Bacteriophage genomes found in a range of bacterial pathogens encode a diverse array of virulence factors ranging from superantigens or pore forming lysins to numerous exotoxins. Recent studies have uncovered an entirely new class of bacterial virulence factors, called effector proteins or effector toxins, which are encoded within phage genomes that reside among several pathovars of Escherichia coli and Salmonella enterica. These effector proteins have multiple domains resulting in proteins that can be multifunctional. The effector proteins encoded within phage genomes are translocated directly from the bacterial cytosol into their eukaryotic target cells by specialized bacterial type three secretion systems (T3SSs). In this review, we will give an overview of the different types of effector proteins encoded within phage genomes and examine their roles in bacterial pathogenesis.
Escherichia coli; Salmonella enterica; phage encoded exotoxins and effector proteins
This review highlights the main strategies available to control phage infection during large-scale milk fermentation by lactic acid bacteria. The topics that are emphasized include the factors influencing bacterial activities, the sources of phage contamination, the methods available to detect and quantify phages, as well as practical solutions to limit phage dispersion through an adapted factory design, the control of air flow, the use of adequate sanitizers, the restricted used of recycled products, and the selection and growth of bacterial cultures.
bacteriophage; dairy industry; milk fermentation; lactic acid bacteria; phage control strategy
The conventional and most accepted method of measuring the lytic activity of a phage against its bacterial host is the plaque assay. This method is laborious, time consuming and expensive, especially in high throughput analyses where multiple phage-bacterial interactions are required to be monitored simultaneously. It can also vary considerably with the experimenter and by the growth and plating conditions. Alternatively, the lytic activity can be measured indirectly by following the decrease in optical density of the bacterial cultures owing to lysis. Here we describe an automated, high throughput, indirect liquid lysis assay to evaluate phage growth using the OmniLogTM system. The OmniLogTM system uses redox chemistry, employing cell respiration as a universal reporter. During active growth of bacteria, cellular respiration reduces a tetrazolium dye and produces a color change that is measured in an automated fashion. On the other hand, successful phage infection and subsequent growth of the phage in its host bacterium results in reduced bacterial growth and respiration and a concomitant reduction in color. Here we show that microtiter plate wells inoculated with Bacillus anthracis and phage show decreased or no growth, compared with the wells containing bacteria only or phage resistant bacteria plus phage. Also, we show differences in the kinetics of bacterial growth and the timing of appearance of phage resistant bacteria in the presence of individual phages or a cocktail of B. anthracis specific phages. The results of these experiments indicate that the OmniLogTM system could be used reliably for indirectly measuring phage growth in high throughput host range and phage and antibiotics combination studies.
Bacillus anthracis; OmniLogTM; bacteriophage; in vitro lytic assay; phage
Five Y. pestis bacteriophages obtained from various sources were characterized to determine their biological properties, including their taxonomic classification, host range and genomic diversity. Four of the phages (YpP-G, Y, R and YpsP-G) belong to the Podoviridae family, and the fifth phage (YpsP-PST) belongs to the Myoviridae family, of the order Caudovirales comprising of double-stranded DNA phages. The genomes of the four Podoviridae phages were fully sequenced and found to be almost identical to each other and to those of two previously characterized Y. pestis phages Yepe2 and φA1122. However, despite their genomic homogeneity, they varied in their ability to lyse Y. pestis and Y. pseudotuberculosis strains. The five phages were combined to yield a “phage cocktail” (tentatively designated “YPP-100”) capable of lysing the 59 Y. pestis strains in our collection. YPP-100 was examined for its ability to decontaminate three different hard surfaces (glass, gypsum board and stainless steel) experimentally contaminated with a mixture of three genetically diverse Y. pestis strains CO92, KIM and 1670G. Five minutes of exposure to YPP-100 preparations containing phage concentrations of ca. 109, 108 and 107 PFU/mL completely eliminated all viable Y. pestis cells from all three surfaces, but a few viable cells were recovered from the stainless steel coupons treated with YPP-100 diluted to contain ca. 106 PFU/mL. However, even that highly diluted preparation significantly (p = < 0.05) reduced Y. pestis levels by ≥ 99.97%. Our data support the idea that Y. pestis phages may be useful for decontaminating various hard surfaces naturally- or intentionally-contaminated with Y. pestis.
