Bacteriophages and their application in food safety

In recent years it has become widely recognized that bacteriophages have several potential applications in the food industry. They have been proposed as alternatives to antibiotics in animal health, as biopreservatives in food and as tools for detecting pathogenic bacteria throughout the food chain. Bacteriophages are viruses that only infect and lyse bacterial cells. Consequently, they display two unique features relevant in and suitable for food safety. Namely, their safe use as they are harmless to mammalian cells and their high host specificity that allows proper starter performance in fermented products and keeps the natural microbiota undisturbed. However, the recent approval of bacteriophages as food additives has opened the discussion about ‘edible viruses’. In this review, we examine the promising uses of phages for the control of foodborne pathogens and the drawbacks on which more research is needed to further exploit these biological entities.     

Natural solution to antibiotic resistance: bacteriophages ‘The Living Drugs’

Antibiotics have been a panacea in animal husbandry as well as in human therapy for decades. The huge amount of antibiotics used to induce the growth and protect the health of farm animals has lead to the evolution of bacteria that are resistant to the drug’s effects. Today, many researchers are working with bacteriophages (phages) as an alternative to antibiotics in the control of pathogens for human therapy as well as prevention, biocontrol, and therapy in animal agriculture. Phage therapy and biocontrol have yet to fulfill their promise or potential, largely due to several key obstacles to their performance. Several suggestions are shared in order to point a direction for overcoming common obstacles in applied phage technology. The key to successful use of phages in modern scientific, farm, food processing and clinical applications is to understand the common obstacles as well as best practices and to develop answers that work in harmony with nature.     

Isolation and partial characterization of Salmonella Gallinarum bacteriophage

Infections caused by Salmonella remain a major public health problem worldwide. Animal food products, including poultry meat and eggs, are considered essential components in the individual’s daily nutrition. However, chicken continues to be the main reservoir for Salmonella spp.Poultry farmers use several types of antibiotics to treat pathogens. This can pose a health risk as pathogens can build antibiotic resistance in addition to the possibility of accumulation of these antibiotics in food products. The use of phages in treating poultry pathogens is increasing worldwide due to its potential use as an effective alternative to antibiotics. Phages have several advantages over antibiotics; phages are very specific to target bacteria, less chances of developing secondary infections, and they only replicate at the site of infection.Here we report the isolation of a bacteriophage from chicken feces. The isolated bacteriophage hosts on Salmonella Gallinarum, a common zoonotic infection that causes fowl typhoid, known to cause major losses to poultry sector. The isolated bacteriophage was partially characterized as a DNA virus resistant to RNase digestion with approximately 20 Kb genome. SDS-PAGE analysis of total viral proteins showed at least five major bands (21, 28, 42, 55 and 68 kDa), indicating that this virus is relatively small compared to other known poultry phages. The isolated bacteriophage has the potential to be an alternative to antibiotics and possibly reducing antibiotic resistance in poultry farms.     

Bacteriophage and their lysins for elimination of infectious bacteria

When phages were originally identified, the possibility of using them as antibacterial agents against pathogens was immediately recognized and put into practise based on the knowledge available at the time. However, with the advent of antibiotics a decline in the use of phage as therapeutics followed. Phages did, however, become more useful in the study of fundamental aspects of molecular biology and in the diagnostic laboratory for the identification of pathogenic bacteria. More recently, the original application of phage as therapeutics to treat human and animal infections has been rekindled, particularly in an era where antibiotic resistance has become so problematic/commonplace. Phage lysins have also been studied and utilized in their own right as potential therapeutics for the treatment of bacterial infections. Indeed the past decade has seen a considerable amount of research worldwide focused on the engineering of phages as antibacterial agents in a wide range of applications. Furthermore, the US Food and Drug Administration and/or the US Department of Agriculture have recently approved commercial phage preparations to prevent bacterial contamination of livestock, food crops, meat and other foods. Such developments have prompted this review into the status of phage research as it pertains to the control of infectious bacteria.     

