts by more than 1 log10 unit, the concomitant administration of a second group III phage (CP81) did not yield any reduction, probably due to the development of resistance induced by this phage. One group of chickens received phage CP14 and, 24 hours later, group II phage CP68. In this group of animals, Campylobacter counts were reduced by more than 3 log10 units. Conclusion The experiments illustrated that Campylobacter phage cocktails have to be carefully composed to achieve the best results. PMID:25490713

  5. A bioinformatic analysis of ribonucleotide reductase genes in phage genomes and metagenomes

    PubMed Central

    2013-01-01

    Background Ribonucleotide reductase (RNR), the enzyme responsible for the formation of deoxyribonucleotides from ribonucleotides, is found in all domains of life and many viral genomes. RNRs are also amongst the most abundant genes identified in environmental metagenomes. This study focused on understanding the distribution, diversity, and evolution of RNRs in phages (viruses that infect bacteria). Hidden Markov Model profiles were used to analyze the proteins encoded by 685 completely sequenced double-stranded DNA phages and 22 environmental viral metagenomes to identify RNR homologs in cultured phages and uncultured viral communities, respectively. Results RNRs were identified in 128 phage genomes, nearly tripling the number of phages known to encode RNRs. Class I RNR was the most common RNR class observed in phages (70%), followed by class II (29%) and class III (28%). Twenty-eight percent of the phages contained genes belonging to multiple RNR classes. RNR class distribution varied according to phage type, isolation environment, and the host’s ability to utilize oxygen. The majority of the phages containing RNRs are Myoviridae (65%), followed by Siphoviridae (30%) and Podoviridae (3%). The phylogeny and genomic organization of phage and host RNRs reveal several distinct evolutionary scenarios involving horizontal gene transfer, co-evolution, and differential selection pressure. Several putative split RNR genes interrupted by self-splicing introns or inteins were identified, providing further evidence for the role of frequent genetic exchange. Finally, viral metagenomic data indicate that RNRs are prevalent and highly dynamic in uncultured viral communities, necessitating future research to determine the environmental conditions under which RNRs provide a selective advantage. Conclusions This comprehensive study describes the distribution, diversity, and evolution of RNRs in phage genomes and environmental viral metagenomes. The distinct distributions of

  6. 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

  7. Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages

    PubMed Central

    Berg, Jordan A.; Merrill, Bryan D.; Crockett, Justin T.; Esplin, Kyle P.; Evans, Marlee R.; Heaton, Karli E.; Hilton, Jared A.; Hyde, Jonathan R.; McBride, Morgan S.; Schouten, Jordan T.; Simister, Austin R.; Thurgood, Trever L.; Ward, Andrew T.; Breakwell, Donald P.; Hope, Sandra; Grose, Julianne H.

    2016-01-01

    Brevibacillus laterosporus is a spore-forming bacterium that causes a secondary infection in beehives following European Foulbrood disease. To better understand the contributions of Brevibacillus bacteriophages to the evolution of their hosts, five novel phages (Jenst, Osiris, Powder, SecTim467, and Sundance) were isolated and characterized. When compared with the five Brevibacillus phages currently in NCBI, these phages were assigned to clusters based on whole genome and proteome synteny. Powder and Osiris, both myoviruses, were assigned to the previously described Jimmer-like cluster. SecTim467 and Jenst, both siphoviruses, formed a novel phage cluster. Sundance, a siphovirus, was assigned as a singleton phage along with the previously isolated singleton, Emery. In addition to characterizing the basic relationships between these phages, several genomic features were observed. A motif repeated throughout phages Jenst and SecTim467 was frequently upstream of genes predicted to function in DNA replication, nucleotide metabolism, and transcription, suggesting transcriptional co-regulation. In addition, paralogous gene pairs that encode a putative transcriptional regulator were identified in four Brevibacillus phages. These paralogs likely evolved to bind different DNA sequences due to variation at amino acid residues predicted to bind specific nucleotides. Finally, a putative transposable element was identified in SecTim467 and Sundance that carries genes homologous to those found in Brevibacillus chromosomes. Remnants of this transposable element were also identified in phage Jenst. These discoveries provide a greater understanding of the diversity of phages, their behavior, and their evolutionary relationships to one another and to their host. In addition, they provide a foundation with which further Brevibacillus phages can be compared. PMID:27304881

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

    PubMed

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

    2015-05-18

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

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

    PubMed Central

    Laanto, Elina; Bamford, Jaana K. H.; Ravantti, Janne J.; Sundberg, Lotta-Riina

    2015-01-01

    Flavobacterium columnare, the causative agent of columnaris disease in fish, causes millions of dollars of losses in the US channel catfish industry alone, not to mention aquaculture industry worldwide. Novel methods are needed for the control and treatment of bacterial diseases in aquaculture to replace traditionally used chemotherapies. A potential solution could be the use of phages, i.e., bacterial viruses, host-specific and self-enriching particles that can be can easily distributed via water flow. We examined the efficacy of phages to combat columnaris disease. A previously isolated phage, FCL-2, infecting F. columnare, was characterized by sequencing. The 47 142 bp genome of the phage had G + C content of 30.2%, and the closest similarities regarding the structural proteins were found in Cellulophaga phage phiSM. Under controlled experimental conditions, two host fish species, rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio), were used to study the success of phage therapy to prevent F. columnare infections. The survival of both fish species was significantly higher in the presence of the phage. Hundred percent of the zebrafish and 50% of the rainbow trout survived in the phage treatment (survival without phage 0 and 8.3%, respectively). Most importantly, the rainbow trout population was rescued from infection by a single addition of the phage into the water in a flow-through fish tank system. Thus, F. columnare could be used as a model system to test the benefits and risks of phage therapy on a larger scale. PMID:26347722

  10. Development of phoH as a Novel Signature Gene for Assessing Marine Phage Diversity▿

    PubMed Central

    Goldsmith, Dawn B.; Crosti, Giuseppe; Dwivedi, Bhakti; McDaniel, Lauren D.; Varsani, Arvind; Suttle, Curtis A.; Weinbauer, Markus G.; Sandaa, Ruth-Anne; Breitbart, Mya

    2011-01-01

    Phages play a key role in the marine environment by regulating the transfer of energy between trophic levels and influencing global carbon and nutrient cycles. The diversity of marine phage communities remains difficult to characterize because of the lack of a signature gene common to all phages. Recent studies have demonstrated the presence of host-derived auxiliary metabolic genes in phage genomes, such as those belonging to the Pho regulon, which regulates phosphate uptake and metabolism under low-phosphate conditions. Among the completely sequenced phage genomes in GenBank, this study identified Pho regulon genes in nearly 40% of the marine phage genomes, while only 4% of nonmarine phage genomes contained these genes. While several Pho regulon genes were identified, phoH was the most prevalent, appearing in 42 out of 602 completely sequenced phage genomes. Phylogenetic analysis demonstrated that phage phoH sequences formed a cluster distinct from those of their bacterial hosts. PCR primers designed to amplify a region of the phoH gene were used to determine the diversity of phage phoH sequences throughout a depth profile in the Sargasso Sea and at six locations worldwide. phoH was present at all sites examined, and a high diversity of phoH sequences was recovered. Most phoH sequences belonged to clusters without any cultured representatives. Each depth and geographic location had a distinct phoH composition, although most phoH clusters were recovered from multiple sites. Overall, phoH is an effective signature gene for examining phage diversity in the marine environment. PMID:21926220

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

    PubMed Central

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

    2011-01-01

    Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem. PMID:21124487

  12. Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages.

    PubMed

    Berg, Jordan A; Merrill, Bryan D; Crockett, Justin T; Esplin, Kyle P; Evans, Marlee R; Heaton, Karli E; Hilton, Jared A; Hyde, Jonathan R; McBride, Morgan S; Schouten, Jordan T; Simister, Austin R; Thurgood, Trever L; Ward, Andrew T; Breakwell, Donald P; Hope, Sandra; Grose, Julianne H

    2016-01-01

    Brevibacillus laterosporus is a spore-forming bacterium that causes a secondary infection in beehives following European Foulbrood disease. To better understand the contributions of Brevibacillus bacteriophages to the evolution of their hosts, five novel phages (Jenst, Osiris, Powder, SecTim467, and Sundance) were isolated and characterized. When compared with the five Brevibacillus phages currently in NCBI, these phages were assigned to clusters based on whole genome and proteome synteny. Powder and Osiris, both myoviruses, were assigned to the previously described Jimmer-like cluster. SecTim467 and Jenst, both siphoviruses, formed a novel phage cluster. Sundance, a siphovirus, was assigned as a singleton phage along with the previously isolated singleton, Emery. In addition to characterizing the basic relationships between these phages, several genomic features were observed. A motif repeated throughout phages Jenst and SecTim467 was frequently upstream of genes predicted to function in DNA replication, nucleotide metabolism, and transcription, suggesting transcriptional co-regulation. In addition, paralogous gene pairs that encode a putative transcriptional regulator were identified in four Brevibacillus phages. These paralogs likely evolved to bind different DNA sequences due to variation at amino acid residues predicted to bind specific nucleotides. Finally, a putative transposable element was identified in SecTim467 and Sundance that carries genes homologous to those found in Brevibacillus chromosomes. Remnants of this transposable element were also identified in phage Jenst. These discoveries provide a greater understanding of the diversity of phages, their behavior, and their evolutionary relationships to one another and to their host. In addition, they provide a foundation with which further Brevibacillus phages can be compared.

  13. 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.

