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1.  Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR 
PLoS ONE  2010;5(6):e11337.
Background
Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics.
Methodology/Principal Findings
The objective of this work was to develop an alternative to conventional phage lysis tests – a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages ϕA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 103 CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR.
Conclusions/Significance
Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.
doi:10.1371/journal.pone.0011337
PMCID: PMC2893161  PMID: 20596528
2.  Diagnostic Bioluminescent Phage for Detection of Yersinia pestis▿  
Journal of Clinical Microbiology  2009;47(12):3887-3894.
Yersinia pestis is the etiological agent of the plague. Because of the disease's inherent communicability, rapid clinical course, and high mortality, it is critical that an outbreak, whether it is natural or deliberate, be detected and diagnosed quickly. The objective of this research was to generate a recombinant luxAB (“light”)-tagged reporter phage that can detect Y. pestis by rapidly and specifically conferring a bioluminescent signal response to these cells. The bacterial luxAB reporter genes were integrated into a noncoding region of the CDC plague-diagnostic phage φA1122 by homologous recombination. The identity and fitness of the recombinant phage were assessed through PCR analysis and lysis assays and functionally verified by the ability to transduce a bioluminescent signal to recipient cells. The reporter phage conferred a bioluminescent phenotype to Y. pestis within 12 min of infection at 28°C. The signal response time and signal strength were dependent on the number of cells present. A positive signal was obtained from 102 cells within 60 min. A signal response was not detectable with Escherichia coli, although a weak signal (100-fold lower than that with Y. pestis) was obtained with 1 (of 10) Yersinia enterocolitica strains and 2 (of 10) Yersinia pseudotuberculosis strains at the restrictive temperature. Importantly, serum did not prevent the ability of the reporter phage to infect Y. pestis, nor did it significantly quench the resulting bioluminescent signal. Collectively, the results indicate that the reporter phage displays promise for the rapid and specific diagnostic detection of cultivated Y. pestis isolates or infected clinical specimens.
doi:10.1128/JCM.01533-09
PMCID: PMC2786668  PMID: 19828743
3.  Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity 
eLife  null;4:e06416.
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.
DOI: http://dx.doi.org/10.7554/eLife.06416.001
eLife digest
Viruses are unable to replicate independently. To generate copies of itself, a virus must instead invade a target cell and commandeer that cell's replication machinery. Different viruses are able to invade different types of cell, and a group of viruses known as bacteriophages (or phages for short) replicate within bacteria. The enormous number and diversity of phages in the world means that they play an important role in virtually every ecosystem.
Despite their importance, relatively little is known about how different phage populations are related to each other and how they evolved. Many phages contain their genetic information in the form of strands of DNA. Using genetic sequencing to find out where and how different genes are encoded in the DNA can reveal information about how different viruses are related to each other. These relationships are particularly complicated in phages, as they can exchange genes with other viruses and microbes.
Previous studies comparing the genomes—the complete DNA sequence—of reasonably small numbers of phages that infect the Mycobacterium group of bacteria have found that the phages can be sorted into ‘clusters’ based on similarities in their genes and where these are encoded in their DNA. However, the number of phages investigated so far has been too small to conclude how different clusters are related. Are the clusters separate, or do they form a ‘continuum’ with different genes and DNA sequences shared between different clusters?
Here, Pope, Bowman, Russell et al. compare the individual genomes of 627 bacteriophages that infect the bacterial species Mycobacterium smegmatis. This is by far the largest number of phage genomes analyzed from a single host species. The large number of genomes analyzed allowed a much clearer understanding of the complexity and diversity of these phages to be obtained. The isolation, sequencing and analysis of the hundreds of M. smegmatis bacteriophage genomes was performed by an integrated research and education program, called the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program. This enabled thousands of undergraduate students from different institutions to contribute to the phage discovery and sequencing project, and co-author the report. SEA-PHAGES therefore shows that it is possible to successfully incorporate genuine scientific research into an undergraduate course, and that doing so can benefit both the students and researchers involved.
The results show that while the genomes could be categorized into 28 clusters, the genomes are not completely unrelated. Instead, a spread of diversity is seen, as genes and groups of genes are shared between different clusters. Pope, Bowman, Russell et al. further reveal that the phage population is in a constant state of change, and continuously acquires genes from other microorganisms and viruses.