The return of the phage

Phages have been used to treat infectious diseases since their discovery nearly a century ago. Modern sequencing and genetic engineering technologies now enable researchers to vastly expand the use of phages as general drug delivery vehicles....it is only in the past five years that the regulatory guidelines for the approval of phage products—both in therapy and food safety—have been createdOver the past decade, bacteriophages have occasionally stirred public and media interest because of their potential as biological weapons against bacterial infections. Such reports have tended to come from Russian or Georgian laboratories, whereas Western research institutes and companies have usually found that phages do not live up to their promise. More than a decade later, however, the view of bacteriophages is set to change. Spurred on by advances in sequencing and other molecular techniques, research into phages has yielded its first applications. Not only are phages proving effective as therapeutic agents, but they are also playing a role in food safety and as delivery vehicles for drugs against a wide range of diseases.Interest in phages as therapeutic agents emerged almost immediately after their discovery nearly a century ago (Twort, 1915; d''Hérelle, 1917). This interest evaporated quickly in the West after the discovery of penicillin, but phage research was kept alive in the old Soviet Union and continued after its collapse in the 1990s. Ongoing studies there, although not always conforming to the most rigorous standards, provided the only evidence of the therapeutic potential of phages.Eventually, especially in the light of the increasing threat from drug-resistant bacteria, Western researchers turned to exploring phages again. However, it is only in the past five years that the regulatory guidelines for the approval of phage products—in both therapy and food safety—have been created. Previously, the US Food and Drug Administration (FDA) had lacked the appropriate regulatory measures; it took them four years to approve the first phage product for use in food safety in 2006. ListShield^TM is a cocktail of several phages that target Listeria monocytogenes, contaminants in meat and poultry products. Approvals for other food safety products have followed with greater speed (Sulakvelidze, 2011). Moreover, in 2008, the FDA approved the first phase 1 clinical trial of phages. This again involved a cocktail of eight phages to target various bacteria including Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, in venous leg ulcers. This trial eventually established the safety of the phage preparation and cleared the way for more phage therapy trials (www.clinicaltrials.gov).The recent acceptance in the West of phages as anti-pathogenic agents was preceded by their use for diagnostic purposes to identify bacteria...The recent acceptance in the West of phages as anti-pathogenic agents was preceded by their use for diagnostic purposes to identify bacteria, according to Martin Loessner from the Institute of Food, Nutrition and Health in Zürich, Switzerland. “It then became possible to [...] harness the specificity of phage for applications such as recognition of the host cell, and also for reporter phage, which is a genetically modified phage with a gene so [you] can easily see the phage''s impact on the target cell,” he explained. “Later on we figured why not go and revisit the idea of using phages against pathogens.”This approach turned out to be highly successful against key food pathogens, Loessner said, because of the way phages work: “[T]he phage has been very finely tuned through zillions of generations in the evolutionary arms race, and is highly specific.” This specificity is important for targeting the few bacteria that cause food poisoning while sparing the bacteria in fermented food—such as soft cheeses—that are harmless and contribute flavour. “The phage is also immune to development of resistance by the host bacteria, because if not it would have become extinct a long time ago,” Loessner said.It is bacterial toxins that cause food poisoning rather than bacteria themselves, so phages are used as a preventive measure to stop the growth of bacteria such as Listeria in the first place. As such, it is important to bombard food products with a large number of phages to ensure that virtually all target bacteria are eradicated. “I always have this magic number of 10⁸, or 100 million per gram of food,” Loessner said. “In 1 g of food there are often only 500 target bacteria, so there is not enough to amplify the phage and you need really high numbers to kill the bacteria in one round of infection.” He added that, in his view, phages would soon become the main treatment for preventing bacterial contamination. “Phage in the near future will be the number one [treatment against] Listeria and Salmonella. It''s becoming number one already, especially in the US.”In Europe, the use of phages in food safety therapy is being held back by the requirement that foods treated with them are labelled as containing viruses, which means they are likely to meet consumer resistance, as happened with foods containing or made from genetically modified organisms. Loessner commented that education is required to raise awareness that the properly controlled use of phages involves minimal risk and could greatly enhance food