Although the study of phage infection has a long history and catalyzed much of our current understanding in bacterial genetics, molecular biology, evolution and ecology, it seems that microbiologists have only just begun to explore the intricacy of phage–host interactions. In a recent manuscript by Cenens et al. we found molecular and genetic support for pseudolysogenic development in the Salmonella Typhimurium–phage P22 model system. More specifically, we observed the existence of phage carrier cells harboring an episomal P22 element that segregated asymmetrically upon subsequent divisions. Moreover, a newly discovered P22 ORFan protein (Pid) able to derepress a metabolic operon of the host (dgo) proved to be specifically expressed in these phage carrier cells. In this addendum we expand on our view regarding pseudolysogeny and its effects on bacterial and phage biology.
Salmonella Typhimurium; phage P22; phage carrier state; phage–host interactions; pseudolysogeny
The control of shigellosis in humans enjoys a prominent position in the history of bacteriophage therapy. d’Herelle first demonstrated the efficacy of phage therapy by curing 4 patients of shigellosis, and several subsequent studies confirmed the ability of phages to reduce Shigella based infection. Shigella spp continue to cause millions of illnesses and deaths each year and the use of phages to control the disease in humans and the spread of the bacteria within food and water could point the way forward to the effective management of an infectious disease with global influence.
shigellosis; control; bacteriophage therapy; food safety; shiga toxins
Anna Sergeyevna Tikhonenko (1925–2010) is to be remembered for the excellency of her electron microscopical work, particularly with bacteriophages. She published 113 articles and one book, Ultrastructure of Bacterial Viruses (Izdadelstvo Nauka, Moscow 1968; Plenum Press, New York, 1972). It included 134 micrographs and a complete overview of the 316 phages then examined by electron microscopy. Most micrographs were of exceptional quality. This book, a rarity in those days of strict separation of Soviet and Western research, was the first bacteriophage atlas in the literature and presented a morphological classification of phages into five categories of family level, similar to a scheme presented in 1965 by D.E. Bradley (J Royal Microsc Soc 84:257–316). Her book remains one of the fundamentals of phage research.
TEM; biography; classification; history; immuno-EM
The circuitry of the phage λ genetic switch determining the outcome of lytic or lysogenic growth is well-integrated and complex, raising the question as to how it evolved. It is plausible that it arose from a simpler ancestral switch with fewer components that underwent various additions and refinements, as it adapted to vast numbers of different hosts and conditions. We have recently identified a new class of genetic switches found in mycobacteriophages and other prophages, in which immunity is dependent on integration. These switches contain only three genes (integrase, repressor and cro) and represent a major departure from the λ-like circuitry, lacking many features such as xis, cII and cIII. These small self-contained switches represent an unrealized, elegant circuitry for controlling infection outcome. In this addendum, we propose a model of possible events in the evolution of a complex λ-like switch from a simpler integration-dependent switch.
genetic switch; genetic circuits; bistable; integration-dependent immunity; lytic and lysogenic growth
Bacteriophage therapy, the use of viruses that infect bacteria as antimicrobials, has been championed as a promising alternative to conventional antibiotics. Although in the laboratory bacterial resistance against phages arises rapidly, resistance so far has been an only minor problem for the effectiveness of phage therapy. Resistance to antibiotics, however, has become a major issue after decades of extensive use. Should we expect similar problems after long-term use of phages as antimicrobials? Like antibiotics, phages are often noted to be drivers of bacterial evolution. Should we expect phage-treated pathogens to develop a general resistance to phages over time, a resistance against which only, for example, hypothetically co-evolved phages might be infective? Here we argue that the global infection patterns of phages suggest that this is not necessarily a concern as environmental phages often can infect bacteria with which those phages lack any recent co-evolutionary history.