bacteriophage; phage; Yersinia pestis; surface decontamination
Foods contaminated with Escherichia coli O157:H7 cause more than 63,000 foodborne illnesses in the United States every year, resulting in a significant economic impact on medical costs and product liabilities. Efforts to reduce contamination with E. coli O157:H7 have largely focused on washing, application of various antibacterial chemicals, and gamma-irradiation, each of which has practical and environmental drawbacks. A relatively recent, environmentally-friendly approach proposed for eliminating or significantly reducing E. coli O157:H7 contamination of foods is the use of lytic bacteriophages as biocontrol agents. We found that EcoShield™, a commercially available preparation composed of three lytic bacteriophages specific for E. coli O157:H7, significantly (p < 0.05) reduced the levels of the bacterium in experimentally contaminated beef by ≥ 94% and in lettuce by 87% after a five minute contact time. The reduced levels of bacteria were maintained for at least one week at refrigerated temperatures. However, the one-time application of EcoShield™ did not protect the foods from recontamination with E. coli O157:H7. Our results demonstrate that EcoShield™ is effective in significantly reducing contamination of beef and lettuce with E. coli O157:H7, but does not protect against potential later contamination due to, for example, unsanitary handling of the foods post processing.
EcoShield™; Escherichia coli O157:H7; bacteriophage; beef; food safety; genomics; ground beef; lettuce; phage; phylogeny
The spread of natural or weaponized drug-resistant plague among humans is a credible high consequence threat to public health that demands the prompt introduction of alternatives to antibiotics such as bacteriophage. Early attempts to treat plague with phages in the 1920s–1930s were sometimes promising but mostly failed, purportedly due to insufficient knowledge of phage biology and poor experimental design. We recently reported the striking stability of plague diagnostic bacteriophages, their safety for animal use, propagation in vivo and partial protection of mice from deadly plague after a single injection of phage. In this addendum we reflect on that article, other recent publications and our unpublished data, and discuss the prospects of phage therapy against plague.
Yersinia pestis; multidrug-resistant plague; bacteriophages; phage safety; pharmacokinetics and pharmacodynamics; plague therapy
The dawn of next generation sequencing technologies has opened up exciting possibilities for whole genome sequencing of a plethora of organisms. The 2nd and 3rd generation sequencing technologies, based on cloning-free, massively parallel sequencing, have enabled the generation of a deluge of genomic sequences of both prokaryotic and eukaryotic origin in the last seven years. However, whole genome sequencing of bacterial viruses has not kept pace with this revolution, despite the fact that their genomes are orders of magnitude smaller in size compared with bacteria and other organisms. Sequencing phage genomes poses several challenges; (1) obtaining pure phage genomic material, (2) PCR amplification biases and (3) complex nature of their genetic material due to features such as methylated bases and repeats that are inherently difficult to sequence and assemble. Here we describe conclusions drawn from our efforts in sequencing hundreds of bacteriophage genomes from a variety of Gram-positive and Gram-negative bacteria using Sanger, 454, Illumina and PacBio technologies. Based on our experience we propose several general considerations regarding sample quality, the choice of technology and a “blended approach” for generating reliable whole genome sequences of phages.
bacteriophage genome; Illumina HiSeq; Roche 454; PacBio; Sanger sequencing; Hybrid genome assembly; SISPA; Scaffolding
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.