DOI: http://dx.doi.org/10.7554/eLife.06416.002
doi:10.7554/eLife.06416
PMCID: PMC4408529  PMID: 25919952
bacteriophage; genomics; evolution; viruses
4.  Bacteriophages and Phage-Derived Proteins – Application Approaches 
Current Medicinal Chemistry  2015;22(14):1757-1773.
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.
doi:10.2174/0929867322666150209152851
PMCID: PMC4468916  PMID: 25666799
Bacteriophage application; endolysins; holins; phage-encoded proteins; polysaccharide depolymerases; spanins.
5.  Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future 
Viruses  2014;6(7):2623-2672.
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.
doi:10.3390/v6072623
PMCID: PMC4113786  PMID: 25010767
(bacterio)phages; Bacillus cereus group; Bacillus anthracis; Bacillus thuringiensis; transducing phages; chromosomal prophages; plasmidial prophages; jumbo-phages; Gamma-like phages
6.  The Genomes, Proteomes, and Structures of Three Novel Phages That Infect the Bacillus cereus Group and Carry Putative Virulence Factors 
Journal of Virology  2014;88(20):11846-11860.
ABSTRACT
This article reports the results of studying three novel bacteriophages, JL, Shanette, and Basilisk, which infect the pathogen Bacillus cereus and carry genes that may contribute to its pathogenesis. We analyzed host range and superinfection ability, mapped their genomes, and characterized phage structure by mass spectrometry and transmission electron microscopy (TEM). The JL and Shanette genomes were 96% similar and contained 217 open reading frames (ORFs) and 220 ORFs, respectively, while Basilisk has an unrelated genome containing 138 ORFs. Mass spectrometry revealed 23 phage particle proteins for JL and 15 for Basilisk, while only 11 and 4, respectively, were predicted to be present by sequence analysis. Structural protein homology to well-characterized phages suggested that JL and Shanette were members of the family Myoviridae, which was confirmed by TEM. The third phage, Basilisk, was similar only to uncharacterized phages and is an unrelated siphovirus. Cryogenic electron microscopy of this novel phage revealed a T=9 icosahedral capsid structure with the major capsid protein (MCP) likely having the same fold as bacteriophage HK97 MCP despite the lack of sequence similarity. Several putative virulence factors were encoded by these phage genomes, including TerC and TerD involved in tellurium resistance. Host range analysis of all three phages supports genetic transfer of such factors within the B. cereus group, including B. cereus, B. anthracis, and B. thuringiensis. This study provides a basis for understanding these three phages and other related phages as well as their contributions to the pathogenicity of B. cereus group bacteria.
IMPORTANCE The Bacillus cereus group of bacteria contains several human and plant pathogens, including B. cereus, B. anthracis, and B. thuringiensis. Phages are intimately linked to the evolution of their bacterial hosts and often provide virulence factors, making the study of B. cereus phages important to understanding the evolution of pathogenic strains. Herein we provide the results of detailed study of three novel B. cereus phages, two highly related myoviruses (JL and Shanette) and an unrelated siphovirus (Basilisk). The detailed characterization of host range and superinfection, together with results of genomic, proteomic, and structural analyses, reveal several putative virulence factors as well as the ability of these phages to infect different pathogenic species.
doi:10.1128/JVI.01364-14
PMCID: PMC4178739  PMID: 25100842
7.  Application of bacteriophages for detection of foodborne pathogens 
Bacteriophage  2014;4:e28137.
Bacterial contamination of food products presents a challenge for the food industry and poses a high risk for the consumer. Despite increasing awareness and improved hygiene measures, foodborne pathogens remain a threat for public health, and novel methods for detection of these organisms are needed. Bacteriophages represent ideal tools for diagnostic assays because of their high target cell specificity, inherent signal-amplifying properties, easy and inexpensive production, and robustness. Every stage of the phage lytic multiplication cycle, from the initial recognition of the host cell to the final lysis event, may be harnessed in several ways for the purpose of bacterial detection. Besides intact phage particles, phage-derived affinity molecules such as cell wall binding domains and receptor binding proteins can serve for this purpose. This review provides an overview of existing phage-based technologies for detection of foodborne pathogens, and highlights the most recent developments in this field, with particular emphasis on phage-based biosensors.