safety. However, he also emphasized that the use of phages should represent an extra level of protection, not replace existing quality control measures....because phage lysins are often specific to a single bacterial genus, they would allow the specific targeting of pathogenic bacteriaThe ability of phages to target specific bacteria while leaving others alone also has great potential for treating bacterial infections, particularly in the light of increasing antibiotic resistance. Such treatments would not necessarily involve the phage themselves, but rather the use of their lysins—the enzymes that weaken the bacterial cell wall to allow newly formed viruses to exit the host cell. Lysins can be administered as antibiotics, at least for gram-positive bacteria that lack a separate outer membrane around the cell wall. Moreover, because phage lysins are often specific to a single bacterial genus, they would allow the specific targeting of pathogenic bacteria. “The fact that phage lysins leave the commensal microflora undisturbed is particularly significant,” commented Olivia McAuliffe, Senior Research Officer at the Teagasc Food Research Centre in Cork, Ireland. “Most of the antibiotics used clinically have broad-activity spectra and treatment with these antibiotics can have devastating effects on the normal flora, in particular for those taking long-term antibiotic courses.”Phages also have another great advantage over most conventional antibiotics in being potent against both dividing and non-dividing cells. “Because most antibiotics target pathways such as protein synthesis, DNA replication, and cell wall biosynthesis, they can only act when the cells are actively growing,” McAuliffe added. “Because lysins are enzymes, they will chew away the peptidoglycan in both viable and non-viable cells, dividing and non-dividing cells. This would be particularly important in the case of slow-growing organisms that cause infection, an example being Mycobacterium species.”This specificity of phages and their lysins is particularly important for treating chronic conditions resulting from persistent bacterial infection, particularly in the respiratory system or digestive tract. Broad-spectrum antibiotics also attack harmless and beneficial commensal bacteria, and can even worsen the condition by encouraging the growth of resistant bacteria. This is the case with Clostridium difficile, a cause of secondary infections and a major nosocomial (hospital-acquired) antibiotic-resistant pathogen, according to McAuliffe. It is a Gram-positive, rod-shaped, spore-forming bacterium that is the most serious and common cause of diarrhoea and other intestinal disease when competing bacteria in the gut flora have been wiped out by antibiotics. The bacterium and its spores, which form in aerobic conditions outside the body, are widespread in the environment and are present in the guts of 3% of healthy individuals and 66% of infants, according to the UK''s Health Protection Agency. Clostridium spreads readily on the hands of healthcare staff and visitors in hospitals. The ability of the bacteria to form spores resistant to heat, drying and disinfectants, which then adhere to surfaces, enables them to persist in the hospital environment.Because Clostridium is resistant to most conventional antibiotics, it has for some years usually been treated with metronidazole, which exploits the fact that Clostridium is anaerobic during infection. Metronidazole has proven particularly appealing as it has relatively little impact on human cells or commensal aerobic bacteria in the gut as it does not work in the presence of oxygen. But metronidazole does not always work, and physicians have therefore been using vancomycin, a stronger but more toxic antibiotic, as a last resort. Moreover, even in cases where antibiotics seem to eliminate Clostridium and cure the associated diarrhoea, infection recurs in as many as 20% of hospital patients (Kelly & LaMont, 2008). About one-fifth of these 20%, or 4% of the total number of patients succumbing to Clostridium, end up with a long-term infection that at present is difficult to eradicate.This is where phages step in, because they are well tolerated by patients and their specificity means that they will not target other gut bacteria. Clostridium phages have already been demonstrated to work selectively and there is the possibility of extracting lysins against Clostridium from the phage itself; an avenue being pursued by Aidan Coffey''s group at the Department of Biological Sciences at the Cork Institute of Technology in Bishoptown, Ireland.There is also growing interest in using phages to tackle various other infections that are resistant to existing drugs—for example, in wounds that fail to heal, which are a major risk for diabetics. The application of phages in such cases is not new—before penicillin it was often the only option—but the difference now is that modern molecular techniques for isolating bacterial strains from biopsies and matching them to phages greatly increases efficiency. One clinical trial, organized by the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, is currently recruiting patients to evaluate the use of phage preparations against a range of drug-resistant bacteria, including MRSA (methicillin-resistant Staphylococcus aureus), Enterococcus, Escherichia, Citrobacter, Enterobacter, Klebsiella, Shigella and