  14. Characterization of a new virulent phage (MLC-A) of Lactobacillus paracasei.

    PubMed

    Capra, M L; Del L Quiberoni, A; Ackermann, H-W; Moineau, S; Reinheimer, J A

    2006-07-01

    A new virulent bacteriophage (MLC-A) was recently isolated in Argentina from a probiotic dairy product containing a strain of Lactobacillus paracasei. Observation of the lysate with an electron microscope revealed bacteriophage particles with an icosahedral capsid of 57 +/- 2 nm; with a collar and a noncontractile tail of 156 +/- 3 nm terminating with a baseplate to which a tail fiber was attached. Therefore, phage MLC-A belongs to the Siphoviridae family. This phage was able to survive the pasteurization process and was resistant to alcohols and sodium hypochlorite (400 mg/kg). Only peracetic acid could inactivate high-titer suspensions of phages in a short time. The maximum rates of phage adsorption to its host cells were obtained at 30 degrees C with a pH between 5 and 7, and in the presence of calcium or magnesium ions. The host range of phage MLC-A encompassed L. paracasei and Lactobacillus casei strains, but it was not able to infect Lactobacillus rhamnosus or Lactobacillus gasseri strains. One-step growth kinetics of its lytic development revealed latent and burst periods of 30 and 135 min, respectively, with a burst size of about 69 +/- 4 plaque-forming units per infected cell. Phage MLC-A had a distinctive restriction profile when compared with the 2 well-studied Lactobacillus phages, PL-1 and J-1. The genome size of the MLC-A phage was estimated to be approximately 37 kb. This study presents the description of the first phage specific for L. paracasei isolated in Argentina. The isolation of phage MLC-A indicates that, beside lactic acid bacteria starters, probiotic cultures can also be sensitive to virulent phages in industrial processes.

  15. Characteristics and complete genome analysis of a novel jumbo phage infecting pathogenic Bacillus pumilus causing ginger rhizome rot disease.

    PubMed

    Yuan, Yihui; Gao, Meiying

    2016-12-01

    Tailed phages with genomes larger than 200 kbp are classified as jumbo phage and exhibit extremely high diversity. In this study, a novel jumbo phage, vB_BpuM_BpSp, infecting pathogenic Bacillus pumilus, the cause of ginger rhizome rot disease, was isolated. Notable features of phage vB_BpuM_BpSp are the large phage capsid of 137 nm and baseplate-attached curly tail fibers. The genome of the phage is 255,569 bp in size with G+C content of 25.9 %, and it shows low similarity to known biological entities. The phage genome contains 318 predicted coding sequences. Among these predicted coding sequences, 26 genes responsible for nucleotide metabolism were found, and seven structural genes could be identified. The findings of this study provide new understanding of the genetic diversity of phages.

  16. Bacteriophage module reshuffling results in adaptive host range as exemplified by the baseplate model of listerial phage A118.

    PubMed

    Cambillau, Christian

    2015-10-01

    Each phage infects its specific bacterial host strain through highly specific interactions between the baseplate-associated receptor binding protein (RBP) at the tip of the phage tail and the receptor at the host surface. Baseplates incorporate structural core modules, Dit and Tal, largely conserved among phages, and peripheral modules anchoring the RBPs. Exploiting structural information from the HHpred program and EM data from the Bielmann et al. (2015) paper, a molecular model of the A118 phage baseplate was generated from different building blocks. This model implies the occurrence of baseplate module reshuffling and suggests that listerial phage A118 may have been derived from lactococcal phage TP901-1 through host species exchange. With the increase of available viral module structures, modelling phage baseplates will become easier and more reliant, and will provide insightful information on the nature of the phage host receptor and its mode of recognition.

  17. Genetic Diversity of Brucella Reference and Non-reference Phages and Its Impact on Brucella-Typing

    PubMed Central

    Hammerl, Jens A.; Göllner, Cornelia; Jäckel, Claudia; Scholz, Holger C.; Nöckler, Karsten; Reetz, Jochen; Al Dahouk, Sascha; Hertwig, Stefan

    2017-01-01

    Virulent phages have been used for many years to type Brucella isolates, but until recently knowledge about the genetic makeup of these phages remains limited. In this work the host specificity and genomic sequences of the original set (deposited in 1960) of VLA Brucella reference phages Tb, Fi, Wb, Bk2, R/C, and Iz were analyzed and compared with hitherto described brucellaphages. VLA phages turned out to be different from homonymous phages in other laboratories. The host range of the phages was defined by performing plaque assays with a wide selection of Brucella strains. Propagation of the phages on different strains did not alter host specificity. Sequencing of the phages TbV, FiV, WbV, and R/CV revealed nucleotide variations when compared to same-named phages previously described by other laboratories. The phages Bk2V and IzV were sequenced for the first time. While Bk2V exhibited the same deletions as WbV, IzV possesses the largest genome of all Brucella reference phages. The duplication of a 301 bp sequence in this phage and the large deletion in Bk2V, WbV, and R/CV may be a result of recombination caused by repetitive sequences located in this DNA region. To identify new phages as potential candidates for lysotyping, the host range and Single Nucleotide Polymorphisms (SNPs) of 22 non-reference Brucella phages were determined. The phages showed lysis patterns different from those of the reference phages and thus represent novel valuable candidates in the typing set. PMID:28360895

  18. Lytic and inhibition responses to bacteriophages among marine bacteria, with special reference to the origin of phage-host systems

    NASA Astrophysics Data System (ADS)

    Moebus, K.

    1983-12-01

    The results of phage-host cross-reaction tests reported by Moebus & Nattkemper (1981) were re-examined using serially diluted bacteriophage suspensions to elicit the actual type of reaction between the bacteria and phage lysates tested. More than 1450 phage-host systems were studied at 25 °C incubation temperature. Among the nearly 300 phage strains used, 29 were identified as temperate ones. In about 65 % of the phage-host systems bacteriophage propagation was indicated by plaque formation. The remaining systems were characterized by the “inhibition” reaction of bacteria to phage lysates indicated by homogenously reduced bacterial growth within the test area without production of progeny phages. Since crude phage lysates had to be used, it remains obscure whether agents other than infective phage particles (defective ones or bacteriocins) caused this reaction. Among 269 systems of the inhibition type which were also tested at 5° and 15 °C, 54 were observed to propagate phages at one of or both the lower temperatures. Plaques produced at 15 °C with several phage-host systems were found to yield only few progeny phages which generally could not be propagated to produce high-titer phage stocks. With one system temperature-sensitive phage mutants were isolated. The probability of inhibition reactions occurring was found to be higher with phage-host systems isolated east of the Azores than with systems derived from the western Atlantic. With systems from the last mentioned area the proportion of inhibition versus lytic responses of bacteria to phages was observed to increase with the distance between the stations where both parts of the systems were derived. The latter findings are discussed in view of the assumption that bacterial and bacteriophage populations undergo genetic changes while being transported from west to east.

  19. Filamentous phages prevalent in Pseudoalteromonas spp. confer properties advantageous to host survival in Arctic sea ice.

    PubMed

    Yu, Zi-Chao; Chen, Xiu-Lan; Shen, Qing-Tao; Zhao, Dian-Li; Tang, Bai-Lu; Su, Hai-Nan; Wu, Zhao-Yu; Qin, Qi-Long; Xie, Bin-Bin; Zhang, Xi-Ying; Yu, Yong; Zhou, Bai-Cheng; Chen, Bo; Zhang, Yu-Zhong

    2015-03-17

    Sea ice is one of the most frigid environments for marine microbes. In contrast to other ocean ecosystems, microbes in permanent sea ice are space confined and subject to many extreme conditions, which change on a seasonal basis. How these microbial communities are regulated to survive the extreme sea ice environment is largely unknown. Here, we show that filamentous phages regulate the host bacterial community to improve survival of the host in permanent Arctic sea ice. We isolated a filamentous phage, f327, from an Arctic sea ice Pseudoalteromonas strain, and we demonstrated that this type of phage is widely distributed in Arctic sea ice. Growth experiments and transcriptome analysis indicated that this phage decreases the host growth rate, cell density and tolerance to NaCl and H2O2, but enhances its motility and chemotaxis. Our results suggest that the presence of the filamentous phage may be beneficial for survival of the host community in sea ice in winter, which is characterized by polar night, nutrient deficiency and high salinity, and that the filamentous phage may help avoid over blooming of the host in sea ice in summer, which is characterized by polar day, rich nutrient availability, intense radiation and high concentration of H2O2. Thus, while they cannot kill the host cells by lysing them, filamentous phages confer properties advantageous to host survival in the Arctic sea ice environment. Our study provides a foremost insight into the ecological role of filamentous phages in the Arctic sea ice ecosystem.

  20. Filamentous phages prevalent in Pseudoalteromonas spp. confer properties advantageous to host survival in Arctic sea ice

    PubMed Central

    Yu, Zi-Chao; Chen, Xiu-Lan; Shen, Qing-Tao; Zhao, Dian-Li; Tang, Bai-Lu; Su, Hai-Nan; Wu, Zhao-Yu; Qin, Qi-Long; Xie, Bin-Bin; Zhang, Xi-Ying; Yu, Yong; Zhou, Bai-Cheng; Chen, Bo; Zhang, Yu-Zhong

    2015-01-01

    Sea ice is one of the most frigid environments for marine microbes. In contrast to other ocean ecosystems, microbes in permanent sea ice are space confined and subject to many extreme conditions, which change on a seasonal basis. How these microbial communities are regulated to survive the extreme sea ice environment is largely unknown. Here, we show that filamentous phages regulate the host bacterial community to improve survival of the host in permanent Arctic sea ice. We isolated a filamentous phage, f327, from an Arctic sea ice Pseudoalteromonas strain, and we demonstrated that this type of phage is widely distributed in Arctic sea ice. Growth experiments and transcriptome analysis indicated that this phage decreases the host growth rate, cell density and tolerance to NaCl and H2O2, but enhances its motility and chemotaxis. Our results suggest that the presence of the filamentous phage may be beneficial for survival of the host community in sea ice in winter, which is characterized by polar night, nutrient deficiency and high salinity, and that the filamentous phage may help avoid over blooming of the host in sea ice in summer, which is characterized by polar day, rich nutrient availability, intense radiation and high concentration of H2O2. Thus, while they cannot kill the host cells by lysing them, filamentous phages confer properties advantageous to host survival in the Arctic sea ice environment. Our study provides a foremost insight into the ecological role of filamentous phages in the Arctic sea ice ecosystem. PMID:25303713

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

    PubMed

    Bull, James J; Vegge, Christina Skovgaard; Schmerer, Matthew; Chaudhry, Waqas Nasir; Levin, Bruce R

    2014-01-01

    The canonical view of phage - bacterial interactions in dense, liquid cultures is that the phage will eliminate most of the sensitive cells; genetic resistance will then ascend to restore high bacterial densities. Yet there are various mechanisms by which bacteria may remain sensitive to phages but still attain high densities in their presence - because bacteria enter a transient state of reduced adsorption. Importantly, these mechanisms may be cryptic and inapparent prior to the addition of phage yet result in a rapid rebound of bacterial density after phage are introduced. We describe mathematical models of these processes and suggest how different types of this 'phenotypic' resistance may be elucidated. We offer preliminary in vitro studies of a previously characterized E. coli model system and Campylobacter jejuni illustrating apparent phenotypic resistance. As phenotypic resistance may be specific to the receptors used by phages, awareness of its mechanisms may identify ways of improving the choice of phages for therapy. Phenotypic resistance can also explain several enigmas in the ecology of phage-bacterial dynamics. Phenotypic resistance does not preclude the evolution of genetic resistance and may often be an intermediate step to genetic resistance.

