Bacteriophages in Food Processing
Foodborne illnesses, despite many advances in food hygiene and pathogen surveillance, remain a leading cause of hospitalizations and deaths worldwide. Conventional antimicrobial methods such as pasteurization, high-pressure processing, irradiation, and chemical disinfectants can reduce microbial populations in food to varying degrees, but also have significant drawbacks, such as high initial investment, potential damage to processing equipment due to their corrosive nature, and a detrimental effect on the organoleptic properties (and possibly the nutritional value) of food. Perhaps most importantly, these decontamination strategies kill indiscriminately, including many—often beneficial—bacteria naturally present in food.One promising technique that addresses several of these shortcomings is bacteriophage biocontrol, an environmentally friendly and natural method that uses lytic bacteriophages isolated from the environment to specifically target and eliminate (or significantly reduce the content of) pathogenic bacteria from food. Since the initial idea of using bacteriophages in food, numerous research reports have described the use of bacteriophage biocontrol to combat a variety of bacterial pathogens in diverse foods, from ready-to-eat deli meats to fresh fruits and vegetables. The number of commercially available products containing bacteriophages approved for food safety applications has also steadily increased.Although some challenges remain, bacteriophage biocontrol is increasingly recognized as an attractive modality in our arsenal of tools for the safe and natural elimination of pathogenic bacteria from food.
1. Introduction
From lettuce leaves to cheddar cheese in a Cobb salad to frozen ready meals, the foods we consume are constantly at risk of contamination by microbial pathogens, which can then be transmitted to the consumer. Recently, the Foodborne Disease Epidemiology Reference Group (FERG) was established by the World Health Organization (WHO) to monitor foodborne diseases globally. FERG monitored the 31 foodborne pathogens that caused the highest morbidity and mortality in humans. In its latest (2015) estimate of the global burden of foodborne diseases, FERG estimated that 600 million foodborne infections occurred in 2010, causing over 400,000 deaths. Among the five most common microorganisms causing foodborne diseases, four were bacteria: Escherichia coli (~111 million), Campylobacter spp. (~96 million), non-typhoidal Salmonella enterica (~78 million), and Shigella spp. (~51 million), with the number of foodborne deaths caused by these bacteria estimated at ~15,000 for Shigella spp. to ~63,000 for E. coli [1]. Notably, children under five years of age were disproportionately affected; they account for 40% of deaths and represent only 9% of the world’s population [1]. These foodborne illnesses also place an enormous burden on national economies. In the United States, for example, the average incident is estimated at approximately $1,500 per person, with the estimated total annual cost of these foodborne diseases exceeding $75 billion [2].
There are various approaches to improving the safety of our food. Heat pasteurization is commonly used to reduce bacterial counts in liquids and dairy products, especially milk. However, pasteurization is not suitable for many fresh foods, as the process causes the products to be cooked. Another method for reducing pathogens in food is high-pressure processing (HPP), which exposes food to high pressure to inactivate microbes. This technique has been successfully applied to liquid products and pre-cooked meals intended for freezing. However, as with heat pasteurization, it is generally not used for fresh meat and produce, as it can affect the appearance (color) and/or nutritional content of these products [3, 4]. Irradiation is also an effective means of reducing the burden of pathogenic organisms in food. However, irradiation can adversely affect the organoleptic properties of food. Furthermore, customer acceptance of this method is low and is exacerbated by mandatory labeling for many radiation-treated foods [5, 6]. Finally, chemical disinfectants such as chlorine and peracetic acid (PAA) are widely used to reduce microbial contamination of many fresh fruit and vegetable products, as well as ready-to-eat (RTE) food products [7, 8]. Although generally effective, many of these chemicals are corrosive and can damage food processing equipment. Chemical disinfectants can also have detrimental effects on the environment (i.e., they are not eco-friendly), and given current trends towards chemical-free organic foods, consumer acceptance of chemical additives in food (especially in fresh produce) is rapidly declining. A common drawback of all these techniques is that they kill microbes indiscriminately. In other words, both pathogenic and potentially beneficial normal flora bacteria are equally affected. Furthermore, despite the multitude of available methods, foodborne outbreaks still occur relatively frequently.These combined factors illustrate the need for a targeted antimicrobial approach that can be used alone or in combination with the techniques described above to create additional barriers in a multi-hurdle approach to prevent foodborne bacterial pathogens from reaching consumers. One such technique is the use of lytic bacteriophages to target specific foodborne bacteria in our food without adversely affecting their normal—and often beneficial—microflora. This approach is referred to as “bacteriophage biocontrol” or “phage biocontrol.”
