Meeting global food demands for a growing human population with finite natural resources is a major challenge. Aquaculture and agriculture are critical to satisfy food requirements, yet suffer significant losses from bacterial diseases. Therefore, there is an urgent need to develop novel antimicrobial strategies, which is heightened by increasing antibiotic resistance. Bacteriophages (phages) are viruses that specifically infect bacteria, and phage-derived therapies are promising treatments in the fight against bacterial diseases. Here, we describe multiple ways that phages and phage-based technologies can be used as antimicrobials. Antimicrobial activity can be achieved through lysis of targeted bacteria by virulent phages or lytic enzymes. Alternatively, phages can be engineered for the delivery of lethal genes and other cargoes to kill bacteria and to manipulate the bacterial response to conventional antibiotics. We also briefly highlight research exploring phages as potential biocontrol agents with examples from agriculture and aquaculture.
The growing population poses critical challenges to global food demands. For example, a 70% increase in food supply is estimated to be required for a population of 9.6 billion people by 2050 [1,2]. The combination of changing demographics and consumer trends for healthier alternatives are likely to result in intensified supplies from fisheries, aquaculture, and agriculture in the coming years. Indeed, agriculture and aquaculture are two of the most rapidly expanding sectors in food production, yet the most vulnerable to losses caused by disease [3,4]. In some aquaculture sectors, infectious diseases can result in losses exceeding 40% of global supply . Similarly, ∼10% of the global agricultural yield is lost annually due to pathogenic organisms (i.e. parasites, viruses, and bacteria), with potential further losses occurring post-harvest . Current measures to control pathogens include the implementation of appropriate management practices and the use of antibiotics and chemicals. However, the increased prevalence of antibiotic and chemical resistance, coupled with concerns about the accumulation of these compounds, has led to efforts to develop environment-friendly and sustainable approaches to control diseases in both agriculture and aquaculture.
Bacteriophages (phages) are viruses that specifically infect bacteria and are the most diverse and abundant biological entities on earth, playing pivotal roles in bacterial community dynamics [7,8], nutrient cycling [9,10], and bacterial genome evolution [11,12]. Phages were discovered independently by Fedrick Twort (1915) and Felix d'Herelle (1917) and were immediately recognized as potential antimicrobial agents . The application of phages as antimicrobials is known as phage therapy and was extensively practiced in the 1920s. While these early studies were promising, phage therapy was abandoned in the West due to the discovery and successful application of antibiotics; although, phage therapy continued in Eastern Europe and the former Soviet Union . Despite the wide success of antibiotics, there are concerns about the paucity of new antibiotics in the drug development pipelines. This issue, coupled to the rise of multidrug resistance in bacteria , has led to a renewed interest in the use, efficacy, and safety of phages to combat bacterial pathogens in human health , veterinary medicine , agriculture , aquaculture , and food safety [20,21] (for recent reviews, see [21–23]). In this mini-review, we first describe the various ways that phages, both engineered and natural, and phage-derived products can act as antimicrobial agents. Finally, we will highlight examples of phage-based biocontrol applications in aquaculture and agriculture.
Phage-based antimicrobial approaches
Although there is extensive morphological and genetic diversity, most characterized phages are members of the Caudovirales, which are dsDNA tailed phages. These phages are either virulent or temperate, depending on their life cycles . Virulent phages enter the lytic cycle, whereas temperate phages can replicate via either the lytic or lysogenic cycles . At the onset of infection, phages attach to specific cell surface receptors on bacteria , and upon irreversible attachment inject their genomic DNA (Figure 1A). The bacterium provides resources for phage genome replication and for the production of the protein components required to assemble new virions. Phage-encoded holins and lysins are expressed near the late stages of the lytic cycle to lyse the bacterium and release the viral progeny (Figure 1A) [10,24]. Since virulent phages often replicate quickly and kill the infected cells, they are used in the development of bacteriolytic-based therapeutics. In contrast, for temperate phages in the lysogenic cycle, they do not immediately undergo phage production. Instead, the phage DNA becomes a prophage, either through integration into the host chromosome or by the formation of an extrachromosomal plasmid-like replicon (Figure 2A) . Within the bacterial lysogen, the prophage is replicated along with the host genome for multiple generations. Certain conditions, such as environmental stress, can trigger prophage excision, initiating the shift to the lytic cycle . Temperate phages can encode virulence genes, thereby impacting the genome evolution of bacterial pathogens. For example, CTXφ is a temperate phage that infects Vibrio cholerae and encodes for the cholera toxin (CT) genes . Likewise, many environmental phages carry antibiotic resistance genes . While it is widely accepted that it is not ideal to use naturally derived temperate phages as therapeutics, they can be engineered to become virulent or to deliver genetic elements that can render pathogens susceptible to antimicrobials or disrupt virulence factors.