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

    PubMed

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

    2015-01-16

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

  3. Spatial vulnerability: bacterial arrangements, microcolonies, and biofilms as responses to low rather than high phage densities.

    PubMed

    Abedon, Stephen T

    2012-05-01

    The ability of bacteria to survive and propagate can be dramatically reduced upon exposure to lytic bacteriophages. Study of this impact, from a bacterium's perspective, tends to focus on phage-bacterial interactions that are governed by mass action, such as can be observed within continuous flow or similarly planktonic ecosystems. Alternatively, bacterial molecular properties can be examined, such as specific phage‑resistance adaptations. In this study I address instead how limitations on bacterial movement, resulting in the formation of cellular arrangements, microcolonies, or biofilms, could increase the vulnerability of bacteria to phages. Principally: (1) Physically associated clonal groupings of bacteria can represent larger targets for phage adsorption than individual bacteria; and (2), due to a combination of proximity and similar phage susceptibility, individual bacteria should be especially vulnerable to phages infecting within the same clonal, bacterial grouping. Consistent with particle transport theory-the physics of movement within fluids-these considerations are suggestive that formation into arrangements, microcolonies, or biofilms could be either less profitable to bacteria when phage predation pressure is high or require more effective phage-resistance mechanisms than seen among bacteria not living within clonal clusters. I consider these ideas of bacterial 'spatial vulnerability' in part within a phage therapy context.

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

    PubMed

    Tazzyman, Samuel J; Hall, Alex R

    2015-03-17

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. The impact of phages on interspecific competition in experimental populations of bacteria

    PubMed Central

    Brockhurst, Michael A; Fenton, Andrew; Roulston, Barrie; Rainey, Paul B

    2006-01-01

    Background Phages are thought to play a crucial role in the maintenance of diversity in natural bacterial communities. Theory suggests that phages impose density dependent regulation on bacterial populations, preventing competitive dominants from excluding less competitive species. To test this, we constructed experimental communities containing two bacterial species (Pseudomonas fluorescens and Pseudomonas aeruginosa) and their phage parasites. Communities were propagated at two environmental temperatures that reversed the outcome of competition in the absence of phage. Results The evenness of coexistence was enhanced in the presence of a phage infecting the superior competitor and in the presence of phage infecting both competitors. This occurred because phage altered the balance of competitive interactions through reductions in density of the superior competitor, allowing concomitant increases in density of the weaker competitor. However, even coexistence was not equally stable at the two environmental temperatures. Conclusion Phage can alter competitive interactions between bacterial species in a way that is consistent with the maintenance of coexistence. However, the stability of coexistence is likely to depend upon the nature of the constituent bacteria-bacteriophage interactions and environmental conditions. PMID:17166259

  7. Quorum sensing influences phage infection efficiency via affecting cell population and physiological state.

    PubMed

    Qin, Xuying; Sun, Qinghui; Yang, Baixue; Pan, Xuewei; He, Yang; Yang, Hongjiang

    2017-02-01

    Bacterial growth phase has been reported affecting phage infection. To underpin the related mechanism, infection efficiency of Pseudomonas aeruginosa phage K5 is characterized. When infecting the logarithmic cells, phage K5 produced significantly more infection centers than the stationary cells, well concordant with the viable cell ratio in the different growth phases. Additionally, the burst size decreased dramatically in the stationary cells, implying that the physiological state of the viable cells contributed to the productivity of phage K5, and it was consistent with the expression variation of the phage RNA polymerase. Quorum sensing inhibitor penicillic acid was applied and could significantly improve the viable cell proportion and the infection center numbers, but had less effect on the corresponding burst sizes. Moreover, the effect of penicillic acid and the quorum sensing regulator mutants on the production of phage C11 was also analyzed. Taken together, our data suggest that quorum sensing is involved in the defense of phage K5 infection by influencing the viable cell population and their physiological state, and it is an efficient and intrinsic pathway allowing bacteria to resist phage attacks in natural environment.

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

    PubMed

    Zhang, Hui; Wang, Ran; Bao, Hongduo

    2013-01-01

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

  9. Role of phages in the control of bacterial pathogens in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophages (phages) are viruses of bacteria and were independently discovered by English bacteriologist Twort in 1915 and Franco-Canadian microbiologist d’Hérelle in 1917 (d'Hérelle, 1919). Since then, phages have been used to treat infectious disease in humans, animals, and plants, but in the ...

  10. An improved method for rapid generation and screening of Bacillus thuringiensis phage-resistant mutants.

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-11-01

    A simple method to isolate, screen and select phage-resistant mutants of Bacillus thuringiensis was developed. The traditional double-layer agar method was improved by a combination of the spotting assay using a lytic phage, to generate the bacterial-resistant mutants, with an inverted spotting assay (ISA), to rapidly screen the candidate-resistant mutants.

  11. Isolation and characterization of novel giant Stenotrophomonas maltophilia phage phiSMA5.

    PubMed

    Chang, Hsiao-Chuan; Chen, Chiy-Rong; Lin, Juey-Wen; Shen, Gwan-Han; Chang, Kai-Ming; Tseng, Yi-Hsiung; Weng, Shu-Fen

    2005-03-01

    Stenotrophomonas maltophilia is one of the most prevalent opportunistic bacteria causing nosocomial infections. It has become problematic because most of the isolates are resistant to multiple antibiotics, and therefore, development of phage therapy has attracted strong attention. In this study, eight S. maltophilia phages were isolated from clinical samples including patient specimens, catheter-related devices, and wastewater. These phages can be divided into four distinct groups based on host range and digestibility of the phage DNAs with different restriction endonucleases. One of them, designated phiSMA5, was further characterized. Electron microscopy showed it resembled Myoviridae, with an isometric head (90 nm in diameter), a tail (90 nm long), a baseplate (25 nm wide), and short tail fibers. The phiSMA5 double-stranded DNA, refractory to digestion by most restriction enzymes, was tested and estimated to be 250 kb by pulsed-field gel electrophoresis. This genome size is second to that of the largest phage, phiKZ of Pseudomonas aeruginosa. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 25 virion proteins were visualized. N-terminal sequencing of four of them suggested that each of them might have had its N terminus cleaved off. Among the 87 S. maltophilia strains collected in this study, only 61 were susceptible to phiSMA5, indicating that more phages are needed toward a phage therapy strategy. Since literature search yielded no information about S. maltophilia phages, phiSMA5 appears to be the first reported.

  12. Coevolutionary diversification creates nested-modular structure in phage-bacteria interaction networks.

    PubMed

    Beckett, Stephen J; Williams, Hywel T P

    2013-12-06

    Phage and their bacterial hosts are the most diverse and abundant biological entities in the oceans, where their interactions have a major impact on marine ecology and ecosystem function. The structure of interaction networks for natural phage-bacteria communities offers insight into their coevolutionary origin. At small phylogenetic scales, observed communities typically show a nested structure, in which both hosts and phages can be ranked by their range of resistance and infectivity, respectively. A qualitatively different multi-scale structure is seen at larger phylogenetic scales; a natural assemblage sampled from the Atlantic Ocean displays large-scale modularity and local nestedness within each module. Here, we show that such 'nested-modular' interaction networks can be produced by a simple model of host-phage coevolution in which infection depends on genetic matching. Negative frequency-dependent selection causes diversification of hosts (to escape phages) and phages (to track their evolving hosts). This creates a diverse community of bacteria and phage, maintained by kill-the-winner ecological dynamics. When the resulting communities are visualized as bipartite networks of who infects whom, they show the nested-modular structure characteristic of the Atlantic sample. The statistical significance and strength of this observation varies depending on whether the interaction networks take into account the density of the interacting strains, with implications for interpretation of interaction networks constructed by different methods. Our results suggest that the apparently complex community structures associated with marine bacteria and phage may arise from relatively simple coevolutionary origins.

  13. Regulation of replication of lambda phage and lambda plasmid DNAs at low temperature.

    PubMed

    Gabig, M; Obuchowski, M; Srutkowska, S; Wegrzyn, G

    1998-06-01

    It was previously demonstrated that while lysogenic development of bacteriophage lambda in Escherichia coli proceeds normally at low temperature (20-25 degrees C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the pE promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage lambda cIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37 degrees C, while no phage progeny are observed at 20 degrees C. Contrary to previous reports, it is possible to demonstrate that pE promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20 degrees C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage lambda is neither inhibited at 20 degrees C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between lambda phage and lambda plasmid DNA at low temperature.

  14. Short communication: Characterization of Salmonella phages from dairy calves on farms with history of diarrhea.

    PubMed

    Dueñas, Fernando; Rivera, Dácil; Toledo, Viviana; Tardone, Rodolfo; Hervé-Claude, Luis P; Hamilton-West, Christopher; Switt, Andrea I Moreno

    2017-03-01

    Salmonella enterica can cause disease and mortality in calves. This pathogen is also a zoonosis that can be transmitted by animal contact or by food. The prevalence of Salmonella in dairy farms has been reported to range from 0 to 64%, and, due to the diversity of Salmonella serovars that can be circulating, Salmonella is an important concern for dairy production. Bacteriophages that infect Salmonella have been documented to be abundant and widely distributed in the dairy environment. The current study investigated the diversity of Salmonella serovars and Salmonella phages in 8 dairy farms with a history of diarrhea in southern Chile. A total of 160 samples from sick calves, healthy calves, and the environment were analyzed for Salmonella and phage. Isolated phages were characterized and classified by their host range using a panel of 26 Salmonella isolates representing 23 serovars. Host ranges were classified according to lysis profiles (LP) and their spatial distribution was mapped. Salmonella-infecting phages were identified, but none of the 160 samples were positive for Salmonella. A total of 45 phage isolates were obtained from sick calves (11), healthy calves (16), or the environment (18). According to their host range, 19 LP were identified, with LP1 being the most common on all 8 farms; LP1 represents phages that only lyse serogroup D Salmonella. The identification of Salmonella phages but not Salmonella in the same samples could suggest that these phages are controlling Salmonella in these farms.