Phage biocontrol is increasingly accepted as a natural and environmentally friendly technology that can specifically target bacterial pathogens in various foods to protect the food chain (Table 1). Bacteriophages were first identified by Felix d’Herelle in 1917, and the usefulness of these “bacteria eaters” in combating bacterial diseases was quickly exploited [9].In the context of food safety, bacteriophages address many consumer concerns. For example, due to the specificity of bacteriophages, phage biocontrol offers a unique way to target pathogenic bacteria in food without disturbing the normal microflora of food. Notably, the U.S. Army recently initiated a project (W911QY-18-C-0010) to further investigate the effects of phage application compared to conventional chemical antibiotics on the normal microbiota of fresh produce and the potential impact of these measures on the nutritional value of food. Furthermore, phage biocontrol is likely the most environmentally friendly antimicrobial intervention currently available. Most, if not all, currently available commercial phage biocontrol products contain natural phages, i.e., phages isolated from the environment that are not genetically modified. Many of these preparations also contain no additives or preservatives; they are typically water-based solutions consisting of purified phages and small amounts of salts. Some phage preparations available on the market are also certified as kosher and halal and are available for use in organic foods (OMRI-listed in the USA; SKAL in the EU) (Table 2). Although there are limited tests, our group’s work suggests that bacteriophages do not alter the organoleptic (i.e., sensory) properties of food [10]. Compared to other food safety measures, the cost of applying bacteriophages is relatively low, typically ranging from 1 to 4 cents per pound of treated food. HPP treatment and irradiation typically cost 10 to 30 cents per pound [11]. It is important to note that these figures represent only the cost of each intervention and do not account for situations where a multi-hurdle approach may be necessary for food safety reasons (e.g., concern that food is contaminated by more than one foodborne pathogen) or for food quality reasons (e.g., food spoilage, which is typically caused by several different microorganisms).
The biological properties of lytic bacteriophages and other characteristics of commercial phage biocontrol products, as discussed above, make phage biocontrol a very attractive method for further improving the safety of our food, and an increasing number of companies worldwide are engaged in its development and commercialization [12] (Table 2). However, phage biocontrol has its limitations and disadvantages. For example, phage preparations require refrigerated storage (usually 2–8°C) and may need to be applied separately from chemical disinfectants, as aggressive chemicals can also inactivate phage particles, making phage biocontrol less effective. Due to their high natural specificity, phage preparations can effectively target pathogens in food. However, if food is accidentally contaminated with two or more foodborne bacterial pathogens, a phage preparation directed against a single pathogen will not be effective in removing non-target pathogens from food. As a final consideration, care must be taken to use lytic phages and to exclude temperate phages from bacteriophage preparations. Temperate phages are typically less effective at killing their bacterial hosts than lytic phages. Furthermore, temperate phages can integrate their DNA into the bacterial chromosome and therefore potentially promote the transfer of virulence genes or other undesirable genes (e.g., antibiotic resistance-encoding genes) between bacterial strains, which could lead to the emergence of new pathogenic strains. The risk of such an occurrence is significantly lower when lytic phages are used.
This review focuses on applications of wild-type bacteriophages to improve food safety. We do not discuss other possible phage-related methods, such as the use of phage endolysins to combat foodborne pathogens or the use of bacteriophages to control food spoilage. These topics have already been discussed by other authors, and relevant reviews are available [13, 14].In the context of food safety applications, wild-type lytic bacteriophages can be used both pre-harvest (e.g., in live animals, administered via animal feed or sprayed before slaughter) and/or post-harvest (e.g., applied directly to food surfaces, either by direct spraying, via packaging materials, or otherwise) to reduce contamination by pathogenic bacteria [12, 15]. Bacteriophage biocontrol could also be a means of disinfecting surfaces used in food production and processing [16, 17]. In previous reviews [12, 14, 18, 19], we and others have compiled a general overview of the industries and products where bacteriophages are used in food safety applications. Here, we provide an updated overview (and an expanded summary table) describing studies in which bacteriophages have been predominantly applied to post-harvest foods, particularly meat, fresh produce, and RTE foods (Table 1). The next section will discuss selected studies from the last five years that have used bacteriophage biocontrol to combat four major foodborne pathogens. Finally, we also discuss the regulation of bacteriophages for food safety applications and some of the challenges of phage biocontrol.