bacteriophages; biofilm; cellular arrangement; lysis inhibition; microcolony; phage; phage ecology; plaque formation; T-even phages
Bacteriophages (phages) have been utilized for decades as a means for uniquely identifying their target bacteria. Due to their inherent natural specificity, ease of use, and straightforward production, phage possess a number of desirable attributes which makes them particularly suited as bacterial detectors. As a result, extensive research has been conducted into the development of phage, or phage-derived products to expedite the detection of human pathogens. However, very few phage-based diagnostics have transitioned from the research lab into a clinical diagnostic tool. Herein we review the phage-based platforms that are currently used for the detection of Mycobacterium tuberculosis, Yersinia pestis, Bacillus anthracis and Staphylococcus aureus in the clinical field. We briefly describe the disease, the current diagnostic options, and the role phage diagnostics play in identifying the cause of infection, and determining antibiotic susceptibility.
clinical diagnosis; bacterial detection; phage; reporter; bioluminescence; infectious disease; species identification
The quality of bacteriophage electron microscopy appears to be on a downward course since the 1980s. This coincides with the introduction of digital electron microscopes and a general lowering of standards, possibly due to the disappearance of several world-class electron microscopists The most important problem seems to be poor contrast. Positive staining is frequently not recognized as an undesirable artifact. Phage parts, bacterial debris, and aberrant or damaged phage particles may be misdiagnosed as bacterial viruses. Digital electron microscopes often seem to be operated without magnification control because this is difficult and inconvenient. In summary, most phage electron microscopy problems may be attributed to human failure. Journals are a last-ditch defense and have a heavy responsibility in selecting competent reviewers and rejecting, or not, unsatisfactory articles.
artifacts; contrast; crystals; digital electron microscopy; dimensions; fake viruses focus; misdiagnosis; monsters; positive staining; purification
Since the enlightenment, scientists have enjoyed a self-image as rational actors, guided only by reason, evidence and logic. When the Royal Society of London was founded in 1660 it chose as its motto “nullius in verba” (often translated as “on the word of no one”) a reference to Horace’s Epistles “Nullius addictus iurare in verba magistri…” (being not obliged swear allegiance to any master). Similar to our 21st century contemporaries who embrace the “new evidenced-based medicine,” the “virtuosi” of the Royal Society proclaimed a new era in science based only on observation and direct experience.
phage therapy; Félix d'Herelle; Gunther Stent; Frederick Twort; lytic principle
New immunoreagents for detection of TNT-derivatives TNP and TNP-Tris were developed using phage display technique. The monovalent and pentavalent recombinant phages carrying scFv specific for TNT were constructed and compared with each other to define the impact of valency and molecule dimension on antibody binding in immunoassay. Also, the bifunctional phages were generated, which carried TNT-specific scFvs as well as enzyme β-lactamase as a model marker on its surface. The most sensitive recombinant phages were selected and used for detection of TNP-Tris in a competitive ELISA based on immobilized antigen. Preincubation and partial phages saturation with a sample containing antigen allowed competition with immobilized hapten and displacement of free antigen. The phages exposing enzyme were used as immunoreagents for single step detection. The other phages were detected with specific marked antibodies. To date, the results presented in this paper are the first ever published regarding the recombinant phages for the detection of TNT.
TNT; phage; scFv; recombinant; immunoassay
Staphylococcus aureus pathogenicity islands (SaPIs) are mobile genetic elements that encode virulence factors and depend on helper phages for their mobilization. Such mobilization is specific and depends on the ability of a phage protein to inactivate the SaPI repressor Stl. Phage 80α can mobilize several SaPIs, including SaPI1 and SaPIbov1, via its Sri and Dut proteins, respectively. In many cases, the capsids formed in the presence of the SaPI are smaller than those normally produced by the phage. Two SaPI-encoded proteins, CpmA and CpmB, are involved in this size determination process. S. aureus strain Newman contains four prophages, named φNM1 through φNM4. Phages φNM1 and φNM2 are very similar to phage 80α in the structural genes, and encode almost identical Sri proteins, while their Dut proteins are highly divergent. We show that φNM1 and φNM2 are able to mobilize both SaPI1 and SaPIbov1 and yield infectious transducing particles. The majority of the capsids formed in all cases are small, showing that both SaPIs can redirect the capsid size of both φNM1 and φNM2.