  15. Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

    PubMed

    Henry, M F; Cronan, J E

    1991-06-01

    We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.

  16. Phage display screen for peptides that bind Bcl-2 protein.

    PubMed

    Park, Hye-Yeon; Kim, Joungmok; Cho, June-Haeng; Moon, Ji Young; Lee, Su-Jae; Yoon, Moon-Young

    2011-01-01

    Bcl-2 family proteins are key regulators of apoptosis associated with human disease, including cancer. Bcl-2 protein has been found to be overexpressed in many cancer cells. Therefore, Bcl-2 protein is a potential diagnostic target for cancer detection. In the present study, the authors have identified several Bcl-2 binding peptides with high affinity (picomolar range) from a 5-round M13 phage display library screening. These peptides can be used to develop novel diagnostic probes or potent inhibitors with diverse polyvalencies.

  17. Biological and genomic analysis of a PBSX-like defective phage induced from Bacillus pumilus AB94180.

    PubMed

    Jin, Tingting; Zhang, Xiaoming; Zhang, Yang; Hu, Zhongsheng; Fu, Zhengwei; Fan, Junpeng; Wu, Ming; Wang, Yi; Shen, Ping; Chen, Xiangdong

    2014-04-01

    Defective prophages, which are found in the genomes of many bacteria, are unable to complete a viral replication cycle and propagate in their hosts as healthy prophages. They package random DNA fragments derived from various sites of the host chromosome instead of their own genomes. In this study, we characterized a defective phage, PBP180, which was induced from Bacillus pumilus AB94180 by treatment with mitomycin C. Electron microscopy showed that the PBP180 particle has a head with a hexagonal outline of ~40 nm in diameter and a long tail. The DNA packaged in the PBP180 head consists of 8-kb DNA fragments from random portions of the host chromosome. The head and tail proteins of the PBP180 particle consist of four major proteins of approximately 49, 33, 16 and 14 kDa. The protein profile of PBP180 is different from that of PBSX, a well-known defective phage induced from Bacillus subtilis 168. A killing activity test against two susceptible strains each of B. subtilis and B. pumilus showed that the defective particles of PBP180 killed three strains other than its own host, B. pumilus AB94180, differing from the host-killing ranges of the defective phages PBSX, PBSZ (induced from B. subtilis W23), and PBSX4 (induced from B. pumilus AB94044). The genome of the PBP180 prophage, which is integrated in the B. pumilus AB94180 chromosome, is 28,205 bp in length, with 40 predicted open reading frames (ORFs). Further genomic comparison of prophages PBP180, PBSX, PBSZ and other PBSX-like prophage elements in B. pumilus strains revealed that their overall architectures are similar, but significant low homology exists in ORF29-ORF38, which presumably encode tail fiber proteins involved in recognition and killing of susceptible strains.

  18. Phages of non-dairy lactococci: isolation and characterization of ΦL47, a phage infecting the grass isolate Lactococcus lactis ssp. cremoris DPC6860

    PubMed Central

    Cavanagh, Daniel; Guinane, Caitriona M.; Neve, Horst; Coffey, Aidan; Ross, R. Paul; Fitzgerald, Gerald F.; McAuliffe, Olivia

    2014-01-01

    Lactococci isolated from non-dairy sources have been found to possess enhanced metabolic activity when compared to dairy strains. These capabilities may be harnessed through the use of these strains as starter or adjunct cultures to produce more diverse flavor profiles in cheese and other dairy products. To understand the interactions between these organisms and the phages that infect them, a number of phages were isolated against lactococcal strains of non-dairy origin. One such phage, ΦL47, was isolated from a sewage sample using the grass isolate L. lactis ssp. cremoris DPC6860 as a host. Visualization of phage virions by transmission electron microscopy established that this phage belongs to the family Siphoviridae and possesses a long tail fiber, previously unseen in dairy lactococcal phages. Determination of the lytic spectrum revealed a broader than expected host range, with ΦL47 capable of infecting 4 industrial dairy strains, including ML8, HP and 310, and 3 additional non-dairy isolates. Whole genome sequencing of ΦL47 revealed a dsDNA genome of 128, 546 bp, making it the largest sequenced lactococcal phage to date. In total, 190 open reading frames (ORFs) were identified, and comparative analysis revealed that the predicted products of 117 of these ORFs shared greater than 50% amino acid identity with those of L. lactis phage Φ949, a phage isolated from cheese whey. Despite their different ecological niches, the genomic content and organization of ΦL47 and Φ949 are quite similar, with both containing 4 gene clusters oriented in different transcriptional directions. Other features that distinguish ΦL47 from Φ949 and other lactococcal phages, in addition to the presence of the tail fiber and the genome length, include a low GC content (32.5%) and a high number of predicted tRNA genes (8). Comparative genome analysis supports the conclusion that ΦL47 is a new member of the 949 lactococcal phage group which currently includes the dairy Φ949. PMID

  19. Detection of E. coli O157:H7 with a reporter phage containing the luxCDABE cassette

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage and reporter phage are used for typing and/or detection of pathogens. The temperate tailed phage fV10 has been utilized for phage-typing E. coli O157:H7. By modifying fV10 to transduce kanamycin resistance and the a luxCDABE cassette, we developed a reporter bacteriophage (fV10-lux) p...

  20. Complete Genome Sequences of Nine Phages Capable of Infecting Paenibacillus larvae, the Causative Agent of American Foulbrood Disease in Honeybees

    PubMed Central

    Yost, Diane G.; Krohn, Andrew; LeBlanc, Lucy; Zhang, Anna; Stamereilers, Casey; Amy, Penny S.

    2015-01-01

    We present here the complete genome sequences of nine phages that infect Paenibacillus larvae, the causative agent of American foulbrood disease in honeybees. The phages were isolated from soil, propolis, and infected bees from three U.S. states. This is the largest number of P. larvae phage genomes sequenced in a single publication to date. PMID:26472825

  1. Transcription of the phage-encoded Panton-Valentine leukocidin of Staphylococcus aureus is dependent on the phage life-cycle and on the host background.

    PubMed

    Wirtz, Christiane; Witte, Wolfgang; Wolz, Christiane; Goerke, Christiane

    2009-11-01

    Panton-Valentine leukocidin (PVL) is a pore-forming, bi-component toxin secreted by Staphylococcus aureus strains epidemiologically associated with diseases such as necrotizing pneumonia and skin and soft-tissue infections. Here we demonstrate that transcription of the phage-encoded PVL (encoded in the luk-PV operon) is dependent on two major determinants: the phage life-cycle and the host chromosomal background. Mitomycin C induction of PVL-encoding prophages from different community-acquired MRSA strains led to an increase in the amount of luk-PV mRNA as a result of read-through transcription from latent phage promoters and an increase in phage copy numbers. Failing prophage excision was reflected in a constant expression of luk-PV as in the case of strain USA300, suggesting that phi Sa2USA300 is a replication-defective prophage. Additionally, we could show that luk-PV transcription is influenced by the S. aureus global virulence regulators agr and sae. We found a strong impact of the host background on prophage induction and replication when analysing PVL phages in different S. aureus strains. For example phage phi Sa2mw was greatly induced by mitomycin C in its native host MW2 and in strain Newman but to a considerably lesser extent in strains 8325-4, RN6390 and ISP479c. This discrepancy was not linked to the SOS response of the bacteria since recA transcription did not vary between the strains. These results suggest a fine tuning between certain phages and their host, with major impact on the expression of phage-encoded virulence genes.

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

    PubMed

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

    1989-01-01

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

  3. Selection of Lung Cancer-Specific Landscape Phage for Targeted Drug Delivery

    PubMed Central

    Gillespie, James W.; Wei, Lixia; Petrenko, Valery A.

    2016-01-01

    Cancer cell-specific diagnostic or therapeutic tools are commonly believed to significantly increase the success rate of cancer diagnosis and targeted therapies. To extend the repertoire of available cancer cell-specific phage fusion proteins and study their efficacy as navigating moieties, we used two landscape phage display libraries f8/8 and f8/9 displaying an 8- or 9-mer random peptide fusion to identify a panel of novel peptide families that are specific to Calu-3 cells. Using a phage capture assay, we showed that two of the selected phage clones, ANGRPSMT and VNGRAEAP (phage and their recombinant proteins are named by the sequence of the fusion peptide), are selective for the Calu-3 cell line in comparison to phenotypically normal lung epithelial cells and distribute into unique subcellular fractions. PMID:27095536

  4. Phage-Enabled Nanomedicine: From Probes to Therapeutics in Precision Medicine.

    PubMed

    Sunderland, Kegan S; Yang, Mingying; Mao, Chuanbin

    2017-02-13

    Both lytic and temperate bacteriophages (phages) can be applied in nanomedicine, in particular, as nanoprobes for precise disease diagnosis and nanotherapeutics for targeted disease treatment. Since phages are bacteria-specific viruses, they do not naturally infect eukaryotic cells and are not toxic to them. They can be genetically engineered to target nanoparticles, cells, tissues, and organs, and can also be modified with functional abiotic nanomaterials for disease diagnosis and treatment. This Review will summarize the current use of phage structures in many aspects of precision nanomedicine, including ultrasensitive biomarker detection, enhanced bioimaging for disease diagnosis, targeted drug and gene delivery, directed stem cell differentiation, accelerated tissue formation, effective vaccination, and nanotherapeutics for targeted disease treatment. We will also propose future directions in the area of phage-based nanomedicines, and discuss the state of phage-based clinical trials.