2. Phage Biocontrol to Combat the Most Common Foodborne Bacterial Pathogens
2.1. Listeria monocytogenes
Listeria monocytogenes is a rod-shaped, Gram-positive, facultative anaerobe. Consumption of food contaminated with L. monocytogenes causes a range of symptoms in humans, such as initial flu-like or gastrointestinal symptoms, which in some cases lead to encephalitis or cervical symptoms and possibly stillbirth in pregnant mothers. It is estimated that in 2010, there were more than 14,000 cases of foodborne infections with L. monocytogenes worldwide, resulting in over 3,000 deaths [1]. L. monocytogenes can survive and grow at refrigerated temperatures (2–8°C), which are commonly used in the distribution and storage of many foods. Therefore, the detection and elimination of L. monocytogenes are crucial for ensuring the safety of the food chain, especially in RTE foods. In this context, several researchers have shown that the application of bacteriophages to various foods (including RTE foods) effectively reduces L. monocytogenes contamination (Table 1). For example, a commercial monophage preparation (i.e., a phage preparation consisting of a single phage) targeting Listeria was reported to effectively reduce levels of L. monocytogenes in sliced ham and was superior to lactate-nisin and sodium at the storage abuse temperature of 6–8°C [47]. A similar study by Chibeu and colleagues (2013) showed that the same monophage preparation could also reduce L. monocytogenes on the surface of other deli meats [44]. The meat (cooked sliced turkey and roast beef) was stored at 4°C and an abuse temperature of 10°C. The Listeria-specific phage was effective against L. monocytogenes when used alone and increased the efficacy of other antimicrobial agents when used in combination with sodium diacetate or potassium lactate. All these studies used a single phage preparation. A phage cocktail prepared with multiple bacteriophages, compared to a single phage preparation, can be superior both in terms of broader coverage of target species and reduction of the risk of resistant bacteria emerging. Such a commercially available six-phage cocktail against L. monocytogenes was tested on a range of foods experimentally contaminated with L. monocytogenes, including lettuce, pasteurized hard cheese, smoked salmon, and Gala apple slices; The application of this bacteriophage cocktail reduced L. monocytogenes levels in all these foods by ~0.7–1.1 logs [10]. The same study investigated the application of the L. monocytogenes-specific cocktail to pre-packaged, frozen meals. The meals were experimentally contaminated with L. monocytogenes, treated with the phage cocktail, and subjected to freeze-thaw cycles. The results showed a 2.2 log reduction of L. monocytogenes, suggesting that phage biocontrol can be an effective means of controlling L. monocytogenes in food under “storage abuse” conditions, where frozen meals are intentionally or unintentionally thawed multiple times during storage [10].
In many of the studies discussed above, despite the initial significant reduction in L. monocytogenes levels in the foods, the target bacterial populations were not completely eradicated, and viable L. monocytogenes cells could still be recovered, albeit in much smaller numbers. However, the bacteriophage preparations were still effective against randomly selected colonies of the recovered bacteria, suggesting that phage resistance was not the primary reason for the incomplete eradication of L. monocytogenes [23, 37, 44]. There are several possible explanations for this observation. For example, the L. monocytogenes cells could exhibit temporary resistance to phage infection, as previously reported [70, 71]. Another possible explanation is that after spraying the phages onto the food (e.g., due to using too low a spray volume, especially on foods with complex topography), the phages did not directly contact some L. monocytogenes cells, resulting in these bacterial cells not being lysed by phages. In this latter scenario, using larger spray volumes, fine (mist-like) sprays, rotating/tumbling foods during phage application, and ensuring thorough surface coverage with phages can help improve the efficacy of phage biocontrol.
2.2. Salmonella spp.
The non-typhoidal serotypes of Salmonella enterica are responsible for many cases of gastroenteritis worldwide each year. The illness caused by these Gram-negative, rod-shaped bacteria is often self-limiting, presenting with symptoms such as abdominal cramps, fever, nausea, and diarrhea. However, life-threatening cases can occur where the bacteria are dehydrated and invade the gastrointestinal tract.It is estimated that in 2010, over 78 million cases of foodborne infections caused by Salmonella occurred worldwide, resulting in nearly 60,000 deaths [1]. During food processing and packaging, Salmonella and other pathogens can adhere to and contaminate surfaces where food is prepared. These factors put RTE foods such as fresh fruits and vegetables, which are not cooked before consumption, at a particularly high risk of transmitting bacterial pathogens and causing food poisoning.