doi:10.4161/bact.28137
PMCID: PMC3919822  PMID: 24533229
foodborne pathogens; bacterial detection; diagnostics; reporter phage; biosensor; phage amplification; cell wall binding domain; receptor binding protein
8.  The habits of highly effective phages: population dynamics as a framework for identifying therapeutic phages 
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.
doi:10.3389/fmicb.2014.00618
PMCID: PMC4235362  PMID: 25477869
bacteriophage; phage therapy; mathematical model; population dynamics; bacterial infections
9.  Landscape Phage as a Molecular Recognition Interface for Detection Devices 
Microelectronics journal  2008;39(2):202-207.
Filamentous phages are thread-shaped bacterial viruses. Their outer coat is a tube formed by thousands equal copies of the major coat protein pVIII. Libraries of random peptides fused to pVIII domains were used for selection of phages probes specific for a panel of test antigens and biological threat agents. Because the viral carrier in the phage borne bio-selective probes is infective, they can be cloned individually and propagated indefinitely without needs of their chemical synthesis or reconstructing. As a new bioselective material, landscape phages combine unique characteristics of affinity reagents and self assembling proteins. Biorecognition layers formed by the phage-derived probes bind biological agents with high affinity and specificity and generate detectable signals in analytical platforms. The performance of phage-derived materials as biorecognition interface was illustrated by detection of Bacillus anthracis spores and Salmonella typhimurium cells. With further refinement, the phage-derived analytical platforms for detecting and monitoring of numerous threat agents may be developed, since phage interface against any bacteria, virus or toxin may be readily selected from the landscape phage libraries. As an interface in the field-use detectors, they may be superior to antibodies, since they are inexpensive, highly specific and strong binders, resistant to high temperatures and environmental stresses.
doi:10.1016/j.mejo.2006.11.007
PMCID: PMC2565273  PMID: 19190724
Biosensor; sensor; molecular recognition; interface; bacteriophage; landscape phage; phage display; phage library; Bacillus anthracis; Salmonella typhimurium; nanotechnology; ELISA; quartz crystal microbalance
10.  Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from Yersinia pestis as Next Generation Plague Vaccines 
PLoS Pathogens  2013;9(7):e1003495.
Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.
Author Summary
Plague caused by Yersinia pestis is a deadly disease that wiped out one-third of Europe's population in the 14th century. The organism is listed by the CDC as Tier-1 biothreat agent, and currently, there is no FDA-approved vaccine against this pathogen. Stockpiling of an efficacious plague vaccine that could protect people against a potential bioterror attack has been a national priority. The current vaccines based on the capsular antigen (F1) and the low calcium response V antigen, are promising against both bubonic and pneumonic plague. However, the polymeric nature of F1 with its propensity to aggregate affects vaccine efficacy and generates varied immune responses in humans. We have addressed a series of concerns and generated mutants of F1 and V, which are completely soluble and produced in high yields. We then engineered the vaccine into a novel delivery platform using the bacteriophage T4 nanoparticle. The nanoparticle vaccines induced robust immunogenicity and provided 100% protection to mice and rats against pneumonic plague. These highly efficacious new generation plague vaccines are easily manufactured, and the potent T4 platform which can simultaneously incorporate antigens from other biothreat or emerging infectious agents provides a convenient way for mass vaccination of humans against multiple pathogens.
doi:10.1371/journal.ppat.1003495
PMCID: PMC3708895  PMID: 23853602
11.  Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection 
BMC Microbiology  2011;11:195.
Background
Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy.
Results
We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S. aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (cat) gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80). Treatment with the endolysin-deficient phage rescued mice from the fatal S. aureus infection.
Conclusions
A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage effectively rescued mice from fatal S. aureus infection. To our knowledge this is the first report of a lysis-deficient staphylococcal phage.
doi:10.1186/1471-2180-11-195
PMCID: PMC3224134  PMID: 21880144
12.  The Tripartite Associations between Bacteriophage, Wolbachia, and Arthropods 
PLoS Pathogens  2006;2(5):e43.
