Use of Bacteriophages to Control Escherichia coli O157:H7 in Domestic Ruminants,Meat Products,and Fruits and Vegetables

Abstract

Escherichia coli O157:H7 is an important foodborne pathogen that causes severe bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Ruminant manure is a primary source of E. coli O157:H7 contaminating the environment and food sources. Therefore, effective interventions targeted at reducing the prevalence of fecal excretion of E. coli O157:H7 by cattle and sheep and the elimination of E. coli O157:H7 contamination of meat products as well as fruits and vegetables are required. Bacteriophages offer the prospect of sustainable alternative approaches against bacterial pathogens with the flexibility of being applied therapeutically or for biological control purposes. This article reviews the use of phages administered orally or rectally to ruminants and by spraying or immersion of fruits and vegetables as an antimicrobial strategy for controlling E. coli O157:H7. The few reports available demonstrate the potential of phage therapy to reduce E. coli O157:H7 carriage in cattle and sheep, and preparation of commercial phage products was recently launched into commercial markets. However, a better ecological understanding of the phage E. coli O157:H7 will improve antimicrobial effectiveness of phages for elimination of E. coli O157:H7 in vivo.

Introduction

E scherichia coli O157:H7 is an important highly virulent zoonotic and foodborne pathogen that can cause severe bloody diarrhea and a life-threatening condition known as hemolytic uremic syndrome (HUS) (Riley et al., 1983). Since the first outbreak reported in 1983 (associated with eating hamburgers at fast-food restaurants located in the United States; Riley et al. 1983), more than 60% of O157 cases require hospitalization (Nelson et al., 2011) and ∼15% of cases may progress to HUS, which is most commonly seen in children less than 10 years of age (Karch et al., 2005). From 1982 to 2002, 325 outbreaks have occurred, and the ages reported at death were between 1–4 years and 61–91 years (Rangel et al., 2005).

Infection by E. coli O157:H7 has become a very alarming foodborne disease throughout the United States, the United Kingdom, Canada, Japan, France, and various other countries (Money et al., 2010; Bavaro, 2012; King et al., 2014; Terajima et al., 2014; Gaulin et al., 2015). Outbreaks are most commonly associated with the consumption of undercooked meat. Ground beef remains the most common pathway among E. coli O157:H7 outbreaks (Rangel et al., 2005). E. coli O157:H7 has also been found in pigs (Doane et al., 2007; Lenahan et al., 2009a; Wyrsch et al., 2015), chickens (Doane et al., 2007), turkeys (Doane et al., 2007), wild animals (Sánchez et al., 2010; Navarro-Gonzalez et al., 2015), seafood (Surendraraj et al., 2010), and leafy vegetables (Marder et al., 2014). However, healthy cattle (Doane et al., 2007; Lenahan et al., 2009b; Cabal et al., 2013) and other ruminants (Lenahan et al., 2009b; Söderlund et al., 2012; Gencay, 2014; Kamel et al., 2015) are also important natural reservoirs of E. coli O157:H7.

E. coli O157:H7 infection is associated with higher risk of HUS than other pathotypes of E. coli (Bavaro, 2012). Antimicrobial drug treatment is not a recommended treatment (Davis et al., 2013) as it causes an increase in stx production, particularly those antimicrobials that interfere with DNA synthesis (Kimmitt et al., 2000). In contrast, antibiotics that are widely used as feed supplements to promote growth and to prevent infection in livestock (Kim et al., 2016) contribute to the horizontal transfer of stx genes and diversification of E. coli O157:H7. Therefore, the need for alternatives to antimicrobial drugs for human and animal use has been further accentuated. In this study, the application of bacteriophage for the control of E. coli O157:H7 has been reviewed, establishing a scientific reference for the application of phages toward improving the safety of animal-derived foods.

