A mini-review on the role of bacteriophages in food safety

ABSTRACT

Despite their serious disadvantages, which include higher upfront costs, the possibility of malfunctions due to corrosiveness, and a negative impact on the organoleptic properties of the food and possibly its nutritional importance, conventional antibacterial techniques such as pasteurization, pressure preparation, and radioactive substances are also valid as synthetic antiseptics, in fact, reduce bacterial growth in food to varying degrees. Most importantly, these cleaning techniques remove all contaminants, including various (often helpful) microorganisms found naturally in food. One potential solution to some of these issues is bacteriophage bio-control, a common and inexpensive method that uses lytic bacteriophages taken from the environment to selectively target harmful bacteria and eliminate significantly reduce their stages of feeding. It has been claimed that using bacteriophages on food is a novel way to prevent the growth of germs in vegetables. This review highlights the role of bacteriophages in food safety and their advantages in detail.

KEYWORDS:

• Phages
• food safety
• antimicrobial
• green method

Introduction

Dieticians and health specialists globally promote the intake of fresh fruits and vegetables owing to their rich content of essential vitamins, minerals, and nutrients (Fan et al., 2009). Though, fresh produce remains a significant source of foodborne illnesses, with over 400 outbreaks related to produce reported since 1990, particularly associated with tomatoes, leafy greens, and sprouted seeds (Murray et al., 2017). Factors like open-field cultivation and handling add to the contamination of fruits and vegetables by microorganisms, leading to spoilage and food waste along the production process (Rawat, 2015). Human welfare and health are regularly impacted by foodborne infections, which can also occasionally result in hospitalization or even death. Between 2018 and 2019, outbreaks of Salmonella illness were detected in several US states, with the infection being linked to raw turkey products (Rashida et al., 2019). Due to the rise in the consumption of raw and undercooked chicken in recent years, particularly in China, the infection rate with Campylobacter has increased quickly (Zeng et al., 2016). A Hangzhou high school reported a severe case of Campylobacter jejuni infection in 2018, resulting in 84 pupils experiencing symptoms of foodborne disease, including vomiting and diarrhea (Yu et al., 2020).

Since the incidence of antibiotic generally drug-resistant microorganisms is increasing and the currently utilized procedures for the removal of foodborne pathogens are imprecise, new strategies against foodborne pathogens are badly needed (Moye et al., 2018). Since the beginning of the 20th century, bacteriophages have been known to be capable of eradicating specific bacteria. The initial focus of phage use was on medical cures, but it swiftly spread to several biotechnological and industrial fields. This relates to the food safety sector, where consumers are expressly put in danger by microbes (Sillankorva et al., 2012). Bacteriophages, which are bacterial viruses capable of infecting and killing specific bacterial hosts, have emerged as potential bio-regulator agents for enhancing food safety and reducing waste (O’Sullivan et al., 2019).

Bacteriophages have numerous advantages that make them valuable weapons in the fight against foodborne diseases. Because they are so specific, the product or the natural microbiota of the consumer organism (such as fermentation processes) are unaffected by the use of bacteria in the production process. Often, a single dose is adequate to achieve the desired therapeutic effect of the bacteriophage preparation. Moreover, phages can be readily isolated from their surroundings using simple, inexpensive techniques. Interestingly, and this is significant for customers, the phage addition had no effect on the product’s organoleptic, rheological, or nutritional qualities. By using bacteriophages, foodborne illnesses can be minimized, and food spoilage can be prevented, offering a promising solution to improving food safety and sustainability. Various studies have explored the application of bacteriophages as antibacterial agents to enhance microbiological food safety and reduce pathogenic and spoilage microorganisms in food products such as milk, poultry, cheeses, vegetables, and fresh fruits (Greer, 2005; Islam et al., 2022). This review highlights the prospective application of bacteriophages in regulating microbial contamination in fresh fruits and vegetables, dairy products, and convenience foods, presenting an innovative approach to ensure food safety and minimize food waste.