By manipulating arthropod reproduction worldwide, the heritable endosymbiont Wolbachia has spread to pandemic levels. Little is known about the microbial basis of cytoplasmic incompatibility (CI) except that bacterial densities and percentages of infected sperm cysts associate with incompatibility strength. The recent discovery of a temperate bacteriophage (WO-B) of Wolbachia containing ankyrin-encoding genes and virulence factors has led to intensifying debate that bacteriophage WO-B induces CI. However, current hypotheses have not considered the separate roles that lytic and lysogenic phage might have on bacterial fitness and phenotype. Here we describe a set of quantitative approaches to characterize phage densities and its associations with bacterial densities and CI. We enumerated genome copy number of phage WO-B and Wolbachia and CI penetrance in supergroup A- and B-infected males of the parasitoid wasp Nasonia vitripennis. We report several findings: (1) variability in CI strength for A-infected males is positively associated with bacterial densities, as expected under the bacterial density model of CI, (2) phage and bacterial densities have a significant inverse association, as expected for an active lytic infection, and (3) CI strength and phage densities are inversely related in A-infected males; similarly, males expressing incomplete CI have significantly higher phage densities than males expressing complete CI. Ultrastructural analyses indicate that approximately 12% of the A Wolbachia have phage particles, and aggregations of these particles can putatively occur outside the Wolbachia cell. Physical interactions were observed between approximately 16% of the Wolbachia cells and spermatid tails. The results support a low to moderate frequency of lytic development in Wolbachia and an overall negative density relationship between bacteriophage and Wolbachia. The findings motivate a novel phage density model of CI in which lytic phage repress Wolbachia densities and therefore reproductive parasitism. We conclude that phage, Wolbachia, and arthropods form a tripartite symbiotic association in which all three are integral to understanding the biology of this widespread endosymbiosis. Clarifying the roles of lytic and lysogenic phage development in Wolbachia biology will effectively structure inquiries into this research topic.
Synopsis
Symbiotic bacteria that are maternally inherited are widespread in terrestrial invertebrates. Such bacteria infect the cells of reproductive tissues and can have important evolutionary and developmental effects on the host. Often these inherited symbionts develop beneficial relationships with their hosts, but some species can also selfishly alter invertebrate reproduction to increase the numbers of infected females (the transmitting sex of the bacteria) in the population. Bacterial-mediated distortions such as male-killing, feminization, parthenogenesis induction, and cytoplasmic incompatibility are collectively known as “reproductive parasitism.” In this article, the investigators show that the associations between the most common reproductive parasite in the biosphere (Wolbachia) and a parasitic wasp host are affected by a mobile element—a temperate bacteriophage of Wolbachia. In contrast to recent reports that suggest bacteriophage WO-B may induce reproductive parasitism, the authors' quantitative and ultrastructural analyses indicate that lytic phage WO-B are lethal and therefore associate with a reduction in both Wolbachia densities and reproductive parasitism. Based on these data, the authors propose a phage density model in which lytic phage development specifically leads to a reduction, rather than induction, of reproductive parisitism. The study is among the first investigations to show that lytic bacteriophage inversely associate with the densities and phenotype of an obligate intracellular bacterium.
doi:10.1371/journal.ppat.0020043
PMCID: PMC1463016  PMID: 16710453
13.  Induction and Properties of a Temperate Bacteriophage from Bacillus stearothermophilus 
Journal of Bacteriology  1965;89(1):175-184.
Welker, N. E. (University of Illinois, Urbana), and L. Leon Campbell. Induction and properties of a temperate bacteriophage from Bacillus stearothermophilus. J. Bacteriol. 89:175–184. 1965.—Bacillus stearothermophilus 1503-4R growing at 55 C was induced to lyse either when 0.05 μg/ml of mitomycin C was added or when it was exposed to ultraviolet light for 30 sec. Lysis of the induced cultures occurred 45 to 60 min after induction. Phage were assayed on B. stearothermophilus 4S giving turbid plaques 0.05 to 0.3 cm in diameter. Noninduced cultures of 1503-4R spontaneously produced one phage per 2.8 × 106 bacterial cells. The optimal temperature for phage production and assay was found to be 55 C. B. stearothermophilus 1503-4R was immune to the isolated temperate phage (TP-1) and to a clear-plaque mutant phage (TP-1C), even when tested at phage mutliplicities of 100. TP-1 and TP-1C phage were identical morphologically having a head 65 mμ in diameter and a tail 240 mμ long and 12 mμ wide. TP-1C phage deoxyribonucleic acid (DNA) had an S20,w value of 24.1 and a calculated molecular weight of 1.21 × 107. DNA base compositions of TP-1 and TP-1C phage were identical (42% guanine + cytosine), but differed significantly from those of the lysogenic or indicator strains of B. stearothermophilus (50% guanine + cytosine). No unusual bases were detected in either the bacterial or phage DNA.