  5. Specific ligands for classical swine fever virus screened from landscape phage display library.

    PubMed

    Yin, Long; Luo, Yuzi; Liang, Bo; Wang, Fei; Du, Min; Petrenko, Valery A; Qiu, Hua-Ji; Liu, Aihua

    2014-09-01

    Classical swine fever (CSF) is a devastating infectious disease caused by classical swine fever virus (CSFV). The screening of CSFV-specific ligands is of great significance for diagnosis and treatment of CSF. Affinity selection from random peptide libraries is an efficient approach to discover ligands with high stability and specificity. Here, we screened phage ligands for the CSFV E2 protein from f8/8 landscape phage display library by biopanning and obtained four phage clones specific for the E2 protein of CSFV. Viral blocking assays indicated that the phage clone displaying the octapeptide sequence DRATSSNA remarkably inhibited the CSFV replication in PK-15 cells at a titer of 10(10) transduction units, as evidenced by significantly decreased viral RNA copies and viral titers. The phage-displayed E2-binding peptides have the potential to be developed as antivirals for CSF.

  6. Using a bacteriocin structure to engineer a phage lysin that targets Yersinia pestis.

    PubMed

    Lukacik, Petra; Barnard, Travis J; Buchanan, Susan K

    2012-12-01

    Purified phage lysins present an alternative to traditional antibiotics and work by hydrolysing peptidoglycan. Phage lysins have been developed against Gram-positive pathogens such as Bacillus anthracis and Streptococcus pneumoniae, where the peptidoglycan layer is exposed on the cell surface. Addition of the lysin to a bacterial culture results in rapid death of the organism. Gram-negative bacteria are resistant to phage lysins because they contain an outer membrane that protects the peptidoglycan from degradation. We solved crystal structures of a Yersinia pestis outer-membrane protein and the bacteriocin that targets it, which informed engineering of a bacterial-phage hybrid lysin that can be transported across the outer membrane to kill specific Gram-negative bacteria. This work provides a template for engineering phage lysins against a wide variety of bacterial pathogens.

  7. Phage therapy of the white plague-like disease of Favia favus in the Red Sea

    NASA Astrophysics Data System (ADS)

    Atad, I.; Zvuloni, A.; Loya, Y.; Rosenberg, E.

    2012-09-01

    Coral disease is a major factor in the global decline of coral reefs. At present, there are no known procedures for preventing or treating infectious diseases of corals. Immunization is not possible because corals have a restricted adaptive immune system and antibiotics are neither ecologically safe nor practical in an open system. Thus, we tested phage therapy as an alternative therapeutic method for treating diseased corals. Phage BA3, specific to the coral pathogen Thalassomonas loyana, inhibited the progression of the white plague-like disease and transmission to healthy corals in the Gulf of Aqaba, Red Sea. Only one out of 19 (5 %) of the healthy corals became infected when placed near phage-treated diseased corals, whereas 11 out of 18 (61 %) healthy corals were infected in the no-phage control. This is the first successful treatment for a coral disease in the sea. We posit that phage therapy of certain coral diseases is achievable in situ.

  8. DNA fusion product of phage P2 with plasmid pBR322 - A new phasmid

    NASA Technical Reports Server (NTRS)

    Nicoletti, M.; Bertani, G.

    1983-01-01

    The chromosome of the temperate bacteriophage P2 and that of the plasmid pBR322 have been joined in vitro after treatment with restriction endonuclease EcoRI. The fusion product - a phasmid - can behave as a plasmid, as a phage and as a prophage. It can replicate its DNA under the control of either the specific replication mechanism of the parent phage in a polA mutant or that of the parent plasmid in a rep mutant. Several interesting interactions between the two replication modes are indicated. In particular, phage particles may be produced even when the phage mode of DNA replication is blocked, and this throws new light on the involvement of the early gene A in the regulation of late gene expression in phage P2.

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

    PubMed

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

    2015-05-15

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

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

    PubMed

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

    2014-01-22

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

  11. The Phage-Inducible Chromosomal Islands: A Family of Highly Evolved Molecular Parasites.

    PubMed

    Penadés, José R; Christie, Gail E

    2015-11-01

    The phage-inducible chromosomal islands (PICIs) are a family of highly mobile genetic elements that contribute substantively to horizontal gene transfer, host adaptation, and virulence. Initially identified in Staphylococcus aureus, these elements are now thought to occur widely in gram-positive bacteria. They are molecular parasites that exploit certain temperate phages as helpers, using a variety of elegant strategies to manipulate the phage life cycle and promote their own spread, both intra- and intergenerically. At the same time, these PICI-encoded mechanisms severely interfere with helper phage reproduction, thereby enhancing survival of the bacterial population. In this review we discuss the genetics and the life cycle of these elements, with special emphasis on how they interact and interfere with the helper phage machinery for their own benefit. We also analyze the role that these elements play in driving bacterial and viral evolution.

  12. The tail-associated depolymerase of Erwinia amylovora phage L1 mediates host cell adsorption and enzymatic capsule removal, which can enhance infection by other phage.

    PubMed

    Born, Yannick; Fieseler, Lars; Klumpp, Jochen; Eugster, Marcel R; Zurfluh, Katrin; Duffy, Brion; Loessner, Martin J

    2014-07-01

    The depolymerase enzyme (DpoL1) encoded by the T7-like phage L1 efficiently degrades amylovoran, an important virulence factor and major component of the extracellular polysaccharide (EPS) of its host, the plant pathogen Erwinia amylovora. Mass spectrometry analysis of hydrolysed EPS revealed that DpoL1 cleaves the galactose-containing backbone of amylovoran. The enzyme is most active at pH 6 and 50°C, and features a modular architecture. Removal of 180 N-terminal amino acids was shown not to affect enzyme activity. The C-terminus harbours the hydrolase activity, while the N-terminal domain links the enzyme to the phage particle. Electron microscopy demonstrated that DpoL1-specific antibodies cross-link phage particles at their tails, either lateral or frontal, and immunogold staining confirmed that DpoL1 is located at the tail spikes. Exposure of high-level EPS-producing Er. amylovora strain CFBP1430 to recombinant DpoL1 dramatically increased sensitivity to the Dpo-negative phage Y2, which was not the case for EPS-negative mutants or low-level EPS-producing Er. amylovora. Our findings indicate that enhanced phage susceptibility is based on enzymatic removal of the EPS capsule, normally a physical barrier to Y2 infection, and that use of DpoL1 together with the broad host range, virulent phage Y2 represents an attractive combination for biocontrol of fire blight.

  13. Application of phage therapy during bivalve depuration improves Escherichia coli decontamination.

    PubMed

    Pereira, Carla; Moreirinha, Catarina; Teles, Luís; Rocha, Rui J M; Calado, Ricardo; Romalde, Jesús L; Nunes, Maria L; Almeida, Adelaide

    2017-02-01

    The present study investigated the potential application of the bacteriophage (or phage) phT4A, ECA2 and the phage cocktail phT4A/ECA2 to decrease the concentration of Escherichia coli during the depuration of natural and artificially contaminated cockles. Depuration in static seawater at multiplicity of infection (MOI) of 1 with single phage suspensions of phT4A and ECA2 was the best condition, as it decreased by ∼2.0 log CFU/g the concentration of E. coli in artificially contaminated cockles after a 4 h of treatment. When naturally contaminated cockles were treated in static seawater with single phage suspensions and the phage cocktail, similar decreases in the concentration of E. coli (∼0.7 log CFU/g) were achieved. However, when employing the phage cocktail, a longer treatment time was required to obtain comparable results to those achieved when using single phage suspensions. When naturally contaminated cockles were depurated with phage phT4A in a recirculated seawater system (mimicking industrial depuration conditions), a 0.6 log CFU/g reduction of E. coli was achieved after a 2 h of treatment. When the depuration process was performed without phage addition, a 4 h treatment was necessary to obtain a similar decrease. By combining phage therapy and depuration procedures, a reduction in bivalves depuration period can be achieved for, thus decreasing the cost associated with this procedure and even enhance the quality and safety of depurated bivalves destined for human consumption.

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

    PubMed

    Laanto, Elina; Bamford, Jaana K H; Laakso, Jouni; Sundberg, Lotta-Riina

    2012-12-20

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

  15. The performance of a multi-sensor detection system based on phage-coated magnetoelastic biosensors

    NASA Astrophysics Data System (ADS)

    Huang, S.; Yang, H.; Lakshmanan, R.; Li, S.; Chen, I.; Petrenko, V. A.; Barbaree, J. M.; Chin, B. A.

    2009-05-01

    In this paper the performance of a magnetoelastic biosensor detection system for the simultaneous identification of B. anthracis spores and S. typhimurium was investigated. This system was also designed for selective in-situ detection of B. anthracis spores in the presence a mixed microbial population. The system was composed of a reference sensor (devoid of phage), an E2 phage sensor (coated with phage specific to S. typhimurium) and a JRB7 phage sensor (coated with phage specific to B. anthracis spores). When cells/spores are bound to the specific phage-based ME biosensor surface, only the resonance frequency of the specific sensor changed. The instantaneous response of the multiple sensor system was studied by exposing the system to B. anthracis spores and S. typhimurium suspensions sequentially. A detection limit of 1.6×103 cfu/mL and 1.1×103 cfu/m was observed for JRB7 phage sensor and E2 phage sensor, respectively. Additionally, the performance of the system was also evaluated by exposure to a flowing mixture of B. anthracis spores (5×101-5×108 cfu/ml) in the presence of B. cereus spores (5×107 cfu/ml). Only the JRB7 phage biosensor responded to the B. anthracis spores. Moreover, there was no appreciable frequency change due to non-specific binding when other microorganisms (spores) were in the mixture. A detection limit of 3×102 cfu/mL was observed for JRB7 phage sensor. The results show that the multi-sensor detection system offers good performance, including good sensitivity, selectivity and rapid detection.

