We recently described a novel, non-host-derived, phage-mediated immunity active at mucosal surfaces, the main site of pathogen entry in metazoans. In that work, we showed that phage T4 adheres to mucus glycoproteins via immunoglobulin-like domains displayed on its capsid. This adherence positions the phage in mucus surfaces where they are more likely to encounter and kill bacteria, thereby benefiting both the phage and its metazoan host. We presented this phage-metazoan symbiosis based on an exclusively lytic model of phage infection. Here we extend our bacteriophage adherence to mucus (BAM) model to consider the undoubtedly more complex dynamics in vivo. We hypothesize how mucus-adherent phages, both lytic and temperate, might impact the commensal microbiota as well as protect the metazoan epithelium from bacterial invasion. We suggest that BAM may provide both an innate and an acquired antimicrobial immunity.
phage; bacteriophage; immune system; mucus; lysogen; lytic
A cocktail of six lytic bacteriophages, SalmoFresh™, significantly (p < 0.05) reduced the number of surface-applied Salmonella Kentucky and Brandenburg from stainless steel and glass surfaces by > 99% (2.1–4.3 log). Both strains were susceptible to SalmoFresh™ in the spot-test assay. Conversely, SalmoFresh™ was unable to reduce surface contamination with a Salmonella Paratyphi B strain that was not susceptible to the phage cocktail in the spot-test assay. However, by replacing two SalmoFresh™ component phages with two new phages capable of lysing the Paratyphi B strain in the spot-test assay, the target range of the cocktail was shifted to include the Salmonella Paratyphi B strain. The modified cocktail, SalmoLyse™, was able to significantly (p < 0.05) reduce surface contamination of the Paratyphi B strain by > 99% (2.1–4.1 log). The data show that both phage cocktails were effective in significantly reducing the levels of Salmonella on hard surfaces, provided the contaminating strains were susceptible in the spot-test (i.e., spot-test susceptibility was indicative of efficacy in subsequent surface decontamination studies). The data also support the concept that phage preparations can be customized to meet the desired antibacterial application.
Salmonella; SalmoFresh™; SalmoLyse™; bacteriophage; food safety; phage; surface decontamination
biophysics; head structure; head-tail joining; self-assembly; symmetry
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
Four novel, closely related podoviruses, which displayed lytic activity against the gamma-proteobacterium Alteromonas macleodii, have been isolated and sequenced. Alterophages AltAD45-P1 to P4 were obtained from water recovered near a fish farm in the Mediterranean Sea. Their morphology indicates that they belong to the Podoviridae. Their linear and dsDNA genomes are 100–104 kb in size, remarkably larger than any other described podovirus. The four AltAD45-phages share 99% nucleotide sequence identity over 97% of their ORFs, although an insertion was found in AltAD45-P1 and P2 and some regions were slightly more divergent. Despite the high overall sequence similarity among these four phages, the group with the insertion and the group without it, have different host ranges against the A. macleodii strains tested. The AltAD45-P1 to P4 phages have genes for DNA replication and transcription as well as structural genes, which are similar to the N4-like Podoviridae genus that is widespread in proteobacteria. However, in terms of their genomic structure, AltAD45-P1 to P4 differ from that of the N4-like phages. Some distinguishing features include the lack of a large virion encapsidated RNA polymerase gene, very well conserved among all the previously described N4-like phages, a single-stranded DNA binding protein and different tail protein genes. We conclude that the AltAD45 phages characterized in this study constitute a new genus within the Podoviridae.