Images
PMCID: PMC315566  PMID: 14255659
14.  Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing Activity against Bacterial Skin Isolates 
mBio  2012;3(5):e00279-12.
ABSTRACT
Investigation of the human microbiome has revealed diverse and complex microbial communities at distinct anatomic sites. The microbiome of the human sebaceous follicle provides a tractable model in which to study its dominant bacterial inhabitant, Propionibacterium acnes, which is thought to contribute to the pathogenesis of the human disease acne. To explore the diversity of the bacteriophages that infect P. acnes, 11 P. acnes phages were isolated from the sebaceous follicles of donors with healthy skin or acne and their genomes were sequenced. Comparative genomic analysis of the P. acnes phage population, which spans a 30-year temporal period and a broad geographic range, reveals striking similarity in terms of genome length, percent GC content, nucleotide identity (>85%), and gene content. This was unexpected, given the far-ranging diversity observed in virtually all other phage populations. Although the P. acnes phages display a broad host range against clinical isolates of P. acnes, two bacterial isolates were resistant to many of these phages. Moreover, the patterns of phage resistance correlate closely with the presence of clustered regularly interspaced short palindromic repeat elements in the bacteria that target a specific subset of phages, conferring a system of prokaryotic innate immunity. The limited diversity of the P. acnes bacteriophages, which may relate to the unique evolutionary constraints imposed by the lipid-rich anaerobic environment in which their bacterial hosts reside, points to the potential utility of phage-based antimicrobial therapy for acne.
IMPORTANCE
Propionibacterium acnes is a dominant member of the skin microflora and has also been implicated in the pathogenesis of acne; however, little is known about the bacteriophages that coexist with and infect this bacterium. Here we present the novel genome sequences of 11 P. acnes phages, thereby substantially increasing the amount of available genomic information for this phage population. Surprisingly, we find that, unlike other well-studied bacteriophages, P. acnes phages are highly homogeneous and show a striking lack of genetic diversity, which is perhaps related to their unique and restricted habitat. They also share a broad ability to kill clinical isolates of P. acnes; phage resistance is not prevalent, but when detected, it appears to be conferred by chromosomally encoded immunity elements within the host genome. We believe that these phages display numerous features that would make them ideal candidates for the development of a phage-based therapy for acne.
doi:10.1128/mBio.00279-12
PMCID: PMC3448167  PMID: 23015740
15.  Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy 
PLoS ONE  2015;10(3):e0118557.
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.
doi:10.1371/journal.pone.0118557
PMCID: PMC4356574  PMID: 25761060
16.  Genomic comparison of 93 Bacillus phages reveals 12 clusters, 14 singletons and remarkable diversity 
BMC Genomics  2014;15(1):855.
Background
The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains.
Results
Whole genome nucleotide and proteome comparison of the 93 extant Bacillus phages revealed 12 distinct clusters, 28 subclusters and 14 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member of the group. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,922 protein families (phams) of which only 951 (19%) had a predicted function. In addition, 3,058 (62%) of phams were orphams (phams containing a gene product from a single phage). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains.
Conclusions
This analysis provides a basis for understanding and characterizing Bacillus phages and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.
doi:10.1186/1471-2164-15-855
PMCID: PMC4197329  PMID: 25280881
Bacteriophage; Phage; Cluster; Bacillus
17.  Synonymous codon usage in forty staphylococcal phages identifies the factors controlling codon usage variation and the phages suitable for phage therapy 
Bioinformation  2012;8(24):1187-1194.