  16. Evolving interactions between diazotrophic cyanobacterium and phage mediate nitrogen release and host competitive ability

    PubMed Central

    Coloma, Sebastián; Sivonen, Kaarina

    2016-01-01

    Interactions between nitrogen-fixing (i.e. diazotrophic) cyanobacteria and their viruses, cyanophages, can have large-scale ecosystem effects. These effects are mediated by temporal alterations in nutrient availability in aquatic systems owing to the release of nitrogen and carbon sources from cells lysed by phages, as well as by ecologically important changes in the diversity and fitness of cyanobacterial populations that evolve in the presence of phages. However, ecological and evolutionary feedbacks between phages and nitrogen-fixing cyanobacteria are still relative poorly understood. Here, we used an experimental evolution approach to test the effect of interactions between a common filamentous, nitrogen-fixing cyanobacterium (Nodularia sp.) and its phage on cellular nitrogen release and host properties. Ecological, community-level effects of phage-mediated nitrogen release were tested with a phytoplankton bioassay. We found that cyanobacterial nitrogen release increased significantly as a result of viral lysis, which was associated with enhanced growth of phytoplankton species in cell-free filtrates compared with phage-resistant host controls in which lysis and subsequent nutrient release did not occur after phage exposure. We also observed an ecologically important change among phage-evolved cyanobacteria with phage-resistant phenotypes, a short-filamentous morphotype with reduced buoyancy compared with the ancestral long-filamentous morphotype. Reduced buoyancy might decrease the ability of these morphotypes to compete for light compared with longer, more buoyant filaments. Together, these findings demonstrate the potential of cyanobacteria–phage interactions to affect ecosystem biogeochemical cycles and planktonic community dynamics. PMID:28083116

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

    PubMed

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

    2014-01-01

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

  18. Plasticity of the gene functions for DNA replication in the T4-like phages.

    PubMed

    Petrov, Vasiliy M; Nolan, James M; Bertrand, Claire; Levy, Dawn; Desplats, Carine; Krisch, H M; Karam, Jim D

    2006-08-04

    We have completely sequenced and annotated the genomes of several relatives of the bacteriophage T4, including three coliphages (RB43, RB49 and RB69), three Aeromonas salmonicida phages (44RR2.8t, 25 and 31) and one Aeromonas hydrophila phage (Aeh1). In addition, we have partially sequenced and annotated the T4-like genomes of coliphage RB16 (a close relative of RB43), A. salmonicida phage 65, Acinetobacter johnsonii phage 133 and Vibrio natriegens phage nt-1. Each of these phage genomes exhibited a unique sequence that distinguished it from its relatives, although there were examples of genomes that are very similar to each other. As a group the phages compared here diverge from one another by several criteria, including (a) host range, (b) genome size in the range between approximately 160 kb and approximately 250 kb, (c) content and genetic organization of their T4-like genes for DNA metabolism, (d) mutational drift of the predicted T4-like gene products and their regulatory sites and (e) content of open-reading frames that have no counterparts in T4 or other known organisms (novel ORFs). We have observed a number of DNA rearrangements of the T4 genome type, some exhibiting proximity to putative homing endonuclease genes. Also, we cite and discuss examples of sequence divergence in the predicted sites for protein-protein and protein-nucleic acid interactions of homologues of the T4 DNA replication proteins, with emphasis on the diversity in sequence, molecular form and regulation of the phage-encoded DNA polymerase, gp43. Five of the sequenced phage genomes are predicted to encode split forms of this polymerase. Our studies suggest that the modular construction and plasticity of the T4 genome type and several of its replication proteins may offer resilience to mutation, including DNA rearrangements, and facilitate the adaptation of T4-like phages to different bacterial hosts in nature.

  19. Deep sequencing of phage display libraries to support antibody discovery.

    PubMed

    Ravn, Ulla; Didelot, Gérard; Venet, Sophie; Ng, Kwok-Ting; Gueneau, Franck; Rousseau, François; Calloud, Sébastien; Kosco-Vilbois, Marie; Fischer, Nicolas

    2013-03-15

    The use of next generation sequencing (NGS) for the analysis of antibody sequences both in phage display libraries and during in vitro selection processes has become increasingly popular in the last few years. Here, our methods developed for DNA preparation, sequencing and data analysis are presented. A key parameter has also been to develop new software designed for high throughput antibody sequence analysis that is used in combination with publicly available tools. As an example of our methods, we provide data from the extensive analysis of five scFv libraries generated using different heavy chain CDR3 diversification strategies. The results not only confirm that the library designs were correct but also reveal differences in quality not easily identified by standard DNA sequencing approaches. The very large number of reads permits extensive sequence coverage after the selection process. Furthermore, as samples can be multiplexed, costs decrease and more information is gained per NGS run. Using examples of results obtained post phage display selections against two antigens, frequency and clustering analysis identified novel antibody fragments that were then shown to be specific for the target antigen. In summary, the methods described here demonstrate how NGS analysis enhances quality control of complex antibody libraries as well as facilitates the antibody discovery process.

  20. Dualities in the analysis of phage DNA packaging motors

    PubMed Central

    Serwer, Philip; Jiang, Wen

    2012-01-01

    The DNA packaging motors of double-stranded DNA phages are models for analysis of all multi-molecular motors and for analysis of several fundamental aspects of biology, including early evolution, relationship of in vivo to in vitro biochemistry and targets for anti-virals. Work on phage DNA packaging motors both has produced and is producing dualities in the interpretation of data obtained by use of both traditional techniques and the more recently developed procedures of single-molecule analysis. The dualities include (1) reductive vs. accretive evolution, (2) rotation vs. stasis of sub-assemblies of the motor, (3) thermal ratcheting vs. power stroking in generating force, (4) complete motor vs. spark plug role for the packaging ATPase, (5) use of previously isolated vs. new intermediates for analysis of the intermediate states of the motor and (6) a motor with one cycle vs. a motor with two cycles. We provide background for these dualities, some of which are under-emphasized in the literature. We suggest directions for future research. PMID:23532204

  1. Micron-scale holes terminate the phage infection cycle.

    PubMed

    Dewey, Jill S; Savva, Christos G; White, Rebecca L; Vitha, Stanislav; Holzenburg, Andreas; Young, Ry

    2010-02-02

    Holins are small phage-encoded proteins that accumulate harmlessly in the cytoplasmic membrane during the infection cycle until suddenly, at an allele-specific time, triggering to form lethal lesions, or "holes." In the phages lambda and T4, the holes have been shown to be large enough to allow release of prefolded active endolysin from the cytoplasm, which results in destruction of the cell wall, followed by lysis within seconds. Here, the holes caused by S105, the lambda-holin, have been captured in vivo by cryo-EM. Surprisingly, the scale of the holes is at least an order of magnitude greater than any previously described membrane channel, with an average diameter of 340 nm and some exceeding 1 microm. Most cells exhibit only one hole, randomly positioned in the membrane, irrespective of its size. Moreover, on coexpression of holin and endolysin, the degradation of the cell wall leads to spherically shaped cells and a collapsed inner membrane sac. To obtain a 3D view of the hole by cryo-electron tomography, we needed to reduce the average size of the cells significantly. By taking advantage of the coupling of bacterial cell size and growth rate, we achieved an 80% reduction in cell mass by shifting to succinate minimal medium for inductions of the S105 gene. Cryotomographic analysis of the holes revealed that they were irregular in shape and showed no evidence of membrane invagination. The unexpected scale of these holes has implications for models of holin function.

  2. Fully human antibodies from transgenic mouse and phage display platforms.

    PubMed

    Lonberg, Nils

    2008-08-01

    Over the past two decades, technologies have emerged for generating monoclonal antibodies (MAbs) derived from human immunoglobulin gene sequences. These fully human MAbs provide an alternative to re-engineered, or de-immunized, rodent MAbs as a source of low immunogenicity therapeutic antibodies. There are now two marketed fully human therapeutic MAbs, adalimumab and panitumumab, and several dozen more in various stages of human clinical testing. Most of the drugs, including adalimumab and panitumumab, were generated using either phage display or transgenic mouse platforms. The reported clinical experience with fully human MAbs demonstrates that these two platforms are, and should continue to be, a significant source of active and well tolerated experimental therapeutics. While this body of reported clinical data does not yet provide a clear distinction between the platforms, the available descriptions of the drug discovery processes used to identify the clinical candidates highlight one difference. It appears that lead optimization is more commonly applied to phage display derived leads than transgenic mouse derived leads.

  3. Ionic switch controls the DNA state in phage λ

    DOE PAGES

    Li, Dong; Liu, Ting; Zuo, Xiaobing; ...

    2015-06-19

    We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid bymore » changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. The results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.« less

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

    SciTech Connect

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

    2012-02-21

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

  5. Dynamics of adaptive immunity against phage in bacterial populations

    NASA Astrophysics Data System (ADS)

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

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

  6. Bacterial sensing using phage-functionalized whispering gallery microcavities

    NASA Astrophysics Data System (ADS)

    Ghali, Hala; Hibli, Hicham; Bianucci, Pablo; Nadeau, Jay; Peter, Yves-Alain

    2012-02-01

    Whispering gallery optical microcavities are structures which can efficiently confine light at the micro scale. This confinement is based on total internal reflection of light at the interface between the cavity and the surrounding medium. Devices based on optical microcavities have a wide range of applications, such as microlasers, quantum optical devices and much more. In this work, we describe a biosensing application of these optical microcavities for the label-free detection of bacteria. In order for the sensor to be specific to a particular species of bacteria, we need to properly functionalize its surface so that only that kind of bacteria will produce a signal. The microcavity surface is first functionalized using PEGylated aminosilane. We then introduce phage-derived proteins that are specific to the bacteria we want to detect. The binding between the bacteria and the phage proteins creates a perturbation to the cavity field that leads to a thermo-optic effect. This effect is then observed as a shift in the resonance features of the transmission spectrum. We performed experimental measurements using a tapered fiber to couple the light from red laser (635 nm) into the resonator.

  7. In vitro incorporation of the phage Phi29 connector complex

    SciTech Connect

    Fu Chiyu; Prevelige, Peter E.