Currently, at least two FDA-approved phage preparations against Salmonella are on the market (Table 2). Several publications are available describing their applications (and those of other non-commercial phage preparations) in various foods. Brief summaries of these studies are provided in Table 1. One study is of particular interest as it demonstrates how phage resistance can be managed when it compromises the efficacy of a bacteriophage preparation. In this study, a GRAS-listed (generally recognized as safe) six-phage cocktail against Salmonella was investigated for its ability to reduce Salmonella levels on surfaces similar to those commonly found in food processing facilities, such as stainless steel and glass [16]. Initial studies showed that the Salmonella-specific bacteriophage cocktail significantly reduced the population of susceptible Salmonella strains on all tested surfaces by ~2–4 logs; At the same time, it was ineffective at reducing the levels of another Salmonella strain (Salmonella Paratyphi B S661) which was resistant to the phage cocktail in vitro [16]. However, when the phage cocktail was adjusted to include phages specifically targeting this resistant strain, the updated preparation showed a significant reduction (~2 log) of S. Paratyphi B S661 from the surfaces, while also maintaining efficacy against the previously susceptible isolates [16]. This study provides compelling evidence that phage cocktails can be easily modified to target specific bacterial strains, for example, when phage-resistant mutants emerge, or to specifically target problem strains prevalent in particular food manufacturing facilities.
In addition to their usefulness in decontaminating food preparation surfaces, bacteriophage cocktails have also removed Salmonella directly from food. For example, the same Salmonella-specific cocktail discussed above reduced Salmonella levels on experimentally contaminated chicken parts when applied alone, and this effect was enhanced when the phage was applied in combination with conventional chemical disinfectants [59]. On chicken breast fillets, the bacteriophage cocktail significantly reduced the number of a mixture of Salmonella species when applied to the surface of the fillets or when the fillets were dipped into a vessel containing the phage solution [60]. Furthermore, this phage cocktail significantly reduced the number of Salmonella when the fillets were stored under aerobic or modified atmospheric conditions [60]. This latter finding can have direct practical implications, as food manufacturers often use modified atmospheric conditions to inhibit bacterial growth and extend the shelf life of food. Another study found that a single phage, SJ2, significantly reduced the amount of Salmonella in liquid egg and ground pork, and this reduction was more pronounced at higher temperatures [62]. The authors examined residual Salmonella colonies for resistance; While there was no difference in the number of resistant clones from phage-treated and untreated ground pork samples, a significantly higher number of resistant clones was found in the phage-treated liquid egg samples [62]. The authors suggested that both the food matrix (solid and liquid) and differences in the microbiome of the two foods could have contributed to this difference in the number of resistant Salmonella isolates [62].
Foodborne illnesses caused by non-typhoidal serotypes of Salmonella also pose a health risk to pets (e.g., dogs and cats), and the close association of these animals with their owners increases the possibility of human illness. Indeed, human Salmonella outbreaks have been linked to contaminated cat and dog food, and it was found that approximately one-third of the commercial raw and natural pet foods sampled contained Salmonella [72, 73]. To counteract this health risk, phage biocontrol has recently been investigated as a technique to reduce or eliminate Salmonella in pet food. The six-phage Salmonella-specific cocktail discussed above was found to reduce Salmonella levels in experimentally contaminated dry dog food by 1 log [74]; When cats and dogs were fed dry kibble treated with the same phage cocktail, it appeared to be safe and had no noticeable impact on any of the key health metrics recorded for any of the animals [61].
An alternative to dry food that is gaining popularity is raw food. These pet meals consist of meats such as chicken, duck, or tuna, combined with vegetables, including lettuce, blueberries, and broccoli, sold and served raw [61]. Raw pet food is gaining popularity due to its excellent nutritional value. At the same time, because they are not cooked, there is an increased likelihood that foodborne pathogens are present in them, which can be transmitted to both pets and unsuspecting consumers during the feeding process.Recently, at least one report has been published in which the authors investigated the value of using phages to control Salmonella in raw ingredients for pet food. The reduction in bacterial contamination ranged from 0.4 log to 1.1 log, efficacy was concentration-dependent, and the greatest reduction was achieved when high doses of the bacteriophage preparation were used [61] (Table 1).
2.3. Escherichia coli
Many strains of the Gram-negative, rod-shaped bacterium Escherichia coli naturally occur in the human gut and are beneficial for our health and well-being. For example, they aid in food digestion and maintaining a robust immune system. However, some E. coli strains can and do cause illness in humans. For example, Shiga toxin-producing E. coli serotype O157:H7, sometimes found in contaminated water or food, especially beef, can enter the human gastrointestinal tract and cause illness, with symptoms such as abdominal cramps and hemorrhagic diarrhea. These infections are usually self-limiting in immunocompetent individuals but can be life-threatening in very young or elderly patients. It has been estimated that worldwide, over one million cases of foodborne illness and over one hundred deaths are attributable to Shiga toxin-producing E. coli, including serotype O157:H7 [1].