Bacteriolytic phages as antimicrobials.
Engineering lysogenic phages as genetic delivery systems.
There are multiple ways that phages and their products can be exploited as antimicrobials. Owing to the specificity of phages to target and kill bacteria, phages can be (i) directly administered as antimicrobials, (ii) indirectly used by exploiting their lytic enzymes, and (iii) engineered as genetic delivery systems. These examples of phage-based antimicrobial approaches are highlighted in the following sections.
The term phage therapy is usually associated with the treatment of bacterial infections using whole or intact phages. Virulent phages are isolated, purified and administered to treat and/or prevent bacterial diseases. Phages are usually highly host-specific and only infect and replicate if appropriate hosts are available, leaving non-hosts unaffected (Figure 1B) . However, there are methods allowing for the isolation of broader host-range phages and some phages can infect different bacterial genera [30,31]. Identification of the disease-causing agent(s) and strain typing for phage susceptibility of the pathogen(s) before phage administration is necessary and time-consuming. Next-generation sequencing and advances in other diagnostics (see ) are likely to reduce these hurdles in the future. Bacteria can also develop phage resistance, mainly when only one type of phage is used during treatment (see [33,34]). Since most phages exhibit a limited host range, cocktails containing several virulent phages that ideally recognize different bacterial cell surface molecules are used to prevent or decrease the emergence of phage resistance . In addition, phage cocktails can also be designed to improve the overall host range of the therapeutic and increase the treatment efficacy. For example, ListShieldTM is a cocktail composed of three phages targeting a wide range of Listeria monocytogenes strains and is used commercially to disinfect food preparations . This represents one of the increasing commercially available phage products that have FDA-approval and Generally Recognized As Safe (GRAS) status. Furthermore, with progress in synthetic biology, phages can now be modified to provide them with additional desirable properties to expand their potential applications . For example, phage T7 was engineered to overexpress dispersin B (DspB), an enzyme that degrades the biofilm matrix (Figure 1C). Rapid replication of T7DspB phage, lysis of bacterial cells and release of more DspB led to biofilm disruption of Escherichia coli TG1 that was three times more effective than the wild-type T7 phage .
Phage proteins can also be exploited per se as antimicrobials . Lysins (i.e. endolysins) are hydrolytic enzymes that degrade bacterial cell wall peptidoglycan to facilitate phage release . Recombinant lysins have been successfully used against Gram-positive bacteria due to their lack of an outer membrane; whereas, chemical permeabilization or peptide sequences that aid outer membrane penetration are usually added with lysins to target Gram-negative bacteria [17,39]. Lysins also exhibit specificity for certain peptidoglycan types, which can enable targeted antimicrobial action to a specific Gram-positive genus or species . In addition, multiple lysins with different cleavage sites can be combined to increase effectivity. For example, a combination of Pal amidase and Cpl-1 lysozyme was more effective in killing Streptococcus pneumoniae than when either was used alone . Interestingly, no lysin resistance has been reported [41,42] and they can also be engineered through directed evolution for increased bacteriocidal activities. Their modular nature has enabled binding and cleavage specificities to be swapped — expanding the possible enzymatic arsenal against target microbes. Chimeric lysins against S. pneumoniae , Listeria spp. , and Staphylococcus spp.  had increased bacteriocidal activity (up to four orders of magnitude) compared with the parental proteins. Lysins can also act against bacteria growing in biofilms, which typically have higher intrinsic tolerance against traditional antibiotics. For example, lysostaphin, at high concentrations, eradicated biofilms formed by multidrug resistant Staphylococcus aureus . Thus, with the rich abundance and diversity of phages in nature, sequencing, bioinformatics, and proteomic studies can aid in the discovery of novel lysins with more potent antimicrobial activities.
Non-lytic antibacterial strategies
Rapid bacterial lysis caused by virulent phages can result in the release of harmful endotoxins. To circumvent this problem, phages can be engineered to cause bacteriostasis by deleting lysin genes [47,48]. Likewise, phages can also be engineered to deliver genes for proteins that can either inhibit the targeted bacteria or re-sensitize them to antibiotics (Figure 2) . Filamentous M13 phages are commonly used vectors for gene delivery, such as via phagemids, to targeted bacteria (Figure 2C). Phagemids are plasmids containing a selective marker, origin of replication and packaging signal, and require co-infection with helper phages to be packaged into new infective, but non-replicative, phage particles . Like plasmids, phagemids can easily be manipulated and can accommodate foreign DNA fragments. For example, a phagemid was engineered to encode toxin genes, such as gef and chpBK . Gef and ChpBK are toxic proteins derived from toxin–antitoxin systems in bacteria. Gef interferes with, and depolarizes, the cell membrane , whereas ChpBK is a ribonuclease that cleaves mRNA and, as such, inhibits protein translation . Phage delivery of genes expressing Gef or ChpBK reduced E. coli viability in vitro and up to 98% of bacterial titers in a bacteremic mouse model of infection . Similarly, antimicrobial peptides such as cecropin and apidaecin that target the cell membrane can also be delivered via phagemids to induce cell death .