Basic knowledge about bacteriophages

Bacteriophages (phages) were first discovered in 1915 by English bacteriologist Fredrick Twort (1915) and again in 1917 by French Canadian Scientist Felix d'Herelle (1917). Following their discovery, phages were applied in clinics as antimicrobial agents during the 1920s (Huff et al., 2005) and were used to control avian typhoid caused by certain serovars of Salmonella such as Salmonella Typhimurium and Salmonella Enteritidis (Atterbury et al., 2007). Human applications soon followed and between 1930 and 1950, phages were commonly used in therapeutics, particularly in the former Soviet Union (Georgia and Russia), Poland, the United States, and China (Summers, 2004; Johnson et al., 2008; Chen, 2010). However, because of the discovery and extensive application of penicillin, phage therapy was substituted throughout the world by antibiotic treatment with exception of some countries in the former Soviet Union, Poland, and to a lesser extent India (Wittebole et al., 2014). In recent years, the increasing occurrence of bacterial resistance to antibiotics such as extended-spectrum-lactamase-producing E. coli (Ahmad et al., 2015), vancomycin-resistant Enterococci (Otawa et al., 2012), and methicillin-resistant Staphylococcus aureus (Abdulamir et al., 2015; Jensen et al., 2015) has stimulated research on phages as novel antibacterial agents.

Phages are the most abundant entities in the world. It is estimated that the global abundance of phages is about 10³¹, which is tenfold more abundant than bacteria (Kutter and Sulakvelidze, 2004). These bacterial viruses have genetic material in the form of either DNA or RNA encapsulated by a protein coat (Clark and March, 2006). Phages can be divided into two groups, including lytic phages and lysogenic phages, according to their replication cycles. Lytic phages multiply by means of a lytic cycle in which the phage particle adheres to the host bacterial cell surface, injects genomic material, and takes over the host's metabolic system, resulting in intracellular phage proliferation. At the end of the cycle, the host bacteria are lysed and the phage progenies are liberated within a very short time. Typically, this will result in the release of hundreds of new, infectious virus particles within minutes or hours (Harper and Enright, 2011). In contrast, lysogenic phages undergo a lysogenic cycle, in which the phage DNA is incorporated directly into the host bacterial chromosome. The bacterial host cells are not lysed and no progeny phages are produced.

Phages have many advantages over antibiotics as antimicrobials (García et al., 2008). These advantages include having a higher specificity on the intended bacteria without disturbing the normal flora in the microenvironment, easier isolation of lytic phages because of their abundance in nature, having exponential proliferation ability, and finally existing and disappearing within their host bacteria. At present, phages are being reassessed for their ability to prevent and treat bacterial infections in humans (Maura and Debarbieux, 2011), livestock (Johnson et al., 2008; Atterbury, 2009), and plants (Balogh et al., 2010), in addition to decontaminating processed foods and agricultural products (Naylor et al., 2003; Teunis et al., 2004). The following review is focused on recent research in the application of phages for the treatment and prevention of E. coli O157:H7 infections in cattle and sheep as well as the contamination of common livestock and agricultural food products such as meats, fruits, and vegetables.

Use of Bacteriophages to Control E. Coli O157:H7

Due to its low infectious dose and high pathogenicity, E. coli O157:H7 is considered to be one of the main pathogens threatening food safety (Teunis et al., 2004). Ruminant animals and their rearing environment are the main sources of E. coli O157:H7 and the origins of infection (Naylor et al., 2003). At present, efforts for reducing E. coli O157:H7 contamination in food products have largely focused on washing with water and the application of numerous antimicrobial chemicals and γ-irradiation, all of which have practical and environmental drawbacks (Carter et al., 2012). As a consequence, lytic phages, as environment-friendly antimicrobial agents, are receiving increased attention.

Use of bacteriophages in ruminant rearing environments

The prevalence of phages can fluctuate with the amount of E. coli O157:H7 presented in cattle feedlots (Niu et al., 2009). Bacteriophages that infect E. coli O157:H7 were detected more frequently (p < 0.001) after 18–20 h of sample enrichment in tryptic soy broth than during initial screening and were recovered in 239 of 855 samples. Manure slurry showed the highest prevalence (p < 0.001) when compared with other environmental sources of pooled fecal pats, fecal grabs, and water trough samples. The likelihood of fecal shedding of E. coli O157:H7 was reduced if the cattle in the pen harbored the phage (Niu et al., 2009).