mobile genetic elements; SaPI1; SaPIbov1; bacteriophage assembly; capsid size determination
A mycobacteriophage-specific repressor with the enhanced operator DNA binding activity at 32°C and no activity at 42°C has not been generated yet though it has potential in developing a temperature-controlled expression vector for mycobacterial system. To create such an invaluable repressor, here we have characterized four substitution mutants of mycobacteriophage L1 repressor by various probes. The W69C repressor mutant displayed no operator DNA binding activity, whereas, P131L repressor mutant exhibited very little DNA binding at 32°C. In contrast, both E36K and E39Q repressor mutants showed significantly higher DNA binding activity at 32°C, particularly, under in vivo conditions. Various mutations also had different effects on the structure, stability and the dimerization ability of L1 repressor. While the W69C mutant possessed a distorted tertiary structure, the P131L mutant dimerized poorly in solution at 32°C. Interestingly, both these mutants lost their two-domain structure and aggregated rapidly at 42°C. Of the native and mutant L1 repressor proteins, W69C and E36K mutants appeared to be the least stable at 32°C. Studies together suggest that the mutants, particularly P131L and E39Q mutants, could be used for creating a high affinity temperature-sensitive repressor in the future.
mycobacteriophage L1; repressor; early promoter; operator DNA; mutant repressor and expression vector
Two inducible temperate bacteriophages ΦS9 and ΦS63 from Clostridium perfringens were sequenced and analyzed. Isometric heads and long non-contractile tails classify ΦS9 and ΦS63 in the Siphoviridae family, and their genomes consist of 39,457 bp (ΦS9) and 33,609 bp (ΦS63) linear dsDNA, respectively. ΦS63 has 3′-overlapping cohesive genome ends, whereas ΦS9 is the first Clostridium phage featuring an experimentally proven terminally redundant and circularly permuted genome. A total of 50 and 43 coding sequences were predicted for ΦS9 and ΦS63, respectively, organized into 6 distinct lifestyle-associated modules typical for temperate Siphoviruses. Putative functions could be assigned to 26 gene products of ΦS9, and to 25 of ΦS63. The ΦS9 attB attachment and insertion site is located in a non-coding region upstream of a putative phosphorylase gene. Interestingly, ΦS63 integrates into the 3′ part of sigK in C. perfringens, and represents the first functional skin-element-like phage described for this genus. With respect to possible effects of lysogeny, we did not obtain evidence that ΦS9 may influence sporulation of a lysogenized host. In contrast, interruption of sigK, a sporulation associated gene in various bacteria, by the ΦS63 prophage insertion is more likely to affect sporulation of its carrier.
Clostridium perfringens; prophage; attachment site; sporulation; skin-element
Quantification of bacteriophages by real-time quantitative PCR (qPCR) is an interesting alternative to the traditional plaque assay. Importantly, the method should in principle be able to discriminate between closely related phages that are indistinguishable by most other means. Here, a method is presented that employs qPCR to discriminate and quantify ten closely related lambdoid phages of Escherichia coli str. K-12. It is shown that (1) treatment of samples with DNase efficiently removes non-encapsidated DNA, while the titer of plaque forming units is not affected, (2) individual phage types can be accurately quantified in mixed lysates, and (3) the detection limit corresponds to that of a plaque assay. The method is used to quantify individual phage types that are released from lysogens that carry up to three different prophages.
multiple infections; real-time quantitative PCR; bacteriophages; Escherichia coli; bacteriophage lambda; lambdoid bacteriophages; detection; discrimination; quantification; polylysogeny
In 1921, Richard Bruynoghe and his student Joséph Maisin published on the first use of bacteriophages in a phage therapy context. At that time, Bruynoghe (a medical doctor) was affiliated as a professor at the KU Leuven (Belgium) for just over a decade, within the Bacteriological Institute which he founded and led. After a distinguished career (he was acting mayor of the city of Leuven-Belgium during the second World War), he received a special medical award in 1951 just before his retirement in 1952. In this perspective, he was asked to provide an overview of his research for a lay-audience within the local University magazine: Onze Alma Mater (Our alma mater). We, as current affiliates of the KU Leuven are honored to present some of his legacy, which to date has been largely overlooked in historical accounts.