antibiotic resistance; ecology; evolution; phage resistance; phage-therapy
The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield™) or a control (phosphate buffered saline, PBS) was applied to lettuce by either; (1) immersion of lettuce in 500 ml of EcoShield™ 8.3 log PFU/ml or 9.8 log PFU/ml for up to 2 min before inoculation with E. coli O157:H7; (2) spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculation with E. coli O157:H7 (4.10 CFU/cm2) following exposure to 50 μg/ml chlorine for 30 sec. After immersion studies, lettuce was spot-inoculated with E. coli O157:H7 (2.38 CFU/cm2). Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzed for E. coli O157:H7 populations for up to 7 d. Immersion of lettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p < 0.05) reduced E. coli O157:H7 populations after 24 h when stored at 4°C compared with controls. Immersion of lettuce in suspensions containing high concentrations of EcoShield™ (9.8 log PFU/ml) resulted in the deposition of high concentrations (7.8 log log PFU/cm2) of bacteriophages on the surface of fresh cut lettuce, potentially contributing to the efficacy of the lytic phages on lettuce. Spraying phages on to inoculated fresh cut lettuce after being washed in hypochlorite solution was significantly more effective in reducing E. coli O157:H7 populations (2.22 log CFU/cm2) on day 0 compared with control treatments (4.10 log CFU/cm2). Both immersion and spray treatments provided protection from E. coli O157:H7 contamination on lettuce, but spray application of lytic bacteriophages to lettuce was more effective in immediately reducing E. coli O157:H7 populations fresh cut lettuce.
bacteriophages; E. coli O157:H7; lettuce; processing
The effect of a bacteriophage cocktail (EcoShield™) that is specific against Escherichia coli O157:H7 was evaluated against a nalidixic acid-resistant enterohemorrhagic E. coli O157:H7 RM4407 (EHEC) strain on leafy greens stored under either (1) ambient air or (2) modified atmosphere (MA; 5% O2/35% CO2/60% N2). Pieces (~2 × 2 cm2) of leafy greens (lettuce and spinach) inoculated with 4.5 log CFU/cm2 EHEC were sprayed with EcoShield™ (6.5 log PFU/cm2). Samples were stored at 4 or 10°C for up to 15 d. On spinach, the level of EHEC declined by 2.38 and 2.49 log CFU/cm2 at 4 and 10°C, respectively, 30 min after phage application (p ≤ 0.05). EcoShield™ was also effective in reducing EHEC on the surface of green leaf lettuce stored at 4°C by 2.49 and 3.28 log units in 30 min and 2 h, respectively (p ≤ 0.05).
At 4°C under atmospheric air, the phage cocktail significantly (p ≤ 0.05) lowered the EHEC counts in one day by 1.19, 3.21 and 3.25 log CFU/cm2 on spinach, green leaf and romaine lettuce, respectively compared with control (no bacteriophage) treatments. When stored under MA at 4°C, phages reduced (p ≤ 0.05) EHEC populations by 2.18, 3.50 and 3.13 log CFU/cm2, on spinach, green leaf and romaine lettuce. At 10°C, EHEC reductions under atmospheric air storage were 1.99, 3.90 and 3.99 log CFU/cm2 (p ≤ 0.05), while population reductions under MA were 3.08, 3.89 and 4.34 logs on spinach, green leaf and romaine lettuce, respectively, compared with controls (p ≤ 0.05). The results of this study showed that bacteriophages were effective in reducing the levels of E. coli O157:H7 on fresh leafy produce, and that the reduction was further improved when produce was stored under the MA conditions.
E. coli O157:H7; bacteriophage; modified atmosphere packaging; MAP; leafy green
Bacteriophage (phage) are among the most diverse and abundant life forms on Earth. Studies have recently used phage diversity to identify novel antimicrobial peptides and proteins. We showed that one such phage protein, Staphylococcus aureus (Sau) phage G1 gp67, inhibits cell growth in Sau by an unusual mechanism. Gp67 binds to the host RNA polymerase (RNAP) through an interaction with the promoter specificity σ subunit, but unlike many other σ-binding phage proteins, gp67 does not disrupt transcription at most promoters. Rather, gp67 prevents binding of another RNAP domain, the α-C-terminal domain, to upstream A/T-rich elements required for robust transcription at rRNA promoters. Here, we discuss additional biochemical insights on gp67, how phage promoters escape the inhibitory function of gp67, and methodological advancements that were foundational to our work.