E. coli O157:H7-specific phages, such as T4- and O1-like phages of Myoviridae and T1-like phage of Siphoviridae, were widely isolated from cattle supershedders who shed high levels (≥10⁴ colony-forming units [CFU]/g of feces) of pathogenic organisms such as E. coli O157:H7 in their manure and also low shedders who shed low levels (<10⁴ CFU/g of feces) of pathogenic organisms (Oot et al., 2007; Hallewell et al., 2014; Munns et al., 2015). T4-like phages were more frequently isolated from the feces of low shedders than that of supershedders, suggesting that endemic phages may impact the shedding dynamics of E. coli O157:H7 in cattle. This indicates that targeting supershedders for mitigation of E. coli O157:H7 may be a means for reducing the incidence and spread into the environment. Thus, adding phages specific for E. coli O157:H7 to the rearing environment has the potential to further reduce the population of the pathogen in cattle.

Hides and gastrointestinal tracts of preslaughter ruminants carry a large number of bacteria and the large amounts of feces shed in livestock farms could further contaminate the hides of the animals. Coffey et al. (2011) used a phage cocktail consisting of e11/2 and e4/1c with multiplicities of infection (MOIs) of 1000 and 10,000 to treat cattle hide pieces (20 × 20 cm) after inoculation of ∼10⁶ CFU/cm² of E. coli O157:H7. When hide samples were left for 1 h post-treatment, the numbers of E. coli O157:H7 for the phage-treated pieces were significantly (p < 0.05) lower than numbers obtained from water washing and no washing treatments (Coffey et al., 2011).

These abovementioned in vitro studies demonstrate that phages are effective on E. coli O157:H7 under various environmental conditions, illustrating that bacteriophages can reduce the prevalence of E. coli O157:H7 in ruminant rearing environments. However, further research is necessary to optimize and improve the application of various phages to environments for the control of E. coli O157:H7.

Use of bacteriophages in cattle

In vitro studies have shown remarkable effects of bacteriophages on E. coli O157:H7 (O'Flynn et al., 2004; Niu et al., 2009; Coffey et al., 2011; Carter et al., 2012). The potential of phage therapy against E. coli O157:H7 in ruminants was further supported by in vivo work in mice (Tanji et al., 2005; Capparelli et al., 2006; Sheng et al., 2006). Subsequently, the effectiveness of bacteriophage treatment to decrease E. coli O157:H7 in cattle or other ruminants has been reported and is shown in Table 1.

Table 1.

Effect of Phage Therapies on Ruminants

Ruminant	Age and weight	E. coli O157:H7strain	Bacterial amount (CFU)	Phage name	Dose of phage (PFU)	Therapeutic method	Phage application time	Examination time	Examining locations	Reduction(CFU)	p	Ref.
Crossbred ewes		ATCC 43895	10¹⁰	CEV1	10¹¹	Orally	On day 3 after bacterial inoculation	2 days after phage treatment	Intestines	10²–10³	>0.05	Raya et al. (2006)
Wether lambs	4 months	E318N	10⁸	DC22	10¹³	Orally	On day 2 after bacterial inoculation	Over 30 days	Fecal	UD	>0.05	Bach et al. (2003)
Sheep	Average 60 kg body weight	ATCC 43895	10¹⁰	Cocktail of eight phage isolates	10⁹	Orally	On day 2 after bacterial inoculation	24 h after phage treatment	Fecal	10⁴	<0.05	Callaway et al. (2008)
Suffolk ewes	7 months	ATCC 43894	3.5 × 10¹⁰	KH1	1.3 × 10¹¹	Orally	On day 0	8 days after phage treatment	Fecal	10⁹–10¹⁰	0.74	Sheng et al. (2006)
Steers	6 months	ATCC 43894, WSU180, WSU400, and WSU588	10⁶	KH1 and SH1	10¹⁰	Rectally	Phages were applied on days 0, 1, 2, and 4	On days 0, 1, and 7	Rectoanal junction	1–2 log, 3.6 log, 4.6 log	<0.05	Sheng et al. (2006)
Steers	Yearling, 386 ± 29 kg	R508N, CO281-31N, E32511, H4420N, and E318N	2 × 10⁹	Cocktail (wV8, rV5, wV7, and wV11)	3.3 × 10¹¹	Orally	On days −2, 0, 2, 6, and 9	On days 13, 48, and 55	Fecal	3.5 log, 4.4 log, 5 log	<0.01	Niu et al. (2008)
Cattle	Yearlings, adult fistulated cattle (8–9 years)	NCTC 12900, DAF454, and 13C1T3	10¹⁰	Cocktail (e11/2 and e4/1c)	10¹¹	Orally	On days 1, 2, and 3	At 92 h	Fecal and rumen	UD	>0.05	Rivas et al. (2010)
Angus steers	386 ± 29 kg	E318N, R508N, CO281-31N, E32511, and H4420N	5 × 10¹⁰	Cocktail (rV5, wV7, wV8, and wV11)	10¹¹	Oral and rectal	On days −2, 0, 2, 6, and 9	Over 83 days	Fecal	10¹⁰	>0.05	Rozema et al. (2009)
Steers	391 ± 24 kg	E318N, R508N, CO281-31N, E32511, and H4420N	10¹¹	Cocktail (wV8, rV5, wV7, and wV11)	10⁹	Orally	On days 1, 1, 3, 6, and 8	Over 10 weeks	Fecal	UD	>0.05	Stanford et al. (2010)