bacteria; bacteriophages; historical overview; phage biology; phage therapy
Bacteriophages offer interesting alternatives to antibodies for the specific capture and detection of pathogenic bacteria onto biosensing surfaces. Procedures for the optimal chemical immobilization of lytic bacteriophages onto surfaces are presented. More specifically, the removal of lysate contaminants from bacteriophage suspensions by size exclusion chromatography significantly increases the resultant planar surface density of immobilized bacteriophages. E. coli T4 and Salmonella enterica serovar Typhimurium P22 phage systems seem to undergo highly heterogeneous adsorption to the surface, possibly explaining the observed phage clustering at higher surface densities. The T4 phage and its E. coli host were initially employed as a model system where we discovered an optimal planar surface density of phages for best bacterial capture: 18.9 ± 0.8 phages/μm2 capturing 18.0 ± 0.3 bacteria/100 μm2. Phage surface clustering ultimately limits the T4 phage-immobilized surface’s ability to specifically capture its host bacteria. Nevertheless, this is to our knowledge the largest surface capture density of E. coli reported using intact T4 bacteriophages. Two additional purified bacteriophage systems (P22 and Campylobacter jejuni phage NCTC 12673) were then similarly studied for their ability to capture their corresponding host bacteria (Salmonella enterica serovar Typhimurium and Campylobacter jejuni respectively) on a surface.
bacteriophage; biosensor; food contamination; pathogenic bacteria; purification; surface adsorption; virus immobilization
We investigate genes of lytic, Bacillus thuringiensis bacteriophage 0305ϕ8-36 that are non-essential for laboratory propagation, but might have a function in the wild. We isolate deletion mutants to identify these genes. The non-permutation of the genome (218.948 Kb, with a 6.479 Kb terminal repeat and 247 identified orfs) simplifies isolation of deletion mutants. We find two islands of non-essential genes. The first island (3.01% of the genomic DNA) has an informatically identified DNA translocation operon. Deletion causes no detectable growth defect during propagation in a dilute agarose overlay. Identification of the DNA translocation operon begins with a DNA relaxase and continues with a translocase and membrane-binding anchor proteins. The relaxase is in a family, first identified here, with homologs in other bacteriophages. The second deleted island (3.71% of the genome) has genes for two metallo-protein chaperonins and two tRNAs. Deletion causes a significant growth defect. In addition, (1) we find by “in situ” (in-plaque) single-particle fluorescence microscopy that adsorption to the host occurs at the tip of the 486 nm long tail, (2) we develop a procedure of 0305ϕ8-36 purification that does not cause tail contraction, and (3) we then find by electron microscopy that 0305ϕ8-36 undergoes tail tip-tail tip dimerization that potentially blocks adsorption to host cells, presumably with effectiveness that increases as the bacteriophage particle concentration increases. These observations provide an explanation of the previous observation that 0305ϕ8-36 does not lyse liquid cultures, even though 0305ϕ8-36 is genomically lytic.