Salmonella. The intention is to isolate bacterial strains from each patient and to identify matching phages from the Institute''s bacteriophage collection in Wrocław.Although the potential of phages or their lysins to combat bacterial pathogens, whether in food or those causing infectious diseases, has long been recognized, more recent work has identified new applications as delivery vehicles for vaccines or cytotoxic drugs to treat cancer. These applications do not exploit the phage''s natural targeting of bacteria, but make use of their ability to carry surface ligands that attract them to specific host cells.Even though phages do not attack human cells, they elicit an immune response and can be used as vectors to carry an engineered antigen on their surface to vaccinate against viral or bacterial disease. This approach has been tested in rabbits with a DNA vaccine against hepatitis B (Clark et al, 2011). The study compared the phage DNA vaccine with Engerix B—a commercially available vaccine based on a homologous recombinant protein—and found that the phage vaccine produced a significantly higher antibody response more quickly, as well as being potentially cheaper to produce and stable at a wider range of temperatures. This hepatitis B vaccine is now being developed by the UK biotech firm BigDNA in Edinburgh, Scotland, which has been granted a European patent, pending future clinical trials in humans.Modified phages could also serve as nanoparticles to deliver cytotoxic drugs straight to tumour cells, bypassing healthy cellsModified phages could also serve as nanoparticles to deliver cytotoxic drugs straight to tumour cells, bypassing healthy cells. Phages are a promising candidate vehicle because they can be readily engineered both to display appropriate ligands for targeting tumour cells specifically, and to carry a cytotoxic payload that is only released inside the target. One Israeli group has developed a technology for manufacturing phage nanoparticles that in principle can be used to target drugs to either tumour cells or pathogens (Bar et al, 2008). The group chose one particular phage family, known as filamentous phages, because of their small size and the relative ease of engineering them. Filamentous phages comprise just 10 genes with a sheath of several thousand identical α-helical coat proteins in a helical array assembled around a single-stranded circular DNA molecule. The Israeli scientists combine genetic modification and chemical engineering to create a phage that is able to attach to its target cell and release cytotoxic molecules. “Genetic engineering makes it possible to convert the phage to a targeted particle by displaying a target-specifying molecule on the phage coat,” explained Itai Benhar from Tel-Aviv University, the lead author of the paper. “Genetic engineering also makes it possible to design a drug-release mechanism. Finally chemical engineering makes it possible to load the particle with a large payload of cargo.”The group has used the same approach to target two bacteria species, Staphylococcus aureus and Escherichia coli, with the antibiotic chloramphenicol, which was first developed in 1949 but has raised concerns over its toxicity. According to the Israeli group, the phage nanoparticle loaded with the drug was 20,000 times more potent against both bacteria than the drug administered on its own. Just as importantly, the phage particles do not affect other cells. The overall advantage of the phage-based delivery approach is that it can deliver highly effective and toxic drugs in a safe way. The other point is that this and other methods in which phages are engineered to reach specific targets have nothing directly to do with the natural ability of phage viruses to attack bacteria. “The phage''s natural ability to infect bacteria is totally irrelevant to their application for targeting non-bacterial cells,” said Benhar. “In fact, they are not relevant for targeting bacteria either in this case, since the chemical modification we subject the phages to renders them non-infective.”However, the phage nanoparticles retain their immunogenic effect, which is a problem if the objective is merely to deliver a drug to the target while minimizing all other impacts. “Phages are immunogenic, and although we found a way to reduce their immunogenicity we did not totally eliminate it,” Benhar said. The other challenge is that, as the particles carry the payload drug on their surface, the physical and chemical properties change every time a new drug is loaded. Although the payload itself is inert until it reaches the target, the varying characteristics could alter the host response and therefore affect regulatory approval for each new phage construct, as safety would have to be demonstarted in each case.The use of phages is no longer confined to directly attacking infectious bacteria, but has vastly expanded in terms of methods, applications and the diseases that can be tackledNevertheless, this approach holds great promise as a novel way of delivering not just new drugs but also existing ones that are effective but too toxic for healthy cells. This is exactly the most exciting aspect of recent therapeutic phage research. The use of phages is no longer confined to directly attacking infectious bacteria, but has vastly expanded in terms of methods, applications and the diseases that can be tackled.     