The immergence and dissemination of multidrug-resistant strains of Staphylococcus aureus in recent years have expedited the research on the discovery of novel anti-staphylococcal agents promptly. Bacteriophages have long been showing tremendous potentialities in curing the infections caused by various pathogenic bacteria including S. aureus. Thus far, only a few virulent bacteriophages, which do not carry any toxin-encoding gene but are capable of eradicating staphylococcal infections, were reported. Based on the codon usage analysis of sixteen S. aureus phages, previously three phages were suggested to be useful as the anti-staphylococcal agents. To search for additional S. aureus phages suitable for phage therapy, relative synonymous codon usage bias has been investigated in the protein-coding genes of forty new staphylococcal phages. All phages appeared to carry A and T ending codons. Several factors such as mutational pressure, translational selection and gene length seemed to be responsible for the codon usage variation in the phages. Codon usage indeed varied phage to phage. Of the phages, phages G1, Twort, 66 and Sap-2 may be extremely lytic in nature as majority of their genes possess high translational efficiency, indicating that these phages may be employed in curing staphylococcal infections.
doi:10.6026/97320630081187
PMCID: PMC3530870  PMID: 23275718
Staphylococcal phage; Synonymous codon usage; Translational selection; Mutational bias; Phage therapy
18.  Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae 
Virology Journal  2013;10:100.
Background
Members of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment. Bacteriophage therapy forms one of these alternate strategies.
Methods
Electron microscopy, burst size, host range, sensitivity of phage particles to temperature, chloroform, pH, and restriction digestion of phage DNA were used to characterize Klebsiella phages.
Results and conclusions
Of the 32 isolated phages eight belonged to the family Myoviridae, eight to the Siphoviridae whilst the remaining 16 belonged to the Podoviridae. The host range of these phages was characterised against 254 clinical Enterobacteriaceae strains including multidrug resistant Klebsiella isolates producing extended-spectrum beta-lactamases (ESBLs). Based on their lytic potential, six of the phages were further characterised for burst size, physicochemical properties and sensitivity to restriction endonuclease digestion. In addition, five were fully sequenced. Multiple phage-encoded host resistance mechanisms were identified. The Siphoviridae phage genomes (KP16 and KP36) contained low numbers of host restriction sites similar to the strategy found in T7-like phages (KP32). In addition, phage KP36 encoded its own DNA adenine methyltransferase. The φKMV-like KP34 phage was sensitive to all endonucleases used in this study. Dam methylation of KP34 DNA was detected although this was in the absence of an identifiable phage encoded methyltransferase. The Myoviridae phages KP15 and KP27 both carried Dam and Dcm methyltransferase genes and other anti-restriction mechanisms elucidated in previous studies. No other anti-restriction mechanisms were found, e.g. atypical nucleotides (hmC or glucosyl hmC), although Myoviridae phage KP27 encodes an unknown anti-restriction mechanism that needs further investigation.
doi:10.1186/1743-422X-10-100
PMCID: PMC3620542  PMID: 23537199
Bacteriophage; Klebsiella spp.; Multidrug resistance; Restriction endonuclease patterns; Myoviridae; Siphoviridae; Podoviridae
19.  Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice 
PLoS ONE  2011;6(9):e25486.
Background
Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy.
Methodology/Principal Findings
The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence.
Conclusions/Significance
We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.
doi:10.1371/journal.pone.0025486
PMCID: PMC3182234  PMID: 21980477
20.  A Multifaceted Study of Pseudomonas aeruginosa Shutdown by Virulent Podovirus LUZ19 
mBio  2013;4(2):e00061-13.
ABSTRACT
In contrast to the rapidly increasing knowledge on genome content and diversity of bacterial viruses, insights in intracellular phage development and its impact on bacterial physiology are very limited. We present a multifaceted study combining quantitative PCR (qPCR), microarray, RNA-seq, and two-dimensional gel electrophoresis (2D-GE), to obtain a global overview of alterations in DNA, RNA, and protein content in Pseudomonas aeruginosa PAO1 cells upon infection with the strictly lytic phage LUZ19. Viral genome replication occurs in the second half of the phage infection cycle and coincides with degradation of the bacterial genome. At the RNA level, there is a sharp increase in viral mRNAs from 23 to 60% of all transcripts after 5 and 15 min of infection, respectively. Although microarray analysis revealed a complex pattern of bacterial up- and downregulated genes, the accumulation of viral mRNA clearly coincides with a general breakdown of abundant bacterial transcripts. Two-dimensional gel electrophoretic analyses shows no bacterial protein degradation during phage infection, and seven stress-related bacterial proteins appear. Moreover, the two most abundantly expressed early and late-early phage proteins, LUZ19 gene product 13 (Gp13) and Gp21, completely inhibit P. aeruginosa growth when expressed from a single-copy plasmid. Since Gp13 encodes a predicted GNAT acetyltransferase, this observation points at a crucial but yet unexplored level of posttranslational viral control during infection.