Several recent studies proposed combination treatments using antibiotics and phages as delivery vehicles to restore the vulnerability of the pathogens to antibiotics. This two-pronged strategy aims to reduce the incidence of antibiotic resistance and enhance bacterial killing . To re-sensitize pathogens, phagemids can encode repressors that inhibit antibiotic resistance genes or encode RNA-guided nucleases to remove resistance determinants. Repression of the SOS response has been implicated in the enhanced killing of bacteria by antibiotics and limiting the emergence of antibiotic resistance . A phagemid that carried a gene encoding LexA, the SOS response repressor, was delivered by M13mp19 phage into E. coli . Combined administration of the M13mp19LexA3 phage with ofloxacin (a fluoroquinolone) resulted in enhanced bacterial cell death compared with unmodified M13mp19 controls and was effective against quinolone-resistant bacteria . Phagemids were also used to deliver engineered CRISPR–Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated) constructs that specifically eliminated plasmids that carried antibiotic resistance and virulence genes [57,58]. Lastly, owing to the nuclease activity of the CRISPR–Cas systems, these can also be exploited to kill bacteria through the introduction of sequence-specific CRISPR RNAs targeting the bacterial chromosome to induce cytotoxicity [59–62]. Thus, now with the application of CRISPR–Cas and phage delivery systems, there is the potential to engineer phages to target and specifically manipulate bacterial pathogens while preserving the natural microbiota within any ecological system .
Phage-based biocontrol strategies in agriculture and aquaculture
Phage-based biocontrol strategies in plants
The concept of using phages for the control of bacterial plant diseases is longstanding. Indeed, the first study that associated phages and plant pathogenic bacteria was published in 1924 . In this early study, Xanthomonas campestris pv. campestris was isolated from plants with cabbage-rot disease, and a filtrate obtained from the decomposing cabbage tissue inhibited pathogen growth . A complementary study confirmed that phages could prevent soft-rot caused by Pectobacterium carotovorum subsp. carotovorum in carrots  and Pectobacterium atrosepticum in potato slices . More recently, there has been an increase in studies investigating the potential of phages in plant pathogen control . Two of the most economically relevant bacterial plant pathogens are Xanthomonas spp. and Pseudomonas syringae , which are discussed below.
Phages were tested for biocontrol of X. campestris pv. vesicatoria, the causative agent of bacterial spot of tomato, in a series of greenhouse and field trials in 1997 and 1998 . Twice weekly foliar applications of a phage cocktail that targeted different strains of the pathogen resulted in reduced disease incidence and severity compared with non-treated plants and the standard copper–mancozeb treatment . Phage treatment was more effective when applied early in the morning and it was proposed that phage damage by UV, and other environmental factors, could play a role. Subsequent formulations designed to extend phage longevity significantly increased disease control efficacy in field trials . Citrus bacterial canker and citrus bacterial spot are two devastating citrus diseases caused by X. citri subsp. citri and Xanthomonas axonopodis pv. citrumelo. Nursery trials demonstrated that phages reduced disease severity; however, phage treatment was less efficient than copper–mancozeb . Recently, successful management of X. citri subsp. citri was achieved in greenhouse and field trials with a phage cocktail combined with a systemic acquired resistance activator and treatment was as effective as standard copper-based control measures .
Phages for the control of Pseudomonas syringae pv. porri, the causal agent of the bacterial blight of leek, were isolated and characterized . While laboratory-based bioassays showed reduced symptoms in phage-treated leaves, no significant effect was observed in a field trial . This highlights the importance of field trials because results obtained can differ from those acquired in the laboratory. Bacterial canker of kiwifruit, caused by P. syringae pv. actinidiae is a destructive disease that is globally distributed . Several groups have reported isolation, morphologic characterization, genome sequence and host-range determination for phages that could be used for biocontrol of the disease [75–78]. However, no trials testing the efficacy of phage therapy against Pseudomonas syringae pv. actinidiae have been reported.