Recent work has shown that E. coli-specific phage preparations were effective in treating fresh vegetables [75] and both ultra-high temperature (UHT) treated and raw milk contaminated with E. coli [33]. In the first study, E. coli O157:H7 levels on green bell pepper slices and spinach leaves were reduced by approximately 1–4 logs by a single phage, and the initial reduction was maintained at 4°C, while some regrowth was observed at 25°C. In the second study, E. coli concentrations in both UHT and raw milk were reduced to undetectable levels when a cocktail of two or three phages was used. Remarkably, this reduction was maintained in all samples treated with the three-phage preparation during storage at both 4 and 25°C; in contrast, the E. coli strain regrew in samples treated with the two-phage cocktail. Although the underlying reasons are not fully understood, it is possible that the three-phage cocktail offers better resistance control than a two-phage cocktail, and the improved efficacy of multi-phage cocktails has already been demonstrated for other phage preparations [76]. Although the underlying reasons for this phenomenon have not been precisely determined, it is possible that the presence of multiple phages in a phage cocktail reduces the risk of phage-resistant mutants emerging, as multiple mutations would be required to make a specific bacterial cell resistant to not one, but several phages in the cocktail, assuming the phages target different cellular structures. This concept essentially aligns with the multi-hurdle approach, which proposes a combination of antibacterial strategies to prevent the development of bacterial resistance [77]. These and some further studies using E. coli-specific phages in food safety applications are briefly summarized in Table 1.
2.4. Shigella spp.
Species of the Gram-negative, rod-shaped bacterial genus Shigella cause a self-limiting gastrointestinal infection with symptoms such as hemorrhagic diarrhea and abdominal pain. Globally, the incidence of foodborne infections caused by Shigella was estimated at over 50 million in 2010, resulting in over 15,000 deaths [1]. The vast majority of these infections occurred in developing countries, with most infections and deaths occurring in children under 5 years of age [1, 78].
Currently, only one FDA-approved phage preparation for food safety against Shigella spp. is available. [66, 69]. This five-phage cocktail received GRAS status (GRN 672) in 2017 (Table 2), and it has been shown to reduce Shigella levels by approximately 1 log in a variety of foods, including melons, lettuce, yogurt, deli corned beef, smoked salmon, and chicken breast meat [66]. In another study, the same Shigella-specific bacteriophage cocktail was used to compare the safety and efficacy of phage administration with antibiotic treatment in mice exposed to a Shigella sonnei strain [69]. This study showed that although the Shigella-specific bacteriophage cocktail was as effective as standard antibiotic treatment in reducing bacterial load in mice, treatment with the antibiotic significantly altered the mouse gut community diversity, whereas phage administration had a much smaller impact on the normal gut microbiota of mice compared to antibiotic treatment [69]. The authors observed no harmful side effects in the mice following the administration of phages; that is, the phage neither altered the composition of the mice’s blood or urine, nor did it have an adverse effect on the morbidity or mortality, weight, or other physiological parameters of the animals [ 69 ]. Although these bacteriophages are not directly relevant for food safety applications, the study found that when administered orally (mimicking a scenario in which they would be consumed when eating food treated with them), they do not interfere with the normal intestinal flora (unlike antibiotics) and did not trigger side effects in any of the animals studied.
2.5. Campylobacter jejuni
Campylobacter spp., Gram-negative, rod-shaped bacteria, are the main pathogens in human food and cause gastrointestinal symptoms, which can include abdominal pain, fever, and diarrhea. In a recently published (2015) report, FERG estimated that in 2010, global cases of Campylobacter spp. exceeded 95 million and led to more than 21,000 deaths [ 1 ]. The intestinal flora of many poultry and other livestock contains species of Campylobacter . Although the route of entry is not fully understood, Campylobacter can often be isolated from both the surface and the interior of chicken liver. Zoonotic infections usually occur in humans when contaminated animal products such as meat are handled or consumed. Therefore, humans are at an increased risk of a Campylobacter infection when preparing minimally cooked preparations, e.g., pâté.