CFU, colony-forming units; DNS, data not shown; NS, not significant; PFU, plaque-forming units; UD, undetectable.

Phage cocktails (e11/2 and e4/1c) of 10¹¹ plaque-forming units (PFU) per milliliter were dosed daily for 3 days after orally inoculating cattle with 10¹⁰ CFU of E. coli O157:H7 (Rivas et al., 2010). The numbers of E. coli O157:H7 shed in cattle fecal samples declined rapidly within 24–48 h, but no significant difference (p > 0.05) was observed between the phage-treated and control animals (simultaneously reported in the ex vivo ruminant model [P < 0.05]). Rivas et al. (2010) suggested that the most likely explanation could be the lack of exposure to phages (MOI = 10). Therefore, a high MOIs (>100) of 10¹⁰ PFU phages (SH1 and KH1) were applied directly to the mucosa of the rectoanal junction of Holstein steers 7 days after rectal application of E. coli O157:H7 (Sheng et al., 2006). Additionally, phages were maintained at 10⁶ PFU/mL in the drinking water of the phage-treated group. This phage therapy reduced the average number of E. coli O157:H7 CFU (counted by rectoanal mucosal swab [RAMS]) among phage-treated steers when compared with control steers (p < 0.05). However, the bacteria were still present within the majority of steers investigated.

According to Rivas et al. (2010), the effect of the administration method on the efficacy of phage strategy for preharvest control of E. coli O157:H7 in feedlot cattle may cause differences in the shedding levels and/or colonization patterns in the treated cattle. Niu et al. (2008) compared sampling methods, both fecal grabs (FECs) and RAMS, for surveillance of nalidixic acid-resistant (NalR) E. coli O157:H7. During the 83-day sampling period, more FEC samples were positive for NalR E. coli O157:H7 than were RAMS samples after oral bacteriophage treatment, both within (p ≤ 0.02) and across (p < 0.01) bacteriophage treatment groups. A trend toward a treatment effect (oral bacteriophage vs. control) on the proportion of positive samples was observed among RAMS (p = 0.08), but not among FEC (p = 0.27), samples, suggesting an interaction between treatment and sampling method. Simultaneously, the effects of oral or rectal administration of a four-strain O157-specific bacteriophage cocktail for mitigating fecal shedding of NalR E. coli O157:H7 were monitored over 83 days in cattle after oral (3.3 × 10¹¹ PFU), rectal (1.5 × 10¹¹ PFU), both oral and rectal (4.8 × 10¹¹ PFU), or no treatment on days −2, 0, 2, 6, and 9 (Rozema et al., 2009). Although there was a brief increase in shedding by each group, levels of NalR E. coli O157:H7 declined from day 49 until completion of the experiment. However, no significant differences were observed between phage treatments or control groups.