IMPORTANCE
Massive genome sequencing has led to important insights into the enormous genetic diversity of bacterial viruses (bacteriophages). However, for nearly all known phages, information on the impact of the phage infection on host physiology and intracellular phage development is scarce. This aspect of phage research should be revitalized, as phages evolved genes which can shut down or redirect bacterial processes in a very efficient way, which can be exploited towards antibacterial design. In this context, we initiated a study of the human opportunistic pathogen Pseudomonas aeruginosa under attack by one its most common predators, the Phikmvlikevirus. By analyzing various stages of infection at different levels, this study uncovers new features of phage infection, representing a cornerstone for future studies on members of this phage genus.
doi:10.1128/mBio.00061-13
PMCID: PMC3604761  PMID: 23512961
21.  Soil-based systemic delivery and phyllosphere in vivo propagation of bacteriophages 
Bacteriophage  2012;2(4):215-224.
Soil-based root applications and attenuated bacterial strains were evaluated as means to enhance bacteriophage persistence on plants for bacterial disease control. In addition, the systemic nature of phage applied to tomato roots was also evaluated. Several experiments were conducted applying either single phages or phage mixtures specific for Ralstonia solanacearum, Xanthomonas perforans or X. euvesicatoria to soil surrounding tomato plants and measuring the persistence and translocation of the phages over time. In general, all phages persisted in the roots of treated plants and were detected in stems and leaves; although phage level varied and persistence in stems and leaves was at a much lower level compared with persistence in roots. Bacterial wilt control was typically best if the phage or phage mixtures were applied to the soil surrounding tomatoes at the time of inoculation, less effective if applied 3 days before inoculation, and ineffective if applied 3 days after inoculation. The use of an attenuated X. perforans strain was also evaluated to improve the persistence of phage populations on tomato leaf surfaces. In greenhouse and field experiments, foliar applications of an attenuated mutant X. perforans 91-118:∆OPGH strain prior to phage applications significantly improved phage persistence on tomato foliage compared with untreated tomato foliage. Both the soil-based bacteriophage delivery and the use of attenuated bacterial strains improved bacteriophage persistence on respective root and foliar tissues, with evidence of translocation with soil-based bacteriophage applications. Both strategies could lead to improved control of bacterial pathogens on plants.
doi:10.4161/bact.23530
PMCID: PMC3594209  PMID: 23532156
bacteriophage; biocontrol; phage; tomato
22.  YpfΦ: a filamentous phage acquired by Yersinia pestis 
Yersinia pestis, the plague bacillus, has an exceptional pathogenicity for humans. The plague bacillus emerged very recently (≈3,000 years ago) from the enteropathogen Y. pseudotuberculosis. Early after its emergence, Y. pestis became infected by a filamentous phage named YpfΦ. During the microevolution of the plague bacillus, the phage remained in the various lineages as an unstable extrachromosomal element. However, in the sub branch that caused the third plague pandemic, YpfΦ integrated itself into the bacterial chromosome to become a stable prophage. The genome of this phage has the same genetic organization as that of other filamentous phages such as the Vibrio cholerae CTXΦ phage, and shares high sequence identity with the CUS-1 filamentous phage of a high-virulence Escherichia coli K1 clone. In addition to genes involved in phage physiology, YpfΦ carries at each extremity of its genome two open reading frames with no predicted functions. This filamentous phage confers some selective properties to Y. pestis during the infectious process, which may explain why it was conserved duringY. pestis microevolution, despite its instability as an extrachromosomal element in most branches.
doi:10.3389/fmicb.2014.00701
PMCID: PMC4266046  PMID: 25566217
filamentous bacteriophage; plague; Yersinia pestis
23.  Using a bacteriocin structure to engineer a phage lysin that targets Yersinia pestis 
Biochemical Society transactions  2012;40(6):1503-1506.