噬菌体在食品工业中的应用与危害防控

近年来,噬菌体由于其特异性侵染细菌的特性,在食品加工及保藏过程中有害微生物的控制和检测方面展现出良好的应用前景。例如在食品表面喷洒噬菌体或将噬菌体与食品包装材料结合,对食源性致病菌及腐败菌加以控制,以及利用基因工程手段构建报告噬菌体对食源性致病菌进行快速检测等。然而,噬菌体也是危害食品发酵的重要因素之一,轻则减产,重则引起整个发酵过程失败,造成巨大的经济损失。目前主要通过噬菌体消毒及灭活、发酵菌种变换等方式防止噬菌体污染。本文综述了食品工业中噬菌体应用及危害的研究现状,以期为拓宽噬菌体在食品工业中的应用途径及开发噬菌体污染防治的新技术提供理论依据。     

Bacteriophages and biotechnology: vaccines, gene therapy and antibacterials

In recent years it has been recognized that bacteriophages have several potential applications in the modern biotechnology industry: they have been proposed as delivery vehicles for protein and DNA vaccines; as gene therapy delivery vehicles; as alternatives to antibiotics; for the detection of pathogenic bacteria; and as tools for screening libraries of proteins, peptides or antibodies. This diversity, and the ease of their manipulation and production, means that they have potential uses in research, therapeutics and manufacturing in both the biotechnology and medical fields. It is hoped that the wide range of scientists, clinicians and biotechnologists currently researching or putting phages to practical use are able to pool their knowledge and expertise and thereby accelerate progress towards further development in this exciting field of biotechnology.     

The future of bacteriophage biology

After an illustrious history as one of the primary tools that established the foundations of molecular biology, bacteriophage research is now undergoing a renaissance in which the primary focus is on the phages themselves rather than the molecular mechanisms that they explain. Studies of the evolution of phages and their role in natural ecosystems are flourishing. Practical questions, such as how to use phages to combat human diseases that are caused by bacteria, how to eradicate phage pests in the food industry and what role they have in the causation of human diseases, are receiving increased attention. Phages are also useful in the deeper exploration of basic molecular and biophysical questions.     

Bacteriophage biocontrol in animals and meat products

Since their discovery almost a century ago, bacterial viruses (bacteriophages or ‘phages’) have been used to prevent and treat a multitude of bacterial infections (phage therapy: PT). In addition, they have been the basis for many advances in genetics and biochemistry. Phage therapy was performed on human subjects in the United States, Europe and Asia in the few decades following their discovery. However, Western countries largely abandoned PT in favour of antibiotics in the 1940s. The relatively recent renaissance of PT in the West can be attributed partly to the increasing prevalence of antibiotic resistance in human and animal pathogens. However, the stringent controls on human trials now required in the United States and Europe have led to a greater number of domestic animal and agricultural applications as an alternative to PT in man. This trend is set to continue, at least in the short term, with recent approval from the Food and Drug Administration allowing commercial phage treatments to be used in human food in the USA. Nevertheless, despite these significant milestones and the growing number of successful PT trials, significant obstacles remain to their widespread use in animals, food and ultimately medicine in many parts of the world. This review will provide a brief overview of the history of PT in the West and will summarize some of the key findings of phage biocontrol studies in animals and meat products.     

Bacteriocins from lactic acid bacteria and their potential in the preservation of fruit products

Bacteriocins produced by lactic acid bacteria (LAB) are well-recognized for their potential as natural food preservatives. These antimicrobial peptides usually do not change the sensorial properties of food products and can be used in combination with traditional preservation methods to ensure microbial stability. In recent years, fruit products are increasingly being associated with food-borne pathogens and spoilage microorganisms, and bacteriocins are important candidates to preserve these products. Bacteriocins have been extensively studied to preserve foods of animal origin. However, little information is available for their use in vegetable products, especially in minimally processed ready-to-eat fruits. Although, many bacteriocins possess useful characteristics that can be used to preserve fruit products, to date, only nisin, enterocin AS-48, bovicin HC5, enterocin 416K1, pediocin and bificin C6165 have been tested for their activity against spoilage and pathogenic microorganisms in these products. Among these, only nisin and pediocin are approved to be commercially used as food additives, and their use in fruit products is still limited to certain countries. Considering the increasing demand for fresh-tasting fruit products and concern for public safety, the study of other bacteriocins with biochemical characteristics that make them candidates for the preservation of these products are of great interest. Efforts for their approval as food additives are also important. In this review, we discuss why the study of bacteriocins as an alternative method to preserve fruit products is important; we detail the biotechnological approaches for the use of bacteriocins in fruit products; and describe some bacteriocins that have been tested and have potential to be tested for the preservation of fruit products.     