Foodborne illnesses: the connection to contaminated food

Foodborne illness caused by food contamination is the chief contributor to disease and mortality around the world (Murray et al., 2017). Every year, contaminated food results in 600 million foodborne illness cases and 420,000 fatalities worldwide; children under the age of five account for 40% of these deaths (Sarno et al., 2021). The widely held of these cases are attributed to specific foodborne pathogens such as Shigella, Salmonella, Campylobacter, Listeria monocytogenes, and Escherichia coli pathotypes, along with other enteric microorganisms (Murray et al., 2017; Scallan et al., 2010). The claim for fresh produce is a significant factor contributing to these incidences, often related to inadequate thermal storage and microbiologic contamination of equipment (Żaczek et al., 2015).

In addressing the emergence of bacterial resistance, bacteriophages, specialized viruses that target bacteria by rupturing their cell walls, are being explored as an alternative to antibiotics. Bacteriophages possess RNA or DNA genomes and can yield endolysin enzymes that split peptidoglycan, leading to cell wall lysis (Woźnica et al., 2015). Furthermore, the bacteriophage genome comprises proteins known as amurins, which inhibit cell wall formation, causing cell wall rupture (Woźnica et al., 2015).

Bio-control capability of bacteriophages against dietary pathogens

Since the discovery of bacteriophages by Francis Type of circuit and Walter d’Herelle a century earlier (Wittebole et al., 2014), researchers have demonstrated their potential in curing microbial enterococcus illnesses like cholera, correctly selected, and giardiasis, as well as a variety of acute or prolonged pathogens in fields such as cardiology, gastroenterology, neonatology, and multiple surgeries. These infectious agents have been used for various agricultural, animal, and human applications, but their application in local food production remains unexplored (Sillankorva et al., 2012). Contaminated food episodes associated with fresh produce have emphasized the need for concrete methods to eradicate harmful bacteria from food. However, traditional commercial sanitizers have been shown to have limitations in removing pathogens from the surfaces of fruits and vegetables (Bhardwaj et al., 2015).

To find better alternatives for ensuring bacterial exclusion on fresh produce, researchers have explored techniques such as radioactivity, consumable covering, nitrogen oxides, ultraviolet, climate-controlled storage, potassium permanganate, water, and viral proteins (Islam et al., 2022; Mahajan et al., 2014). Phages, as operative and reasonable options for organic management, do not destroy the flavor of fresh food like conventional cleaning methods do. Investigating viral formulations for the overall bio-control capability of bacteriophages against dietary pathogens linked to bug of fruits and vegetables has been a focus. However, the unexpected outcomes have posed challenges in the application of phages for phytoremediation in the native food sector, which is attributed to inadequate treatment during viral concentration and limited knowledge of bacteriophage ecology (McCallin et al., 2013). Addressing these concerns can unlock the potential of phage-based bio-control strategies in ensuring food safety.

Bacteriophage treatment in antimicrobial resistance and infectious diseases

Bacteriophages, outnumbering bacterial cells by a factor of 10 in the environment as well as the intestines of both animal and human species, comprising a vast range of host organisms. Their genome sizes vary from 3.4 kilobases (kb) to around 500 kilobases (kb), containing numerous uncharacterized genes and proteins (Romero-Calle et al., 2019). Phages undergo two discrete life cycles: lysogenic and lytic. Lysogenic phages integrate their viral genome into the host’s genetic material, while lytic phages kill infected host cells (Garvey, 2020) (Figure 1). They exhibit improved selectivity and a restricted host range, binding to host cells through various receptors, such as proteins, sugars, and lipopolysaccharides (Batinovic et al., 2019).

Figure 1. Application of bacteriophages as Antimicrobial.

Phages have a long history of being considered for the management of infectious diseases before the development of antibiotics. Recognized by microbiologist Felix d’Herelle in 1917, phages were primarily explored for their antibacterial activity against dangerous germ cells (Fruciano & Bourne, 2006). Phage therapy showed promising results in controlling diseases like bacillary dysentery and cholera outbreaks (Braga et al., 2020). However, the discovery of antibiotics in the 1930s and 1940s led to a decline in phage research. Challenges such as inconsistent findings, dosages, repeatability, and limited genetic information hindered further exploration (Garvey, 2020) (Figure 1).

Recent resurgence in phage research comes as an unconventional route to combat the growing threat of antimicrobial resistance (AMR). In Eastern Europe, phage treatment has been effectively used for around 90 years without posing health risks to patients (Tang et al., 2019). In countries like Poland, bacteriophage treatment has proven effective against AMR diseases (Selle et al., 2020). As current therapeutic methods falter, bacteriophage therapy offers a promising option, particularly in managing infections like Clostridioides difficile infection (CDI) with high fatality rates. While challenges remain, bacteriophage treatment presents a solution to combat AMR and infectious diseases.