The results gained by the different research groups on the efficacy of oral phage therapy against E. coli O157:H7 in cattle were unexpected. Since bacteriophages lose activity and effectiveness at a low pH, protecting phages from gastric acidity may enhance the efficacy of orally administered phages. Two polymer-encapsulated phages (Ephage) were developed to evaluate their efficacies in eliminating NalR E. coli O157:H7. However, Ephage did not reduce the shedding of NalR E. coli O157:H7, although the duration of shedding was reduced by 14 days (p < 0.1) in gelatin capsule-fed steers compared with control steers (Stanford et al., 2010). These studies indicate that further research is necessary to improve the efficacy of bacteriophages for the control of E. coli O157:H7 in cattle.

Use of bacteriophages in sheep

Several studies regarding E. coli O157:H7 phage therapy intended for sheep serve as a convenient model for controlling this organism. While Sheng et al. (2006) investigated the effect of E. coli O157:H7 phage on cattle (Use of bacteriophages in cattle), during the same period, phage KH1 was also used to reduce E. coli O157:H7 inoculation in sheep. Results showed that phage KH1 was not effective in three 7-month-old Suffolk ewes when given oral doses (1.3 × 10¹¹ PFU) at days 1, 9, 10, and 11 after infection with E. coli O157:H7, despite the presence of 10⁵ to 10⁶ PFU/g phage excreted in feces during this time (Sheng et al., 2006). Similarly, Bach et al. (2003), although using 10⁹ PFU of E. coli O157:H7-specific phage DC22, completely eliminated 10⁴ CFU of E. coli O157:H7 from the artificial rumen system (Rusitec) after 4 h of administration compared with 168 h for the control group (p < 0.05). In vivo tests showed that there was no difference (p > 0.05) in the levels of E. coli O157:H7 (10⁸ CFU) shed by lambs in the DC22-treated group (10¹³ PFU) or control group over the 30-day period (Bach et al., 2003).

Furthermore, Raya et al. (2006) additionally reported a reduction in the level of novobiocin/NalR E. coli O157:H7 by phage CEV1. Four sheep were treated with a single oral dose of CEV 1 (∼10¹¹ PFU) 3 days after being given with ∼10¹⁰ CFU of E. coli O157:H7. Two days post, bacterial counts of ruminal, cecal, and rectal contents showed that E. coli O157:H7 levels had reduced 2–3 log units in the cecum and rectum, but not in the rumen of CEV1-treated sheep when compared with controls. Unfortunately, no significant reductions of E. coli O157:H7 (p > 0.05) were found in this study. Five years later, the same group (Raya et al., 2011) reported that the combined application of E. coli O157:H7-specific phages, CEV1 and CEV2, led to a significant reduction (>99.9%; p < 0.05) in E. coli O157:H7 levels throughout the lower intestinal tract when compared with the untreated control. The greatest reduction (nearly 4 logs) was observed in those sheep in which CEV2 was naturally present and where they had been exposed for 5 days to high levels of host E. coli O157:H7. Moreover, none of the sheep showed any adverse effects from phage administration or colonization. Similarly, Callaway et al. (2008) reported that a cocktail of eight phages could significantly reduce the populations of fecal E. coli O157:H7 strain 24 h after phage treatment. However, the reduction did not approach statistical significance (p < 0.09) in the rumen.

These few available reports demonstrate the potential for the use of phage therapy to reduce E. coli O157:H7 carriage in cattle and sheep. Several studies have reported significant reductions in E. coli O157:H7 levels in ruminants after phage treatment (Sheng et al., 2006; Callaway et al., 2008; Niu et al., 2008; Raya et al., 2011). However, other unsuccessful attempts to use oral or rectal phage therapy to clear intestinal E. coli O157:H7 from ruminants suggest that a better understanding of phage E. coli O157:H7 ecology is required to improve the antimicrobial effectiveness of phages for elimination of E. coli O157:H7 in vivo. Unfortunately, the commercial application of bacteriophage therapy is likely still several years away (Sheng et al., 2006), and widespread use of bacteriophages for treatment of foodborne pathogens in animals will additionally require both regulatory approval and consumer acceptance.