bacteriophage; deletion mutant; DNA sequencing; electron microscopy; fluorescence microscopy; informatics; long-genome; microbial biofilm
Elongated trimeric adhesins are a distinct class of proteins employed by phages and viruses to recognize and bind to their host cells, and by bacteria to bind to their target cells and tissues. The tailspikes of E. coli phage K1F and Bacillus phage Ø29 exhibit auto-chaperone activity in their trimeric C-terminal domains. The P22 tailspike is structurally homologous to those adhesins. Though there are no disulfide bonds or reactive cysteines in the native P22 tailspikes, a set of C-terminal cysteines are very reactive in partially folded intermediates, implying an unusual local conformation in the domain. This is likely to be involved in the auto-chaperone function. We examined the unusual reactivity of C-terminal tailspike cysteines during folding and assembly as a potential reporter of auto-chaperone function. Reaction with IAA blocked productive refolding in vitro, but not off-pathway aggregation. Two-dimensional PAGE revealed that the predominant intermediate exhibiting reactive cysteine side chains was a partially folded monomer. Treatment with reducing reagent promoted native trimer formation from these species, consistent with transient disulfide bonds in the auto-chaperone domain. Limited enzymatic digestion and mass spectrometry of folding and assembly intermediates indicated that the C-terminal domain was compact in the protrimer species. These results indicate that the C-terminal domain of the P22 tailspike folds itself and associates prior to formation of the protrimer intermediate, and not after, as previously proposed. The C-terminal cysteines and triple β-helix domains apparently provide the staging for the correct auto-chaperone domain formation, needed for alignment of P22 tailspike native trimer.
auto-chaperone; cysteines; folding intermediates; tailspike; transient disulfide bond
Recombineering, a recently developed technique for efficient genetic manipulation of bacteria, is facilitated by phage-derived recombination proteins and has the advantage of using DNA substrates with short regions of homology. This system was first developed in E. coli but has since been adapted for use in other bacteria. It is now widely used in a number of different systems for a variety of purposes, and the construction of chromosomal gene knockouts, deletions, insertions, point mutations, as well as in vivo cloning, mutagenesis of bacterial artificial chromosomes and phasmids, and the construction of genomic libraries has been reported. However, these methods also can be effectively applied to the genetic modification of bacteriophage genomes, in both their prophage and lytically growing states. The ever-growing collection of fully sequenced bacteriophages raises more questions than they answer, including the unknown functions of vast numbers of genes with no known homologs and of unknown function. Recombineering of phage genomes is central to addressing these questions, enabling the simple construction of mutants, determination of gene essentiality, and elucidation of gene function. In turn, advances in our understanding of phage genomics should present similar recombineering tools for dissecting a multitude of other genetically naïve bacterial systems.
bacteriophage; BRED; recombineering
Vertebrate animals possess multiple anti-pathogen defenses. Individual mechanisms usually are differentiated into those that are immunologically adaptive vs. more “primitive” anti-pathogen phenomena described as innate responses. Here I frame defenses used by bacteria against bacteriophages as analogous to these animal immune functions. Included are numerous anti-phage defenses in addition to the adaptive immunity associated with CRISPR/cas systems. As these other anti-pathogen mechanisms are non-adaptive they can be described as making up an innate bacterial immunity. This exercise was undertaken in light of the recent excitement over the discovery that CRISPR/cas systems can serve, as noted, as a form of bacterial adaptive immunity. The broader goal, however, is to gain novel insight into bacterial defenses against phages by fitting these mechanisms into considerations of how multicellular organisms also defend themselves against pathogens. This commentary can be viewed in addition as a bid toward integrating these numerous bacterial anti-phage defenses into a more unified immunology.
abortive infection; adsorption resistance; antigen presentation; CRISPR; innate immunity; restriction-modification
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.
bibliography; cultural theory; ethnic origin; family names; linguistics
Viral codon usage is shaped by the conflicting forces of mutational pressure and selection to match host patterns for optimal expression. We examined whether genomic architecture (single- or double-stranded DNA) influences the degree to which bacteriophage codon usage differ from their primary bacterial hosts and each other. While both correlated equally with their hosts’ genomic nucleotide content, the coat genes of ssDNA phages were less well adapted than those of dsDNA phages to their hosts’ codon usage profiles due to their preference for codons ending in thymine. No specific biases were detected in dsDNA phage genomes. In all nine of ten cases of codon redundancy in which a specific codon was overrepresented, ssDNA phages favored the NNT codon. A cytosine to thymine biased mutational pressure working in conjunction with strong selection against non-synonymous mutations appears be shaping codon usage bias in ssDNA viral genomes.
bacteriophage; codon usage bias; evolution; genome; genomic adaptation; genomic architecture; single-stranded DNA