Several Campylobacter bacteriophages have been isolated from chickens, including feces as well as the surface and internal tissue of chicken liver, and some of them have been studied for their ability to reduce Campylobacter contamination of various foods [ 79 , 80 , 81] . 82 ]. For example, Hammerl and colleagues [ 80 ] used the phages as a pre-harvest treatment and demonstrated a significant reduction (~ 3 log) in Campylobacter fecal counts when 20-day-old chickens were treated sequentially with two phages (a Group III phage, then a Group II phage). Interestingly, dosing the Group III phage alone or in conjunction with another Group III phage was not effective, suggesting that a combination of different phages (Group II and III) was required for optimal efficacy. The isolation of Campylobacter-specific phages has historically been performed with a limited number of Campylobacter isolates, with many studies using only one C. jejuni NCTC 12662 isolate as the host strain for phage isolation. Phages isolated with this single strain are almost exclusively Group III phages that target a specific receptor, the capsular polysaccharide [ 83 ]. In contrast, phages isolated on C. jejuni RM1221 are typically Group II phages that use the flagella as a route of entry [ 83 ]. As evident from the study above [ 80 ], a phage cocktail consisting of phages targeting different receptors could potentially lead to a broader target range and more effective cocktails.
3. Bacteriophage Preparations as Commercial Products
3.1. Regulation of Bacteriophage Preparations
In the last approximately 12 years, the number of regulatory approvals for bacteriophage preparations and their use to improve food safety has steadily increased ( Table 2 ). In 2006, the FDA granted the first approval for a bacteriophage preparation for direct use in the food supply for the L. monocytogenes-specific cocktail ListShield™ as a food additive (the FDA does not “approve” products based on phages or otherwise; however, the term “approval” is commonly used to denote obtaining FDA clearance for the use of products for their intended applications). Later that year, the FDA issued a no-objection letter for the Listeria-specific preparation Listex™ (currently PhageGuard Listex™) as a Generally Recognized as Safe (GRAS) substance. In recent years, a number of phage products (e.g., SalmoFresh™ and PhageGuard S™) have been granted GRAS approval by the FDA. Applying for GRAS approval now appears to be the standard regulatory route for phage products used to treat post-harvest food. Since wild-type (i.e., non-genetically modified) lytic bacteriophages are all-natural and already present in the food supply, the GRAS designation seems to be an appropriate regulatory path for such preparations. Furthermore, the USDA has included several phage preparations in its issued guidelines for safe and suitable ingredients for the production of meat, poultry, and egg products. For example, according to FSIS Directive 7120.1, the application of phages to livestock before slaughter (e.g., E. coli O157:H7-targeted phages on cattle hides) and to food (e.g., Salmonella-targeted phages on poultry or meat) is permitted. These guidelines were developed using specific phage preparations. In general, however, any phage product that meets the description in the directive can be considered compliant. Following the lead of regulatory authorities in the US, several health authorities in countries around the world have granted approvals for phage products for use in food. Some examples include Israel, Canada, Switzerland, Australia, New Zealand, and the European Union ( Table 2 ).
3.2. Challenges for Bacteriophage Biocontrol
As described in the preceding sections, bacteriophage biocontrol is increasingly being used to combat specific pathogenic bacteria in various foods, with a growing body of professional literature documenting the utility of bacteriophages in reducing or eradicating their targeted pathogenic bacteria in food. However, several challenges remain before bacteriophage biocontrol is generally accepted, including technical limitations and general consumer acceptance of phage application to food. Some of these challenges are discussed briefly below.
3.2.1. Technical Challenges
Arguably the greatest technical challenge in phage biocontrol is its efficacy. A common observation in studies with bacteriophages on food is that the level of contaminating bacteria initially decreases and thereafter the bacteria are hardly or not at all further reduced [ 54 , 56 ]. In other words, phages can effectively reduce the level of their target bacteria in food, but they do not always eliminate them completely. Bacteriophages must come into contact with susceptible bacterial cells to lyse them. Considering the nature of the phage replication cycle (which begins with a phage infecting a bacterial cell and ends with 100–200 progeny phages bursting out of that cell at the end of each replication cycle, i.e., an exponential effect), one might expect this reduction in bacterial cells to increase exponentially with more replication cycles, as more progeny phages are generated as a result of ongoing phage-mediated lysis of the target bacteria. However, several reports have noted that the phage concentration does not increase significantly after application to food [ 43 , 44 , 45 ], strongly suggesting that “auto-dosing” (exponential increase in the phage population due to repetitive lytic replication cycles) does not occur, at least under the conditions tested so far. It is likely that the progeny phages are unable to reach and penetrate additional bacteria in food, particularly in drier food matrices where the passive movement of phages across food surfaces is limited due to the lack of moisture. In this context, it has been suggested that fewer phage particles may be required to significantly reduce bacterial contamination on moist food surfaces and in liquids compared to drier food matrices, presumably due to the increased “mobility” of phages in the presence of moisture (e.g., natural juices of some foods) [ 84 ]. One possible answer to this challenge is the use of a phage solution with higher concentrations of phage particles to increase the likelihood of phages coming into contact with their target bacteria upon application [ 17 , 21 , 36 , 66 ]; However, a more concentrated solution is more expensive, so implementation may be prohibitive for food processors. Another option is the use of larger spray volumes applied via fine mist to distribute the phage particles more efficiently over the surface of the food and increase the likelihood of them encountering a target bacterium, which could be particularly important under circumstances where pathogens are present in food in very low concentrations or when the infectious dose of the pathogen is extremely low. The correct application of bacteriophages to food to ensure thorough surface coverage and optimal efficacy is one of the most important technical challenges for phage biocontrol and involves a range of aspects, from the dosage of the phages (i.e., the effective concentration of the delivered phages in an optimal volume and how these can be verified in food processing plants) to obtaining the correct equipment (both to ensure accurate dosing, as just mentioned, and to ensure appropriate mixing or tumbling during phage application so that the entire surface of the food is thoroughly treated with the phage solution).