Harvest Control in Meat, Processed Meat, and Fresh Produce

During the process of meat production, carcasses are exposed to the ware, conveyer belt, and chopping board and any contamination of the above devices will affect overall sanitation of the meat. At the same time, freshly processed fruits and vegetables can also be contaminated by E. coli O157:H7 from similar means, becoming the source of foodborne disease.

Use of bacteriophages on meat

Research studies have shown that lytic bacteriophages specific for E. coli O157:H7 are effective in reducing pathogen populations on meat products. The effectiveness of phage treatment on raw and ready-to-eat meat is shown in Table 2, and a low MOI means that fewer bacteriophages are needed to cause bacterial cell reductions.

Table 2.

Effect of Phages on Raw and Ready-to-Eat Meat

Commodity	E.coli O157:H7 strain	Phage	MOI	Application method	Temperature, °C	Examination time (showed significant reducing)	Pathogen reduction	p	Ref.
Beef	Mixture of Ec229, Ec230, and Ec231	ECP-100 cocktail (ECML-4, ECML-i 17, and ECML-134)	1	Spray	10	24 h after inoculation	95%	<0.05	Abuladze et al.(2008)
Raw beef	C918	Cocktail (EcoM-AG2, EcoM-AG3, and EcoM-AG10)	10⁶	MCM^a	10	4 days after inoculation	<1 log CFU	DNS	Anany et al. (2011)
Raw beef	C918	Cocktail (EcoM-AG2, EcoM-AG3, and EcoM-AG10)	10⁶	MCM	4	6 and 9 days; 12 and 15 days after inoculation	1 log CFU; below the detection limit (≈2 log CFU)	<0.05	Anany et al. (2011)
Raw and cooked ready-to-eat beef	NZRM 3647	FAHEc1	10⁴	Spotted	5 and 24	24 h after inoculation	>4 log10 CFU/cm²	<0.01	Hudson et al. (2015)
Steak meat	P1432	Cocktail (e11/2 e4/1c and pp01)	10⁵	Spotted	Room temperature	1 h after inoculation	5 log CFU	DNS	O'Flynn et al. (2004)
Ground beef	309	Cocktail (FFH1, FFH2, and FFH3)	1	Drop application	24, 4, and 46	24 h after inoculation	1.97, 0.48, and 0.56 log CFU/mL	<0.05	Hong et al. (2014)
Beef	464	Cocktail (DT1 and DT6)	3.33 × 10⁵	Spotted	24	3, 6, and 24 h after inoculation	1.43 ± 0.24, 2.58 ± 0.21, and 2.20 ± 0.22 log 10 CFU/mL	0.01	Tomat et al. (2013)
Beef	464	Cocktail (DT1 and DT6)	1.56 × 10⁵	Spotted	5	3, 6, and 24 h after inoculation	4 log CFU	NS	Tomat et al. (2013)
Raw beef	ERL 022447	FAHEc1	2 × 10⁷ PFU/piece/1.4 × 10⁴ CFU/piece	Spotted	37	1 h after inoculation	>2 log 10 CFU/piece	<0.01	Hudson et al. (2013)
Beef	Mixture of 2886–75, G5101, and 93–111	EcoShield™	1000	Spray	4	5-min contact time	>94%	<0.05	Carter et al. (2012)

MCM containing immobilized phages.

CFU, colony-forming units; DNS, data not shown; MCM, modified cellulose membranes; MOIs, multiplicities of infection; NS, not significant; PFU, plaque-forming units.

A combination of three E. coli O157:H7-specific Myoviridae bacteriophages (ECP-100) and a control of sterile phosphate-buffered saline (PBS) buffer was sprayed on ground beef samples that were contaminated by a mixture of E. coli O157:H7. The application of ECP-100 significantly reduced (p = 0.007) the initial E. coli O157:H7 populations by ∼3400 CFU on the ground beef samples and nearly 95% reduction during storage at 10°C for 24 h after spray application (Abuladze et al., 2008). This same research group later found that bacteriophages specific for E. coli O157:H7 significantly (p < 0.05) reduced the level of E. coli O157:H7 on contaminated beef by ≥94% after 5 min of contact time (Carter et al., 2012). These reduced levels of bacteria were maintained for at least 1 week at refrigerated temperatures.