Life cycles of bacteriophages

Through certain receptors on the bacterial host cell, bacteriophages identify their host. The bacteriophage injects its genetic material into the host after attaching. Phage genome can either stay as unintegrated extragenomic genetic material or integrate into the host genetic material once it becomes intracytosolic. Whereas the latter life cycle is lytic, the first is lysogenic. Viral phages have a lytic life cycle and seize control of the host’s replication processes in order to multiply into mature virus particles and eventually cause the host cell to lyse. Reverting to lysogeny, temperate phages incorporate into the host genetic material until a mutagenic stimulus induces them into a lytic life cycle. Because they are restricted to the host in which they are incorporated, they are also highly host specific than virulent ones as shown in Figure 2.

Figure 2. Bacteriophages life cycles.

Phage-based biocontrol of food pathogens

Several experimental studies have investigated the use of phages to control pathogens in various food products and leafy green vegetables as described in Table 1. This indicates that bacteriophages are a promising regulator of food-borne pathogens. The table below describes the process of phage-regulation and their effects on pathogenic bacteria.

Table 1. Application of bacteriophages in regulating food-borne pathogens.

Food	Bacteria targeted	Bacteriophage used	Effects	References
Beef	E. coli O157	EP75 and EP335	Reductions of 0.8–1.1 log10 CFU/cm^2 and 0.9–1.3 log10 CFU/cm^2, respectively.	Witte et al. (2022)
Raw meatball	E. coli O157:H7	M8AEC16	A reduction of 0.69–2.09 log10 CFU/g after 5 h of application.	Gencay et al. (2016)
Beef and lettuce	E. coli O157:H7	EcoShield™	Reduced the level of bacteria by ≥94% and 87% after 5 min contact time in meet and lettuce, respectively.	Carter et al. (2012)
Beef	E. coli O157:H7	PS5/Myoviridae	A 2.4 log10 CFU/piece after 24 h post application at 4°C, whereas a 3.5 log10 CFU/piece after 6 h post application at 24°C.	Duc et al. (2020)
Chicken	S. Typhimurium	PS5/Myoviridae	A 1.2 log10 CFU/piece after 24 h post application at 4°C and a 1.6 log10 CFU/piece after 6 h post application at 24°C.	Duc et al. (2020)
Beef (coarse and fine ground)	S. enterica (ATCC 51,741), S. Heidelberg (ATCC 8326), S. Newport (ATCC 27,869), and S. Enteritidis C (Se 13)	Salmonelex™ (S16 and the FO1a)/Myoviridae	Overall, a reduction of 1.6 log10 CFU/g was observed after the application of 10^9 phage.	Shebs-Maurine et al. (2021)
Ground red meat trim and poultry	S. Infantis (ATCC 51,741), S. Heidelberg (ATCC 8326), S. Newport (ATCC 27,869), and S. Enteritidis (SE13)	Salmonelex™ (S16 and the FO1a)/Myoviridae	Overall, phage application on trim reduced 0.8 and 1 log10 CFU/g of Salmonella in ground pork and beef, respectively, whereas a reduction of 0.9 and 1.1 log10 CFU/g occurred in ground turkey and chicken, respectively.	Yeh et al. (2017)
Chicken skin	Cocktail of S. Typhimurium, S. Heidelberg, and S. Enteritidis	SalmoFresh™	A reduction of 0.9–1 log10 CFU/cm^2 with phage only. Whereas a greater reduction of 1.6 and 1.8 log10 CFU/cm^2 after 2 and 24 h. after chlorine and phage treatment.	Sukumaran et al. (2015)
Chicken	S. Typhimurium, S. Newport, S., and Thompson	Salmonelex™	A reduction of 0.39 log10 CFU/cm^2 and 0.67 log10 CFU/cm^2 after 30 min and 8 h post-inoculation, respectively.
Meat	L. monocytogenes	Halal-certified List-shield	A reduction of 2.3 log10 was recorded in phage-treated beef samples during the storage period of 15 days.	Ishaq et al. (2020)