Another issue related to efficacy is that phage biocontrol typically reduces the concentration of target bacteria by 1–3 log (with rare exceptions: one study reported a reduction in Listeria of up to 5 log as a result of phage treatment [ 36] ]), and this is significantly lower than the reduction of up to 5 logs reported for some other, harsher interventions, e.g., irradiation. Although this is more of a perception issue than a real technical problem (since very few, if any, foods are contaminated with 5 logs of foodborne pathogens per gram), the lower reduction is classified as inferior by the food industry. Even if the target bacterium is not completely eliminated from food and is only reduced by 1 or 2 logs, it can still make the food safer for consumption. For example, the FDA and the USDA’s FSIS jointly produced a risk assessment study in 2003 in which they modeled a series of “what-if” scenarios, including a scenario where a reduction in contamination of deli meats would affect the mortality rate of the elderly. According to this analysis, a 10-fold reduction (1 log) and a 100-fold reduction (2 log) in contamination before sale with L. monocytogenes would reduce the mortality rate by approximately 10-fold. 50% and 74% respectively in this population segment [ 85 ]. Therefore, the implementation of phage biocontrol protocols—even if they do not eradicate (i.e., do not completely eliminate) the pathogens contained in food, but reduce them by 1–3 logs—can lead to significant improvements in food safety and public health.
Another technical challenge concerns the implementation of phage biocontrol. Phage biocontrol is an effective tool for improving food safety, but it does not make safe food handling redundant. For example, regrowth of bacteria has been observed after phage treatment when the food was stored at abuse temperatures [ 33 , 48 , 54 ]. Furthermore, some planning is required to maintain the optimal efficacy of phage biocontrol when bacteriophages are combined with some other food safety measures, e.g., the use of phages in conjunction with chemical disinfectants [ 59 ]. For example, a number of chemical disinfectants are capable of inactivating phages, and therefore they must be applied separately to ensure that the phages retain their viability to achieve the greatest bacterial reductions [ 59 ]. In this context, some researchers have reported that combinations of bacteriophages and preservatives are less effective than either treatment alone [ 86 ]. However, if suitable synergistic combinations of phage preparations with other disinfectants are identified, the efficacy of each could be improved. For example, in the presence of high organic loads, the efficacy of a wash with levulinic acid products was increased (by up to 2 log) when the fruit and vegetables were pre-treated with a bacteriophage preparation [ 34 ].
Another application-related (and efficacy-affecting) technical challenge is the potential emergence of phage-resistant bacterial isolates. Researchers are recovering bacteria that are resistant to phage treatments [ 62 ], and there is concern that the widespread application of this treatment could ultimately lead to selection for phage-resistant bacteria.Phages use a variety of bacterial structures to initiate the invasion of bacterial cells, including surface polysaccharides and proteins as well as the flagella [ 87 , 88 , 89 ]. The use of phage cocktails containing multiple, distinct phages (e.g., phages that use different receptors on the surface of bacteria) over a single monophage can provide a mechanism to reduce the risk/likelihood of bacterial resistance. The intervention strategy itself can also play a key role in the emergence of phage-resistant mutants. For example, applying phages at the end of the food processing cycle (e.g., when phages are sprayed onto food immediately before packaging) reduces the “overall selective pressure” in the environment, as bacterial exposure to the phages is limited. As a result, there is a lower risk of phage-resistant mutants emerging than if, for example, chicken coops or similar complex environments were sprayed with phages to reduce the contamination of livestock. Finally, if resistance occurs, phage cocktails could be modified to contain phages that target previously resistant bacteria. An example of such an approach has already been published and discussed elsewhere in this essay [ 16 ].