Hong et al. (2014) selected two Myoviridae and one Siphoviridae-specific phage for E. coli O157:H7 to prepare a cocktail (MOI = 1). The concentrations of E. coli O157: H7 (10⁷ CFU) in the ground beef treated with phages were reduced by 1.97 log CFU/mL when stored at room temperature (24°C) for 24 h (p < 0.05), 0.48 log CFU/mL with refrigeration (4°C) (p < 0.05), and 0.56 log CFU/mL in an undercooked condition (internal temperature of 46°C) (p < 0.05). Similarly, Hudson et al. (2013) isolated a Myoviridae phage (FAHEc1) that lysed 28 of 30 E. coli O157 tested. When this phage was applied to beef pieces under conditions simulating hot deboning and conventional carcass cooling, a reduction in E. coli O157:H7 (∼2 log) was observed under optimal conditions with phages used at 8 × 10⁶ PFU/cm² per piece. Additionally, E. coli O157:H7 phage FAHEc1 was added to both raw and cooked beef pieces at concentrations ranging from 10¹ to 10⁸ PFU/cm² to either low (<10² CFU/cm²) or high (10⁴ CFU/cm²) concentrations of host bacterial cells. After incubation for 24 h, the concentration of bacterial cells was significantly reduced by >4 log CFU/cm² at both 5°C and 24°C in comparison with the control, with a general observed trend for a greater reduction of bacterial cells with the higher MOI (Hudson et al., 2015).

Additional studies have also evaluated the use of phage and phage cocktails to decrease E. coli O157:H7 in meat. O'Flynn et al. (2004) examined the effectiveness of three bacteriophages (e11/2, pp01, and e4/1c) that could lyse 12 distinct toxigenic E. coli O157:H7 and 2 nontoxigenic E. coli O157:H7 strains. The results showed that phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5 log reduction of pathogen numbers after 1 h at 37°C. In an initial meat trial with the phage cocktail, E. coli O157:H7 on the surface of the meat (beef) in seven of nine cases were below the limit of detection. Tomat et al. (2013) isolated two stable phages (DT1 and DT6) that were negative for all the virulence factors assayed, where the reduction of E. coli O157:H7 on meat was influenced by the phage concentration, with the highest concentrations of 1.7 × 10¹⁰ PFU/mL for DT1 and 1.4 × 10¹⁰ PFU/mL for DT6 (diluted bacterial suspension ranged from 5.9 × 10⁵ to 3.9 × 10⁷ CFU/mL). The cocktail of these two phages reduced E. coli O157:H7 in meat with a high MOI of 3.33 × 10⁵ at 24°C after 3, 6, and 24 h of incubation. Anany et al. (2011) earlier observed that phage cocktails active against E. coli immobilized on modified cellulose membranes were shown to effectively control the growth of E. coli O157:H7 in ready-to-eat and raw meat under different storage temperatures and packaging conditions. These results indicate that the effectiveness of phages can significantly reduce populations of E. coli O157:H7 contamination on meat.

Use of bacteriophages on fruits and vegetables

Bacteriophages specific for E. coli O157:H7 have effectively reduced pathogens on a variety of fresh fruits and vegetables. The effects of bacteriophages against E. coli O157:H7 on different fruits and vegetables can be seen in Table 3.

Table 3.