Purified phage lysins present an alternative to traditional antibiotics and work by hydrolyzing 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.
doi:10.1042/BST20120209
PMCID: PMC3679646  PMID: 23176506
24.  Isolation and Characterization of a Virulent Bacteriophage AB1 of Acinetobacter baumannii 
BMC Microbiology  2010;10:131.
Background
Acinetobacter baumannii is an emerging nosocomial pathogen worldwide with increasing prevalence of multi-drug and pan-drug resistance. A. baumannii exists widely in natural environment, especially in health care settings, and has been shown difficult to be eradicated. Bacteriophages are often considered alternative agent for controlling bacterial infection and contamination. In this study, we described the isolation and characterization of one virulent bacteriophage AB1 capable of specifically infecting A. baumannii.
Results
A virulent bacteriophage AB1, specific for infecting a clinical strain A. baumannii KD311, was first isolated from marine sediment sample. Restriction analysis indicated that phage AB1 was a dsDNA virus with an approximate genome size of 45.2 kb to 46.9 kb. Transmission electron microscopy showed that phage AB1 had an icosahedral head with a non-contractile tail and collar or whisker structures, and might be tentatively classified as a member of the Siphoviridae family. Proteomic pattern of phage AB1, generated by SDS-PAGE using purified phage particles, revealed five major bands and six minor bands with molecular weight ranging from 14 to 80 kilo-dalton. Also determined was the adsorption rate of phage AB1 to the host bacterium, which was significantly enhanced by addition of 10 mM CaCl2. In a single step growth test, phage AB1 was shown having a latent period of 18 minutes and a burst size of 409. Moreover, pH and thermal stability of phage AB1 were also investigated. At the optimal pH 6.0, 73.2% of phages survived after 60 min incubation at 50°C. When phage AB1 was used to infect four additional clinical isolates of A. baumannii, one clinical isolate of Stenotrophomonas maltophilia, and Pseudomonas aeruginosa lab strains PAK and PAO1, none of the tested strains was found susceptible, indicating a relatively narrow host range for phage AB1.
Conclusion
Phage AB1 was capable of eliciting efficient lysis of A. baumannii, revealing its potential as a non-toxic sanitizer for controlling A. baumannii infection and contamination in both hospital and other public environments.
doi:10.1186/1471-2180-10-131
PMCID: PMC2874798  PMID: 20426877
25.  Isolation and Host Range of Bacteriophage with Lytic Activity against Methicillin-Resistant Staphylococcus aureus and Potential Use as a Fomite Decontaminant 
PLoS ONE  2015;10(7):e0131714.
Staphylococcus aureus (SA) is a commensal bacterium and opportunistic pathogen commonly associated with humans and is capable of causing serious disease and death including sepsis, pneumonia, and meningitis. Methicillin-resistant SA (MRSA) isolates are typically resistant to many available antibiotics with the common exception of vancomycin. The presence of vancomycin resistance in some SA isolates combined with the current heavy use of vancomycin to treat MRSA infections indicates that MRSA may achieve broad resistance to vancomycin in the near future. New MRSA treatments are clearly needed. Bacteriophages (phages) are viruses that infect bacteria, commonly resulting in death of the host bacterial cell. Phage therapy entails the use of phage to treat or prevent bacterial infections. In this study, 12 phages were isolated that can replicate in human SA and/or MRSA isolates as a potential way to control these infections. 5 phage were discovered through mitomycin C induction of prophage and 7 others as extracellular viruses. Primary SA strains were also isolated from environmental sources to be used as tools for phage discovery and isolation as well as to examine the target cell host range of the phage isolates by spot testing. Primary isolates were tested for susceptibility to oxacillin in order to determine which were MRSA. Experiments were performed to assess the host range and killing potential of newly discovered phage, and significant reductions in bacterial load were detected. We explored the utility of some phage to decontaminate fomites (glass and cloth) and found a significant reduction in colony forming units of MRSA following phage treatment, including tests of a phage cocktail against a cocktail of MRSA isolates. Our findings suggest that phage treatment can be used as an effective tool to decontaminate human MRSA from both hard surfaces and fabrics.
doi:10.1371/journal.pone.0131714
PMCID: PMC4488860  PMID: 26131892

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