3.2.2. Customer Acceptance
In recent years, consumers have increasingly shown an aversion to purchasing food treated with chemical disinfectants and antibiotics or “genetically modified” foods, while at the same time, demand for locally produced organic foods and products, such as those at local farmers’ markets and in community-supported agriculture (CSA), is on the rise [ 90 , 91 ]. This trend bodes well for phage biocontrol, which offers a non-chemical, environmentally friendly, and targeted antimicrobial approach to improving food safety. However, the public may not be ready to buy food processed with unknown techniques, and the idea of “spraying viruses on their food” could lead to discomfort. Furthermore, food manufacturers are generally hesitant to change their practices, especially when there is a possibility that the public will react negatively. For phage biocontrol to be used more widely, it is crucial to educate the public and food processors about the safety, efficacy, and ubiquity of bacteriophages.
Phages are the most abundant organisms on the planet, with about 1031 particles (ten times as many as the entire global bacterial population) [ 92 ] and about 1015 phage particles colonizing the human gut [ 93 ]. Phages are part of the normal microflora of all fresh foods [ 94 ] and have been isolated from a variety of foods, from fruit and vegetables to meat and dairy products, often in very high numbers, e.g., up to 1 × 109 PFU/ml in yogurt [ 95 , 96 ]. Phage biocontrol is also likely one of the most environmentally friendly interventions available. In a previous review [ 18 ], we estimated that if phages were applied at the maximum approved amount (109 PFU/g for one phage product, all other current approvals for up to 107–108 PFU/g) for all approved food that an average American consumes in a day, the phages consumed would account for <0.2% of the number of phages already present in the human gut. This calculation is a gross overestimation, especially considering several GRAS approvals that allow an application of up to 108 PFU/g (reducing daily phage intake to ~0.02% of the phages in the human intestinal tract). This estimate also assumes that (1) all possible foods are treated, (2) all applied phages survive stomach acid and enter the small intestine (most phages, however, are normally destroyed when exposed to the acidic pH of the stomach), (3) the maximum approved amount of phages is applied, and (4) bacteriophage biocontrol is used universally by all relevant food industries in the United States. In short, the number of phages added to the environment and introduced into the human gut as a result of phage biocontrol is negligible, especially compared to naturally occurring phage populations. Furthermore, the phages in all currently available commercial products ( Table 2 ) are not genetically modified and originally come from the environment, possibly even from food. However, the public is often unaware of these facts. Therefore, a proper understanding of the safety and ubiquity of lytic phages, as well as the pros and cons of phage biocontrol among consumers and food processors, is crucial for the continued successful implementation of this promising approach. In at least one recent study, consumers appeared willing to pay more for fresh produce treated with bacteriophages after the science behind phage biocontrol and the benefits of this technique were explained to them [ 97 ].
4. Concluding Remarks
Although some challenges remain, bacteriophage biocontrol is increasingly being accepted as a safe and effective method for eliminating or significantly reducing the levels of specific bacterial pathogens from food. Commercial bacteriophage products are currently available and approved for use in a growing number of countries. These products can be used to combat contamination by specific bacterial pathogens at various points during food production, including spraying onto produce, application to livestock before processing, rinsing food contact surfaces in processing plants, and treating food after harvest, including RTE foods.Despite progress in improving food safety, foodborne illnesses remain a constant threat, particularly for individuals with weaker immune systems, e.g., children, the elderly, and pregnant women. Bacteriophage biocontrol can serve as an additional tool in a multi-hurdle approach to prevent foodborne pathogens from reaching consumers. This method is particularly promising when food processors wish to preserve the natural and often beneficial microbial population of food while removing the bacteria that can cause disease in humans.”
Acknowledgments
This material is based on work supported in part by the US Army Contracting Command (APG), Natick Contracting Division, Natick, MA, USA, under contract number # W911QY-18-C-0010 (to Alexander Sulakvelidze). The funders were not involved in the design of this literature review, the decision to publish, or the preparation of the manuscript.
Source: Zachary D. Moye, Joelle Woolston, and Alexander Sulakvelidze
https://www.mdpi.com/1999-4915/10/4/205/htm
https://www.mdpi.com/1999-4915/10/4/205/htm