Staphylococcus aureus; RNA polymerase; bacteriophage; gp67; transcription
The use of bacteriophages as an effective phage therapy strategy faces significant challenges for controlling plant diseases in the phyllosphere. A number of factors must be taken into account when considering phage therapy for bacterial plant pathogens. Given that effective mitigation requires high populations of phage be present in close proximity to the pathogen at critical times in the disease cycle, the single biggest impediment that affects the efficacy of bacteriophages is their inability to persist on plant surfaces over time due to environmental factors. Inactivation by UV light is the biggest factor reducing bacteriophage persistence on plant surfaces. Therefore, designing strategies that minimize this effect are critical. For instance, application timing can be altered: instead of morning or afternoon application, phages can be applied late in the day to minimize the adverse effects of UV and extend the time high populations of phage persist on leaf surfaces. Protective formulations have been identified which prolong phage viability on the leaf surface; however, UV inactivation continues to be the major limiting factor in developing more effective bacteriophage treatments for bacterial plant pathogens. Other strategies, which have been developed to potentially increase persistence of phages on leaf surfaces, rely on establishing non-pathogenic or attenuated bacterial strains in the phyllosphere that are sensitive to the phage(s) specific to the target bacterium. We have also learned that selecting the correct phages for disease control is critical. This requires careful monitoring of bacterial strains in the field to minimize development of bacterial strains with resistance to the deployed bacteriophages. We also have data that indicate that selecting the phages based on in vivo assays may also be important when developing use for field application. Although bacteriophages have potential in biological control for plant disease control, there are major obstacles, which must be considered.
bacteriophage; biocontrol; phage; tomato
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.
bacteriophage; biocontrol; phage; tomato
Prophage-encoded genes can provide a variety of benefits for their bacterial hosts. These beneficial genes are often contained within “moron” elements. Morons, thus termed as the insertion of the DNA encoding them adds “more on” the genome in which they are found, are independent transcriptional units disseminated among phage genomes through horizontal gene transfer. Morons have been identified in the majority of phage genomes and they have been found to play diverse roles in bacterial physiology. At present, we are only beginning to ascribe functions to the many proteins encoded within these ubiquitous genetic elements. Recently, we discovered that the first described moron-encoded protein, gp15 of phage HK97, is expressed from the HK97 prophage and functions as a superinfection exclusion protein, protecting its host from genome injection by other phages. This work and the growing body of data pertaining to other morons challenges the traditional view of phages as purely parasites of bacteria and emphasizes the symbiotic relationship between bacteria and prophages.
lysogen; lysogenic conversion; microbiome; phage HK97; phage moron; prophage; superinfection exclusion
We recently described the targeting of O104:H4 Escherichia coli in mouse gut by several virulent bacteriophages, highlighting several issues relating to virus-host interactions, which we discuss further in this addendum to the original publication.
bacteriophage infection; gut; permissivity; pseudolysogeny; phage therapy
The global interplay between bacteria and bacteriophages has generated many macromolecules useful in biotechnology, through the co-evolutionary see-saw of bacterial defense and viral counter-attack measures. Bacteria can protect themselves using abortive infection systems, which induce altruistic suicide in an infected cell and therefore protect the clonal population at the expense of the infected individual. Our recent paper describes how bacteriophage ΦTE successfully subverted the activity of a plasmid-borne abortive infection system. ΦTE evolved mimics of the small RNA antitoxin that naturally inhibits the active toxin component of this anti-viral mechanism. These mutant phages further manipulated the behavior of the host population, through transduction of the plasmid encoding the abortive infection system. Transductants thereby became enslaved by the abortive infection system, committing suicide in response to infection by the original phage population. In effect, the new host was infected by an “addictive altruism,” to the advantage of the resistant bacteriophage.
bacteriophage; toxin-antitoxin; abortive infection; mimicry; transduction; Pectobacterium atrosepticum; RNA pseudoknot; phage resistance
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.
ATPase; DNA packaging; bacteriophage genetics; bacteriophage structure; biological motor; cryo-electron microscopy; single-molecule analysis
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