Application of bacteriophages for detection and control of foodborne pathogens

The incidence of foodborne infectious diseases is stable or has even increased in many countries. Consequently, our awareness regarding hygiene measures in food production has also increased dramatically over the last decades. However, even today’s modern production techniques and intensive food-monitoring programs have not been able to effectively control the problem. At the same time, increased production volumes are distributed to more consumers, and if contaminated, potentially cause mass epidemics. Accordingly, research directed to improve food safety has also been taken forward, also exploring novel methods and technologies. Such an approach is represented by the use of bacteriophage for specific killing of unwanted bacteria. The extreme specificity of phages renders them ideal candidates for applications designed to increase food safety during the production process. Phages are the natural enemies of bacteria, and can be used for biocontrol of bacteria without interfering with the natural microflora or the cultures in fermented products. Moreover, phages or phage-derived proteins can also be used to detect the presence of unwanted pathogens in food or the production environments, which allows quick and specific identification of viable cells. This review intends to briefly summarize and explain the principles and current standing of these approaches.     

Phagotherapy in terms of bacteriophage genetics: hopes, perspectives, safety, limitations

The appearance and spreading of multidrug-resistant bacterial pathogens is a consequence of the large-scale use of antibiotics in medicine. In view of this, claims for the phage therapy were renewed: in recent studies, the natural phages and their products neutralizing various proteins, as well as the bacterial products often controlled by defective prophages (bacteriocins) were applied for treatment of bacterial infections. Constructs obtained by gene engineering are increasingly used to change some bacteriophage: properties to expand the spectrum of their lytic activity and to eliminate therapeutic drawbacks of some natural phages. In this review, the problem of phage therapy is discussed in general with respect to bacteriophage properties, their genetics, structure, evolution, taking into account long-term experience of the author in the field of bacteriophage genetics. Note that the general concept of phage therapy should be developed to ensure long-term, efficient and harmless phage therapy.     

Phage Therapy in Terms of Bacteriophage Genetics: Hopes, Prospects, Safety, Limitations

The appearance and spreading of multidrug-resistant bacterial pathogens is a consequence of the large-scale use of antibiotics in medicine. In view of this, claims for the phage therapy were renewed: in recent studies, the natural phages and their products neutralizing various proteins, as well as the bacterial products often controlled by defective prophages (bacteriocins) were applied for treatment of bacterial infections. Constructs obtained by gene engineering are increasingly used to change bacteriophage properties to expand the spectrum of their lytic activity and to eliminate therapeutic drawbacks of some natural phages. In this review, the problem of phage therapy is discussed in general with respect to bacteriophage properties, their genetics, structure, evolution, taking into account long-term experience of the author in the field of bacteriophage genetics. Note that the general concept of phage therapy should be developed to ensure long-term, efficient and harmless phage therapy.     

Novel broad-spectrum bacteriophages against Xanthomonas oryzae and their biocontrol potential in rice bacterial diseases

Bacterial leaf blight (BLB) and bacterial leaf streak (BLS)—caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively—are two major bacterial diseases that threaten the safe production of rice, one of the most important food crops. Bacteriophages are considered potential biocontrol agents against rice bacterial pathogens, due to their host specificity and environmental safety. It is common for BLB and BLS to occur together in fields, which highlights the need for broad-spectrum phages capable of infecting both Xoo and Xoc. In this study, two lytic broad-spectrum phages (pXoo2106 and pXoo2107) that can infect various strains of Xoo and Xoc were assessed. Both phages belong to the class Caudoviricetes and one of them to the family Autographiviridae, while the other belongs to an unclassified family. Two phages alone or combined in a phage cocktail could effectively inhibit Xoo and Xoc growth in vitro. In an in vivo biocontrol experiment, the phage cocktail reduced the total CFU and significantly eased the symptoms caused by Xoo or Xoc. Our results suggest that pXoo2106 and pXoo2107 have a broad-spectrum host range targeting different X. oryzae strains, and have strong biocontrol potential in field applications against both BLB and BLS.     