Fresh salmon meat	L. monocytogenes	SH3–3/Myoviridae	A reduction of 2.67, 4.14, and 4.54 log10 after 24, 48, and 72 h of phage addition, respectively.	Zhou et al. (2020)
Chicken	Cocktail of L. monocytogenes strains ATCC 19,113, ATCC19115, and ATCC 13,932	ListShield	A mean reduction of 0.56, 0.84, 0.46, and 0.10 log cycles in viable counts was observed at 0, 24, 48, and 72 h after phage treatment, respectively.	Yang et al. (2017)
Cooked turkey and roast beef	A cocktail of L. monocytogenes (serotypes; 1/2a, 1/2b, and 4b)	LISTEX™P100	An initial reduction of 2.1 and 1.7 log10 CFU/cm^2, respectively, for cooked turkey and roast beef at 4°C, while an initial reduction of 1.5 and 1.7 log10 CFU/cm^2, at 10°C.	Chibeu et al. (2013)
Raw chicken and pork meat	C. jejuni (NCTC 11,168) and C. coli (NCTC 12,668)	NCTC group II phage 12,684 or CP81	No reduction at 4°C after 7 days of inoculation.	Orquera et al. (2012)
Raw and cooked beef	C. jejuni	Cj6/Myoviridae	No reduction at 5°C compared to control with low MOI. However, a 2 log10 CFU/cm^2 reduction on raw and cooked meat at high host density and a high MOI of 10,000.	Bigwood et al. (2008)
Chicken	C. jejuni (NCTC12662 or RM1221)	F356 and F357	A 0.73 log10 reduction at 5°C after 24 h post-treatment.	Zampara et al. (2017)
Chicken liver	C. jejuni (HPC5 and 81–176)	Phages ϕ3 or ϕ15/Myoviridae	A 0.2 to 0.7 log10 CFU/g reduction 48 h post-treatment.	Firlieyanti et al. (2016)
Lettuce	Salmonella ser. Enteritidis (ATCC13076) and Salmonella ser. Typhimurium (ATCC14028)	BP 1369 and BP 1370/Myoviridae and Podoviridae, respectively	A reduction of >1.0 log10 CFU/cm^2 after 2 h of post-treatment.	Sadekuzzaman et al. (2018)
Romaine lettuce	Individual strains of STEC (EDL933; O157:H7, SN061; O26: H11, SN576; O111:NM and SN608; and O103:H2)	VE04, VE05, and VE07	A reduction of 2.6–6 log10 CFU/cm^2 after 3 days of storage at a temperature of 10°C.	Lu et al. (2022)
Romaine lettuce, mung bean sprouts, and seeds	Cocktail of Salmonella strains (Newport, Braenderup, Typhimurium, Kentucky, and Heidelberg	SalmoFresh™/Myoviridae	Overall reduction by spraying SalmoFresh™ onto lettuce and sprouts reduced Salmonella by 0.76 and 0.83 log10 CFU/g, respectively, whereas a reduction of 2.43 and 2.16 log10 CFU/g by immersion was observed on lettuce and sprouts, respectively.	Zhang et al. (2019)
Romaine and iceberg lettuce	E. coli O157:H7	AYO26, AXO111, AXO121, AYO145A/Myoviridae, AXO103, AKFV33/Siphoviridae, and AXO45B	A reduction of 2.6–3.2 and 1.7–2.3 log10 CFU/g for low and high contamination, respectively.	Ding et al. (2023)

Benefits and drawbacks of using phages as antimicrobials

Phages, as antimicrobials, have both strengths and limitations as shown in Table 2. Their specificity in targeting diverse bacterial strains can be challenging, especially when dealing with illnesses caused by multiple strains. While some trials have shown the safety of oral phage administration, a key concern is ensuring proper translocation of phages through the intestinal epithelium. Studies have indicated that this translocation can be beneficial by directing the immune response to innate microbial antigens and averting the development of certain inflammatory factors (Ranveer et al., 2024). However, other research did not observe significant changes in cytokine levels after phage therapy. In spite of limited data on phage treatment, it appears to have fewer side effects than conventional antibiotics and can reduce pathogenic flora in the gut.

Table 2. Benefits and drawbacks of using bacteriophages as antimicrobials.