    2009-11-10

    The incorporation of the DNA packaging connector complex during lambdoid phage assembly in vivo is strictly controlled-one and only one of the twelve identical icosahedral vertices is differentiated by the inclusion of a portal or connector dodecamer. Proposed control mechanisms include obligate nucleation from a connector containing complex, addition of the connector as the final step during assembly, and a connector-mediated increase in the growth rate. The inability to recapitulate connector incorporation in vitro has made it difficult to obtain direct biochemical evidence in support of one model over another. Here we report the development an in vitro assembly system for the well characterized dsDNA phage Phi29 which results in the co-assembly of connector with capsid and scaffolding proteins to form procapsid-like particles (PLPs). Immuno-electron microscopy demonstrates the specific incorporation of connector vertex in PLPs. The connector protein increases both the yield and the rate of capsid assembly suggesting that the incorporation of the connector in Phi29 likely promotes nucleation of assembly.

  8. Ionic switch controls the DNA state in phage λ

    SciTech Connect

    Li, Dong; Liu, Ting; Zuo, Xiaobing; Li, Tao; Qiu, Xiangyun; Evilevitch, Alex

    2015-06-19

    We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid by changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. The results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.

  9. Characterization of verotoxin-encoding phages from Escherichia coli O103:H2 strains of bovine and human origins.

    PubMed

    Karama, Musafiri; Gyles, Carlton L

    2008-08-01

    The objectives of this study were to induce and characterize verotoxin-encoding phages from a collection of 91 verotoxin-producing Escherichia coli (VTEC) O103:H2 strains of human and bovine origins. All the strains carried the vt1 gene, and two carried the vt2 gene as well. The phages were induced by UV irradiation and characterized by DNA restriction fragment length polymorphism (RFLP), genome size, morphology, and Q and P genes, characteristic of lambdoid phages. A total of 32 vt-positive phages were induced and isolated from 31 VTEC O103:H2 strains. Thirty phages were vt1 positive, and two were vt2 positive. Ten of the 30 vt1-positive phages (33.3%) were from cattle strains, and 20 (66.6%) were from human strains. The two vt2-positive phages were from human strains. Phages belonged to 21 RFLP profiles, of which 17 were single-phage profiles and 4 were multiple-phage profiles. The estimated genome size of the phages ranged from 34 to 84 kb. Two phages that were examined by electron microscopy possessed hexagonal heads with long tails, and one had an elongated head with a long tail. The Q and P genes were amplified in all 32 phages, and the Q-stxA(1) gene region yielded an amplicon in 19 phages (59.3%). It is concluded that the VTEC O103:H2 strains of human origin were more readily inducible than those of bovine origin and that the genotypic profiles of verotoxin-encoding phages were highly diverse, as revealed by their RFLP profiles.

  10. Magnetically-assisted impedimetric detection of bacteria using phage-modified carbon microarrays.

    PubMed

    Shabani, Arghavan; Marquette, Christophe A; Mandeville, Rosemonde; Lawrence, Marcus F

    2013-11-15

    This study presents an investigation on the possibility of improving the detection limit of bacteria with an inexpensive electrochemical, impedimetric sensor platform, by integrating the sensor with magnetic manipulation. The approach uses T4 bacteriophage coated Dynabeads to selectively capture and concentrate E. coli K12 cells from samples, to increase the sensitivity of detection at the surface of functionalized screen-printed carbon microarrays. Fluorescence and flow cytometry measurements indicate that the surface modification of the magnetic beads, with phages, and binding with the bacteria, were successful. Integration of the screen-printed carbon-based impedimetric sensor, with a magnetic manipulation system, was found to improve the sensitivity of the device, decreasing the limit of detection of E. coli K12 from 10(4) to 10(3) cfu/mL. We have also demonstrated that this approach provides for more specific detection of bacteria, enabling the operator to account for non-specific adsorption, and detection of bacteria in more complex (real) samples (milk).

  11. Characterization of Streptococcus gordonii prophage PH15: complete genome sequence and functional analysis of phage-encoded integrase and endolysin.

    PubMed

    van der Ploeg, Jan R

    2008-10-01

    Streptococcus gordonii OMZ1039, isolated from supragingival dental plaque, was found to harbour a prophage, PH15, whose excision could be induced by mitomycin treatment. Phage PH15 belongs to the Siphoviridae. The complete genome sequence of PH15 was determined. The genome was 39 136 bp in size and contained 61 ORFs. The genome of PH15 was most similar in the structural module to the temperate bacteriophages MM1 and phiNIH1.1 from Streptococcus pneumoniae and Streptococcus pyogenes, respectively. In strain OMZ1039, PH15 was found to reside as a prophage in the cysteinyl-tRNA gene. A plasmid, harbouring the attP site and the integrase gene downstream of a constitutive promoter, was capable of site-specific integration into the genomes of different oral streptococcal species. The phage endolysin was purified after expression in Escherichia coli and found to inhibit growth of all S. gordonii strains tested and several different streptococcal species, including the pathogens Streptococcus mutans, S. pyogenes and Streptococcus agalactiae.

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

    PubMed

    Mikalová, Lenka; Bosák, Juraj; Hříbková, Hana; Dědičová, Daniela; Benada, Oldřich; Šmarda, Jan; Šmajs, David

    2017-01-01

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

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

    PubMed Central

    Hříbková, Hana; Dědičová, Daniela; Benada, Oldřich; Šmarda, Jan; Šmajs, David

    2017-01-01

    Forty strains of Salmonella enterica (S. enterica) subspecies salamae (II), arizonae (IIIa), diarizonae (IIIb), and houtenae (IV) were isolated from human or environmental samples and tested for bacteriophage production. Production of bacteriophages was observed in 15 S. enterica strains (37.5%) belonging to either the subspecies salamae (8 strains) or diarizonae (7 strains). Activity of phages was tested against 52 pathogenic S. enterica subsp. enterica isolates and showed that phages produced by subsp. salamae had broader activity against pathogenic salmonellae compared to phages from the subsp. diarizonae. All 15 phages were analyzed using PCR amplification of phage-specific regions and 9 different amplification profiles were identified. Five phages (SEN1, SEN4, SEN5, SEN22, and SEN34) were completely sequenced and classified as temperate phages. Phages SEN4 and SEN5 were genetically identical, thus representing a single phage type (i.e. SEN4/5). SEN1 and SEN4/5 fit into the group of P2-like phages, while the SEN22 phage showed sequence relatedness to P22-like phages. Interestingly, while phage SEN34 was genetically distantly related to Lambda-like phages (Siphoviridae), it had the morphology of the Myoviridae family. Based on sequence analysis and electron microscopy, phages SEN1 and SEN4/5 were members of the Myoviridae family and phage SEN22 belonged to the Podoviridae family. PMID:28118395

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

    SciTech Connect

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

    2013-09-01

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

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

    PubMed

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

    2014-09-01

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

  16. Genome sequence of the phage clP1, which infects the beer spoilage bacterium Pediococcus damnosus.

    PubMed

    Kelly, David; O'Sullivan, Orla; Mills, Susan; McAuliffe, Olivia; Ross, R Paul; Neve, Horst; Coffey, Aidan

    2012-08-01

    Pediococcus damnosus (P. damnosus) bacteriophage (phage) clP1 is a novel virulent phage isolated from a municipal sewage sample collected in Southern Ireland. This phage infects the beer spoilage strain P. damnosus P82 which was isolated from German breweries. Sequencing of the phage has revealed a linear double stranded DNA genome of 38,013 base pairs (bp) with an overall GC content of 47.6%. Fifty seven open reading frames (ORFs) were identified of which 30 showed homology to previously sequenced proteins, and as a consequence 20 of these were assigned predicted functions. The majority of genes displayed homology with genes from the Lactobacillus plantarum phage phiJL-1. All genes were located on the same coding strand and in the same orientation. Morphological characterisation placed phage clP1 as a member of the Siphoviridae family with an isometric head (59 nm diameter) and non-contractile tail (length 175 nm; diameter 10nm. Interestingly, the phage clP1 genome was found to share very limited identity with other phage genome sequences in the database, and was hence considered unique. This was highlighted by the genome organisation which differed slightly to the consensus pattern of genomic organisation usually found in Siphoviridae phages. With the genetic machinery present for a lytic lifecycle and the absence of potential endotoxin factors, this phage may have applications in the biocontrol of beer spoilage bacteria. To our knowledge, this study represents the first reported P. damnosus phage genome sequence.

  17. Phage-mediated cloning of bldA, a region involved in Streptomyces coelicolor morphological development, and its analysis by genetic complementation.

    PubMed Central

    Piret, J M; Chater, K F

    1985-01-01

    Streptomyces coelicolor bald (bld) mutants form colonies of vegetative substrate mycelium, but do not develop aerial hyphae or spore chains. The bldA strains form none of the four antibiotics known to be produced by the parent strain. With a vector derived from the temperate bacteriophage phi C31, a 5.6-kilobase fragment of wildtype DNA was cloned which restored sporulation to five independent bldA mutants when lysogenized with the recombinant phage. The cloned gene(s) was dominant over the mutant alleles. Phage integration by recombination of the cloned bldA+ DNA with the bldA region of each mutant produced mainly sporulating colonies, presumably heterozygous bldA+/bldA partial diploids for the insert DNA. However, a minority of these primary transductants were bald and were apparently homozygous bldA/bldA mutant partial diploids, formed by some homogenetization process. The phages released from the bald lysogens carried bldA mutations and were used to show that bldA+ sequences had been cloned and that fine mapping of the region could be performed. Images PMID:2993254

  18. Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.

    PubMed

    Ren, Z J; Baumann, R G; Black, L W

    1997-08-22

    A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA ligase. Intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end terminated linear plasmid DNAs following transformation. Recombinant plasmids could be constructed in vivo by co-transformation with linearized vector plus insert DNAs, followed by intermolecular ligation in the T4 ligase strains to yield clones without deletions or rearrangements. Thus, in vitro packaged lox-site terminated plasmid DNAs injected from phage T4 were recircularized by T4 ligase in vivo with an efficiency comparable to CRE recombinase. Clones that expressed a capsid-binding 14-aa N-terminal peptide extension derivative of the HOC (highly antigenic outer capsid) protein for T4 phage hoc gene display were constructed by co-transformation with a linearized vector and a PCR-synthesized hoc gene. Therefore, the T4 DNA ligase strains are useful for cloning linear DNAs in vivo by transformation or transduction of DNAs with nonsequence-specific but compatible DNA ends.