Alteromonas macleodii; Podoviridae; N4-like virus; lytic phage; marine phages
Psu, a 20-kD bacteriophage P4 capsid decorating protein moonlights as a transcription antiterminator of the Rho-dependent termination. Psu forms specific complex with E.coli Rho protein, and affects the latter's ATP-dependent translocase activity along the nascent RNA. It forms a unique knotted dimer to take a V-shaped structure. The C-terminal helix of Psu makes specific contacts with a disordered region of Rho, encompassing the residues 139–153. An energy minimized structural model of the Rho–Psu complex reveals that the V-shaped Psu dimer forms a lid over the central channel of the Rho hexamer. This configuration of Psu causes a mechanical impediment to the translocase activity of Rho. The knowledge of structural and mechanistic basis of inhibition of Rho action by Psu may help to design peptide inhibitors for the conserved Rho-dependent transcription termination process of bacteria.
transcription termination; Psu; Rho; bacteriophage
Foodborne illnesses resulting from the consumption of produce commodities contaminated with enteric pathogens continue to be a significant public health issue. Lytic bacteriophages may provide an effective and natural intervention to reduce bacterial pathogens on fresh and fresh-cut produce commodities. The use of multi-phage cocktails specific for a single pathogen has been most frequently assessed on produce commodities to minimize the development of bacteriophage insensitive mutants (BIM) in target pathogen populations. Regulatory approval for the use of several lytic phage products specific for bacterial pathogens such as Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes in foods and on food processing surfaces has been granted by various agencies in the US and other countries, possibly allowing for the more widespread use of bacteriophages in the decontamination of fresh and minimally processed produce. Research studies have shown lytic bacteriophages specific for E. coli O157:H7, Salmonella spp. and Listeria monocytogenes have been effective in reducing pathogen populations on leafy greens, sprouts and tomatoes.
bacteriophages; lytic; leafy greens; melons; sprouts; produce; vegetables; Escherichia coli O157:H7; Salmonella; Listeria monocytogenes
Bacterial resistance to antibiotics poses a serious health threat. Since research into new antibiotics is not progressing at the same rate as the development of bacterial resistance, widespread calls for alternatives to antibiotics have been made. Phage therapy is an ideal alternative candidate to be investigated. However the success of phage therapy may be hampered by a lack of investment support from large pharmaceutical companies, due to their narrow spectrum of activity in antibiotics, very large costs associated with clinical trials of the variety of phages needed, and regulatory requirements remaining unclear. Intellectual property is difficult to secure for therapeutic phage products for a variety of reasons, and patenting procedures vary widely between the US and the EU. Consequently, companies are more likely to invest in phage products for decontamination or veterinary use, rather than clinical use in humans. Some still raise questions as to the safety of phage therapy overall, suggesting the possibility of cytotoxicity and immunogenicity, depending on the phage preparation and route. On the other hand, with patients dying because of infections untreatable with conventional antibiotics, the question arises as to whether it is ethical not to pursue phage therapy more diligently. A paradigm shift about how phage therapy is perceived is required, as well as more rigorous proof of efficacy in the form of clinical trials of existing medicinal phage products. Phage therapy potential may be fulfilled in the meantime by allowing individual preparations to be used on a named-patient basis, with extensive monitoring and multidisciplinary team input. The National Health Service and academia have a role in carrying out clinical phage research, which would be beneficial to public health, but not necessarily financially rewarding.
phage therapy; limitations; safety; ethics; paradigm changes; clinical use
The bacteriophage EL is a virus that specifically attacks the human pathogen Pseudomonas aeruginosa. This phage carries a large genome that encodes for its own chaperonin which presumably facilitates the proper folding of phage proteins independently of the host chaperonin system. EL also encodes a lysin enzyme, a critical component of the lytic cycle that is responsible for digesting the peptidoglycan layer of the host cell wall. Previously, this lysin was believed to be a substrate of the chaperonin encoded by phage EL. In order to characterize the activity of the EL lysin, and to determine whether lysin activity is contingent on chaperonin-mediated folding, a series of peptidoglycan hydrolysis activity assays were performed. Results indicate that the EL-encoded lysin has similar enzymatic activity to that of the Gallus gallus lysozyme and that the EL lysin folds into a functional enzyme in the absence of phage chaperonin and should not be considered a substrate.
bacteriophage EL; chaperonin; lysozyme; protein folding; lysin
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