Benefits	Drawbacks	References
Phages infect only one type of bacteria, making consumer dysbiosis unlikely due to their extreme specificity.	To meet the rising demands of the food industry, phage, and phage cocktail manufacturing must be scaled significantly.	Garvey (2020); Culot et al. (2019)
Phages barely affect the organoleptic qualities of food.	To guarantee the right administration of the proper phage or phage cocktail, accurate prediction of the current pathogens is essential.	Moye et al. (2018); Thanki et al. (2021)
A rather high level of resistance to various food preservation techniques is displayed by phages.	There is still disagreement on the stability of phages during food storage.	Garvey (2020); Greer (2005)
Phages have tremendous efficacy, requiring just a tiny amount to kill germs.	Bacterial phage resistance is frequently erratic and can evolve over time.	Garvey (2022); Salmond and Fineran (2015)
Against bacterial biofilms, phages have demonstrated effectiveness.	At high temperatures, phages are prone to denaturation.	Liu et al. (2022); Garvey (2022)
The term “Generally Recognised as Safe” (GRAS) is now used to describe a few goods.	The amount of chlorine in water can have an impact on the effectiveness of phages.	Vikram et al. (2021); Zhang et al. (2019)
Phages have the ability to replicate themselves, making minimal dosages necessary for effectiveness.	Pathogens can emit pro-inflammatory substances such endotoxins and peptidoglycans when they are lysed.	Tang et al. (2019)
Phages are appropriate for both pre-and post-harvest situations due to their wide variety of applications.	Toxins carried by bacteriophages include pathogenicity islands, diphtheria toxin, cholera toxin, botulism toxin, and diphtheria toxin.	Imran et al. (2023); Sisakhtpour et al. (2022)
Phages are thought of as eco-friendly technology that is biocompatible with both people and animals.	The structure and properties of the food matrix may have an impact on how well bacteriophages work.	Garvey (2020); Vikram et al. (2021)
The efficacy of phages against MDR bacterial species has been established.	Bacterial endotoxins might possibly be present in crude phage lysates.	Sisakhtpour et al. (2022); Villa et al. (2019)
Bacterial endotoxins might possibly be present in crude phage lysates.		Lawpidet et al. (2021)
Possible cures and preventative measures for Clostridioides difficile (C. difficile) infections might be provided by phages.		Giau et al. (2019)

The regional specificity of phages allows for the selection of phages with the best infectivity contrary to target pathogens, making them potentially valuable in combating antibiotic-resistant bacteria, particularly in hospital settings (Ali et al., 2023). Additionally, phages carry enzymes that can break down bacterial biofilms and extracellular polymeric materials, providing an advantage over antibiotics, which are often ineffective against biofilm-forming bacteria (Liu et al., 2022). Nevertheless, further investigation is needed to completely understand and harness the potential of phages as antimicrobials.

Conclusion and future perspectives

Despite improvements in safety procedures for food, adulteration of fresh fruits and vegetables stays a significant issue. Pathogenic and deteriorative bacteria can undermine product quality and contribute to food waste. Current research in food microbiology has highlighted the effectiveness of bacteriophages in averting the growth of harmful bacteria on fresh produce. Bacteriophages are advantageous at various points of the food manufacture chain. They offer a promising auxiliary tool to combat foodborne infections, particularly concerning vulnerable populations like children, the elderly, and expectant mothers. Phages are strong, definite, and self-replicating pillagers of bacterial species, making them valuable for disease mitigation, farm-level disinfection, and food preservation. They are increasingly recognized as GRAS-Generally Recognized as Safe for use in food products and are considered organic and legitimate. Phage cocktails, in particular, have shown remarkable activity to counter Multi-Drug Resistant (MDR) species and can be combined with other safe antimicrobials, like bacteriocins, to augment effectiveness and selectivity. However, issues that require further investigation include phage resistance mechanisms, potential transmission of pathogenic genes, phage traceability in the environment, and formulation and stability challenges for therapeutic applications. Overall, Bacteriophages are essential components of ecosystems, playing a crucial character in bacterial evolution. Their use as biocontrol agents during before-harvest, harvest, and after-harvest stages provides several benefits for refining food safety and sustainability, aligning with the Sustainable Development Goals (SDGs).

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All authors are willing for publication of this manuscript.

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Acknowledgements

Authors are thankful to Government College University for providing literature collection facilities.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets generated used and/or analyzed during the current study available from the corresponding author on reasonable request.

Additional information

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.