Phage lysins are also effective at eliminating plant pathogens. Peptidoglycan hydrolases of phages Atu_ph02 and Atu_ph03 blocked cell division and subsequently lysed Agrobacterium tumefaciens, the etiological agent of crown gall disease . Similarly, exogenous application of purified lysins of CMP1 and CN77 phages specifically lysed Clavibacter michiganensis subsp. michiganensis, the causative agent of bacterial wilt and canker of tomato . As the widespread administration of lysins to vast numbers of plants may lead to production challenges, transgenic crops that express phage lysins may also be developed to provide protection from pathogens. Indeed, transgenic tomato plants expressing the lys gene of CMP1 showed no symptoms after C. michiganesis challenge . Overall, phages and their enzymes provide valuable additions to the tools available in agriculture for plant pathogen control. Although the advances highlighted here provide evidence that this is a promising technology, more work is required to provide consistent protection of plant crops. Of particular importance, it will be critical to develop delivery strategies and formulations that ensure high persistence of phage particles and lysins in the rhizosphere and phyllosphere of treated plants.
Phage-based biocontrol strategies in aquaculture
A wide array of bacterial pathogens are associated with infections in fish and shellfish . Several phages have been isolated and studied for their therapeutic effects against various pathogens in aquaculture, namely Aeromonas hydrophila in loaches, Aeromonas salmonicida in trout and salmon, Edwardsiella tarda in eel, Flavobacterium columnare in catfish, Streptococcus iniae in flounder, and Vibrio harveyi in shrimp . Studies on the usage of phages as biocontrol agents in aquaculture have been positive, with phage administration providing protection with no reported side effects [82,83]. For example, two Aeromonas phages, pAh1-C and pAh6-C, were isolated and tested against A. hydrophila, a Gram-negative pathogen that causes tail and fin rot, and hemorrhagic septicemia in fish . Each phage was administered either orally through food pellets or intraperitoneally via injection to infected loaches. Both delivery methods were effective against A. hydrophila with significantly increased survival rates with some trials exhibiting full protection . Furthermore, no negative effects were reported when healthy loaches were treated with phages alone, indicating the safety of phages in aquaculture . Many studies have also demonstrated successful treatment of vibriosis caused by V. harveyi in shrimp hatcheries [85–87]. Phage application, both single- or cocktail-based, resulted in elevated shrimp survival rates, even better than the antibiotic-treated trials [85,86,88]. Interestingly, phage Viha10 was also effective against V. harveyi biofilms and showed a 3-log reduction in bacterial counts after 18 h of treatment . All these studies suggest that whole phages can be effective biocontrol agents in aquaculture, perhaps highlighting the benefits of phage therapy in aqueous environments. However, the success of phage therapy in aquaculture may also be influenced by environmental factors such as salt concentrations, pH and temperature, among others . Thus, further research about the delivery, stability, efficacy, and safety of these phages should be conducted before these can be applied commercially. Currently, only natural phages have been tested in aquaculture, despite the advances in engineered phages as antimicrobials — possibly due to the possible reluctance of consumers for synthetic phages in the food-chain. However, there is potential to use lysins as biocontrol agents. For example, a recent study demonstrated that the endolysin LysVPMS1 from vibriophage VPMS1 had a broad muralytic activity against Vibrio species when administered with EDTA to permeabilize the gram-negative outer membrane . Although this is an in vitro study, it shows that there is potential to develop lysin-based methods for biocontrol in aquaculture in the future. Lysins might provide biocontrol benefits, such as the ability to inhibit biofilms, their broader spectrum than phages against gram-negative bacteria and the reduced resistance development.
Food supplies from agriculture and aquaculture will need to intensify as a result of the anticipated growth in the human population. Current efforts have depended on husbandry practices to improve yields; however, biocontrol agents to manage disease outbreaks are still limited. Phages and phage-based technologies are promising approaches for the treatment and biocontrol of bacterial diseases, including drug-resistant pathogens. Research thus far has validated phages and their enzymes as effective and specific killers of bacterial pathogens, mostly under controlled conditions. To fully implement phage-based applications, research into their efficacy in the real-world control of diseases in agriculture and aquaculture are needed. It is likely that as phage application in the medical sector intensifies, a clear demonstration of positive outcomes will be crucial in helping promote the potential of phage therapy in agriculture and aquaculture. For example, in a recent high profile case, US researchers used phages to treat a patient afflicted with multidrug-resistant Acinetobacter baumannii, which has heightened the public interest in phage therapy . Biotechnology companies are also investigating the use of phage-derived and engineered lysins in various applications, but their potential in agriculture and aquaculture is, as yet, relatively unexplored.
Work in the Fineran laboratory on plant pathogens, phages, and phage resistance mechanisms are supported by the Marsden Fund (Royal Society of New Zealand), ZESPRI Ltd, the Bio-protection Research Centre (Tertiary Education Commission) and the School of Biomedical Sciences Dean's Bequest Fund, University of Otago. Luciano Ariel Rigano is funded by Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Argentina. Work in the Dy's laboratory is supported by the University of the Philippines, National Institute of Molecular Biology and Biotechnology (NIMMB) Grant.
The Authors declare that there are no competing interests associated with the manuscript.