Bacteria between protists and phages: from antipredation strategies to the evolution of pathogenicity

Bacteriophages and protists are major causes of bacterial mortality. Genomics suggests that phages evolved well before eukaryotic protists. Bacteria were thus initially only confronted with phage predators. When protists evolved, bacteria were caught between two types of predators. One successful antigrazing strategy of bacteria was the elaboration of toxins that would kill the grazer. The released cell content would feed bystander bacteria. I suggest here that, to fight grazing protists, bacteria teamed up with those phage predators that concluded at least a temporary truce with them in the form of lysogeny. Lysogeny was perhaps initially a resource management strategy of phages that could not maintain infection chains. Subsequently, lysogeny might have evolved into a bacterium-prophage coalition attacking protists, which became a food source for them. When protists evolved into multicellular animals, the lysogenic bacteria tracked their evolving food source. This hypothesis could explain why a frequent scheme of bacterial pathogenicity is the survival in phagocytes, why a significant fraction of bacterial pathogens have prophage-encoded virulence genes, and why some virulence factors of animal pathogens are active against unicellular eukaryotes. Bacterial pathogenicity might thus be one playing option of the stone-scissor-paper game played between phages-bacteria-protists, with humans getting into the crossfire.     

Biofilm control with natural and genetically-modified phages

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

Phages in the global fruit and vegetable industry

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

Application of filamentous phages in environment: A tectonic shift in the science and practice of ecorestoration

Theories in soil biology, such as plant–microbe interactions and microbial cooperation and antagonism, have guided the practice of ecological restoration (ecorestoration). Below‐ground biodiversity (bacteria, fungi, invertebrates, etc.) influences the development of above‐ground biodiversity (vegetation structure). The role of rhizosphere bacteria in plant growth has been largely investigated but the role of phages (bacterial viruses) has received a little attention. Below the ground, phages govern the ecology and evolution of microbial communities by affecting genetic diversity, host fitness, population dynamics, community composition, and nutrient cycling. However, few restoration efforts take into account the interactions between bacteria and phages. Unlike other phages, filamentous phages are highly specific, nonlethal, and influence host fitness in several ways, which make them useful as target bacterial inocula. Also, the ease with which filamentous phages can be genetically manipulated to express a desired peptide to track and control pathogens and contaminants makes them useful in biosensing. Based on ecology and biology of filamentous phages, we developed a hypothesis on the application of phages in environment to derive benefits at different levels of biological organization ranging from individual bacteria to ecosystem for ecorestoration. We examined the potential applications of filamentous phages in improving bacterial inocula to restore vegetation and to monitor changes in habitat during ecorestoration and, based on our results, recommend a reorientation of the existing framework of using microbial inocula for such restoration and monitoring. Because bacterial inocula and biomonitoring tools based on filamentous phages are likely to prove useful in developing cost‐effective methods of restoring vegetation, we propose that filamentous phages be incorporated into nature‐based restoration efforts and that the tripartite relationship between phages, bacteria, and plants be explored further. Possible impacts of filamentous phages on native microflora are discussed and future areas of research are suggested to preclude any potential risks associated with such an approach.     

Marine phage genomics

Marine phages are the most abundant biological entities in the oceans. They play important roles in carbon cycling through marine food webs, gene transfer by transduction and conversion of hosts by lysogeny. The handful of marine phage genomes that have been sequenced to date, along with prophages in marine bacterial genomes, and partial sequencing of uncultivated phages are yielding glimpses of the tremendous diversity and physiological potential of the marine phage community. Common gene modules in diverse phages are providing the information necessary to make evolutionary comparisons. Finally, deciphering phage genomes is providing clues about the adaptive response of phages and their hosts to environmental cues.     

Illuminating the host – How RNAi screens shed light on host-pathogen interactions

Over millions of years pathogens have coevolved with their respective hosts utilizing host cell functions for survival and replication. Despite remarkable progress in developing antibiotics and vaccination strategies in the last century, infectious diseases still remain a severe threat to human health. Meanwhile, genomic research offers a new era of data-generating platforms that will dramatically enhance our knowledge of pathogens and the diseases they cause. Improvements in gene knockdown studies by RNA interference (RNAi) combined with recent developments in instrumentation and image analysis enable the use of high-throughput screening approaches to elucidate host gene functions exploited by pathogens. Although only a few RNAi-based screens focusing on host genes have been reported so far, these studies have already uncovered hundreds of genes not previously known to be involved in pathogen infection. This review describes recent progress in RNAi screening approaches, highlighting both the limitations and the tremendous potential of RNAi-based screens for the identification of essential host cell factors during infection.