  19. Phage Therapy is Effective in Protecting Honeybee Larvae from American Foulbrood Disease.

    PubMed

    Ghorbani-Nezami, Sara; LeBlanc, Lucy; Yost, Diane G; Amy, Penny S

    2015-01-01

    American foulbrood disease has a major impact on honeybees (Apis melifera) worldwide. It is caused by a Gram-positive, spore-forming bacterium, Paenibacillus larvae. The disease can only affect larval honeybees, and the bacterial endospores are the infective unit of the disease. Antibiotics are not sufficient to combat the disease due to increasing resistance among P. larvae strains. Because of the durability and virulence of P. larvae endospores, infections spread rapidly, and beekeepers are often forced to burn beehives and equipment. To date, very little information is available on the use of bacteriophage therapy in rescuing and preventing American foulbrood disease, therefore the goal of this study was to test the efficacy of phage therapy against P. larvae infection. Out of 32 previously isolated P. larvae phages, three designated F, WA, and XIII were tested on artificially reared honeybee larvae infected with P. larvae strain NRRL B-3650 spores. The presence of P. larvae DNA in dead larvae was confirmed by 16S rRNA gene-specific polymerase chain reaction amplification. Survival rates for phage-treated larvae were approximately the same as for larvae never infected with spores (84%), i.e., the phages had no deleterious effect on the larvae. Additionally, prophylactic treatment of larvae with phages before spore infection was more effective than administering phages after infection, although survival in both cases was higher than spores alone (45%). Further testing to determine the optimal combination and concentration of phages, and testing in actual hive conditions are needed.

  20. Phage Therapy is Effective in Protecting Honeybee Larvae from American Foulbrood Disease

    PubMed Central

    Ghorbani-Nezami, Sara; LeBlanc, Lucy; Yost, Diane G.; Amy, Penny S.

    2015-01-01

    American foulbrood disease has a major impact on honeybees (Apis melifera) worldwide. It is caused by a Gram-positive, spore-forming bacterium, Paenibacillus larvae. The disease can only affect larval honeybees, and the bacterial endospores are the infective unit of the disease. Antibiotics are not sufficient to combat the disease due to increasing resistance among P. larvae strains. Because of the durability and virulence of P. larvae endospores, infections spread rapidly, and beekeepers are often forced to burn beehives and equipment. To date, very little information is available on the use of bacteriophage therapy in rescuing and preventing American foulbrood disease, therefore the goal of this study was to test the efficacy of phage therapy against P. larvae infection. Out of 32 previously isolated P. larvae phages, three designated F, WA, and XIII were tested on artificially reared honeybee larvae infected with P. larvae strain NRRL B-3650 spores. The presence of P. larvae DNA in dead larvae was confirmed by 16S rRNA gene-specific polymerase chain reaction amplification. Survival rates for phage-treated larvae were approximately the same as for larvae never infected with spores (84%), i.e., the phages had no deleterious effect on the larvae. Additionally, prophylactic treatment of larvae with phages before spore infection was more effective than administering phages after infection, although survival in both cases was higher than spores alone (45%). Further testing to determine the optimal combination and concentration of phages, and testing in actual hive conditions are needed. PMID:26136497

  1. Screening specific polypeptides of breast cancer stem cells from a phage display random peptide library

    PubMed Central

    Liu, Fei; Qi, Chun-Ling; Kong, Mian; Liu, Ting-Ting; Li, Lei; Li, Bao-Jiang

    2016-01-01

    The present study aimed to identify polypeptides that specifically bond to breast cancer stem cells from a phage display random 12 peptide library, in addition to the affinity and specificity of polypeptides. A phage display random 12 peptide library was screened using breast cancer stem cells as targets isolated from the MDA-MB-231 cell line using the serum-free culture technique with hs578bst and MDA-MB-231 cells as subtract-screening cells. Positive and specific binding clones were amplified and sent for sequencing. The affinity and specificity of the positive clones were subsequently identified by ELISA and 3,3′-diaminobenzidine staining. The results demonstrated that phages were gathered ~500 times following three rounds of biopanning. ELISA identified that the affinity to breast cancer stem cells of the no. 6 phage was 6.14 times higher than that in the control group. In addition, immunohistochemistry observed that the no. 6 phage exhibited high-specificity bonding to breast cancer stem cells, and the peptide sequence of the positive phage was GYSASRSTIPGK following DNA sequencing and translation. Thus, the present study isolated a specific peptide that bonds to breast cancer stem cells from a phage display random peptide library, which may facilitate further studies regarding the stem cell-targeted therapy of breast cancer. PMID:28105180

  2. Phage resistance of a marine bacterium, Roseobacter denitrificans OCh114, as revealed by comparative proteomics.

    PubMed

    Huang, Chunxiao; Zhang, Yongyu; Jiao, Nianzhi

    2010-08-01

    Roseobacter is a dominant lineage in the marine environment. This group of bacteria is diverse in terms of both their phylogenetic composition and their physiological potential. Roseobacter denitrificans OCh114 is one of the most studied bacteria of the Roseobacter lineage. Recently, a lytic phage (RDJLPhi1) that infects this bacterium was isolated and a mutant strain (M1) of OCh114 that is resistant to RDJLPhi1 was also obtained. Here, we investigate the mechanisms supporting phage resistance of M1. Our results excluded the possibilities of several phage resistance mechanisms, including abortive infection, lysogeny, and the clustered regularly interspaced short palindromic repeats (CRISPRs) related mechanism. Adsorption kinetics assays revealed that adsorption inhibition might be a potential cause for the phage resistance of M1. Comparative proteomic analysis of M1 and OCh114 revealed significant changes in the membrane protein compliment of these bacteria. Five membrane proteins with important biological functions were significantly down-regulated in the phage-resistant M1. Meanwhile, several outer membrane porins with different modifications and an OmpA family domain protein were markedly up-regulated. We hypothesize that the down-regulated membrane proteins in M1 may serve as the potential phage receptors, whose absence prevented the adsorption of phage RDJLPhi1 to host cells and subsequent infection.

  3. Antibiotic treatment expands the resistance reservoir and ecological network of the phage metagenome.

    PubMed

    Modi, Sheetal R; Lee, Henry H; Spina, Catherine S; Collins, James J

    2013-07-11

    The mammalian gut ecosystem has considerable influence on host physiology, but the mechanisms that sustain this complex environment in the face of different stresses remain obscure. Perturbations to the gut ecosystem, such as through antibiotic treatment or diet, are at present interpreted at the level of bacterial phylogeny. Less is known about the contributions of the abundant population of phages to this ecological network. Here we explore the phageome as a potential genetic reservoir for bacterial adaptation by sequencing murine faecal phage populations following antibiotic perturbation. We show that antibiotic treatment leads to the enrichment of phage-encoded genes that confer resistance via disparate mechanisms to the administered drug, as well as genes that confer resistance to antibiotics unrelated to the administered drug, and we demonstrate experimentally that phages from treated mice provide aerobically cultured naive microbiota with increased resistance. Systems-wide analyses uncovered post-treatment phage-encoded processes related to host colonization and growth adaptation, indicating that the phageome becomes broadly enriched for functionally beneficial genes under stress-related conditions. We also show that antibiotic treatment expands the interactions between phage and bacterial species, leading to a more highly connected phage-bacterial network for gene exchange. Our work implicates the phageome in the emergence of multidrug resistance, and indicates that the adaptive capacity of the phageome may represent a community-based mechanism for protecting the gut microflora, preserving its functional robustness during antibiotic stress.

  4. On size-dependent stability and infectivity of λ bacterial phages

    NASA Astrophysics Data System (ADS)

    Li, Long; Wang, Jizeng

    2015-02-01

    The elastic icosahedral capsid of λ phages plays an important role in the life cycle of these phages, such as holding the viral genome and releasing confinement for DNA ejection. Understanding how a nanosized elastic capsid guarantees the stability and infectivity of λ phages is challenging. In this article, we propose a combined nonlinear continuum and statistical mechanics model by considering the effects of DNA bending deformation, electrostatic repulsion between DNA-DNA strands, and elastic deformation of phage capsid to investigate the coupled process between capsid and DNA in packaging and ejection. Based on this model, we show that packaging DNA into immature λ phage capsid uses less force than packaging DNA into mature λ phage because of the deformability and softness of the former. Consequently, resistance to DNA packaging inside capsid decreases compared with mature ones. We also observe relationships between phage capsid size and the maximum shear stress on the inner surface of capsid and required osmotic pressure for the complete inhibition of DNA ejection. An optimized radius of capsid, i.e., around 30 nm, is found for both stable DNA packaging and effective viral infection from mechanical standpoints, which may result from physical evolution. All these findings may be interesting to toxicologists, nanotechnologists, and virologists.

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

    PubMed

    Lee, Young-Duck; Park, Jong-Hyun

    2016-02-01

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

  6. Prospects of Phage Application in the Treatment of Acne Caused by Propionibacterium acnes

    PubMed Central

    Jończyk-Matysiak, Ewa; Weber-Dąbrowska, Beata; Żaczek, Maciej; Międzybrodzki, Ryszard; Letkiewicz, Sławomir; Łusiak-Szelchowska, Marzanna; Górski, Andrzej

    2017-01-01

    Propionibacterium acnes is associated with purulent skin infections, and it poses a global problem for both patients and doctors. Acne vulgaris (acne) remains a problem due to its chronic character and difficulty of treatment, as well as its large impact on patients' quality of life. Due to the chronic course of the disease, treatment is long lasting, and often ineffective. Currently there are data regarding isolation of P. acnes phages, and there have been numerous studies on phage killing of P. acnes, but no data are available on phage application specifically in acne treatment. In this review, we have summarized the current knowledge on the phages active against P. acnes described so far and their potential application in the treatment of acne associated with P. acnes. The treatment of acne with phages may be important in order to reduce the overuse of antibiotics, which are currently the main acne treatment. However, more detailed studies are first needed to understand phage functioning in the skin microbiome and the possibility to use phages to combat P. acnes. PMID:28228751