Effect of Phages on Fruits and Vegetables

Commodity	E. coli O157:H7 strain	Phage	MOI	Application method	Temperature	Examination time	Pathogen reduction	Ref.
Lettuce	Mixture of 2886–75, G5101, and 93–111	EcoShield	100	Spray	4°C	5-min contact time	87%	Carter et al. (2012)
Iceberg lettuce	gfp86	EcoShield	8.3 log PFU/mL/5.1 log CFU/mL	Immersion^a	4°C	7 days after inoculation	DNS	Ferguson et al. (2013)
Iceberg lettuce	gfp86	EcoShield	9.8 log PFU/mL/5.1 log CFU/mL	Immersion	4°C	On days 1, 3, and 7 after inoculation	DNS	Ferguson et al. (2013)
Iceberg lettuce	gfp86	EcoShield	8.3 log PFU/mL/5.1 log CFU/mL	Immersion	4°C	6 days after inoculation	DNS	Ferguson et al. (2013)
Iceberg lettuce	gfp86	EcoShield	1000	Spray	4°C	0 day (immediately after spraying test)	1.88 log CFU/cm²	Ferguson et al. (2013)
Iceberg lettuce	B6914, gfp 86	ECP-100 cocktail (ECML-4, ECML-i 17, and ECML-134)	10,000	Spray	4°C	1 day after inoculation	3.54 log CFU/cm²	Sharma et al. (2009)
Green leaf lettuce	RM4407	EcoShield	100	Spray	4°C	30 min after inoculation	2.49 log CFU/cm²	Boyacioglu et al. (2013)
Green leaf lettuce	RM4407	EcoShield	100	Spray	4°C	2 h after inoculation	3.28 log CFU/cm²	Boyacioglu et al. (2013)
Baby romaine lettuce	Mixture of EK27, ATCC 43895, and 472	BEC8 (bacteriophage cocktail38, 39, 41, CEV2, AR1, 42, ECA1, and ECB7)	100	Spot	8, 23, and 37°C	24 h after inoculation	At least 2 log CFU/cm²	Viazis et al. (2011)
Spinach	RM4407	EcoShield	100	Spray	4°C	30 min after inoculation	2.38 log CFU/cm²	Boyacioglu et al. (2013)
Spinach	RM4407	EcoShield	100	Spray	10°C	30 min after inoculation	2.49 log CFU/cm²	Boyacioglu et al.
Spinach	309	Cocktail (FFH1, FFH2, and FFH3)	1	Drop application	Room temperature	24, 48, and 72 h after inoculation	3.28, 2.88, and 2.77 log CFU/mL	Hong et al. (2014)
Spinach	Mixture of Ec229, Ec230, and Ec231	ECP-100	>1000	Spray	10°C	24 h after inoculation	4.15 log CFU/cm²	Abuladze et al. (2008)
Baby spinach leaves	Mixture of EK27, ATCC 43895, and 472	BEC8	100	Spot	8, 23, and 37°C	24 h after inoculation	At least 3 log CFU/cm²	Viazis et al. (2011)
Cut pieces of cantaloupe	B6914, gfp86	ECP-100	100	Spray	4°C	2 days after inoculation	3.27 log CFU/cm²	Sharma et al. (2009)
Broccoli	Mixture of Ec229, Ec230, and Ec231	ECP-100	>100	Spray	10°C	24 h after inoculation	2.85 log CFU/cm²	Abuladze et al.(2008)
Tomatoes	Mixture of Ec229, Ec230, and Ec231	ECP-100	>100	Spray	10°C	24 h after inoculation	2.81 log CFU/cm²	Abuladze et al. (2008)

p < 0.05 except the study about iceberg lettuce in row 2.

Immersion for 2 min in solution before bacterial inoculation.

CFU, colony-forming units; DNS, data not shown; MOIs, multiplicities of infection; PFU, plaque-forming units.

The previously mentioned phage cocktail ECP-100 was also tested for reducing the contamination of E. coli O157:H7 on broccoli, tomatoes, and spinach (Abuladze et al., 2008). Application of ECP-100 significantly reduced (p ≤ 0.05) the concentration of viable E. coli O157:H7 organisms on broccoli by 2.85, 2.85, and 2.84 log CFU/cm², on tomato slices by 2.81, 2.79, and 2.80 log CFU/cm², and on spinach samples by 4.15, 4.15, and 4.14 log CFU/cm² during storage at 10°C for 24, 120, or 168 h, respectively. The same ECP-100 bacteriophage cocktail was later examined for its ability to reduce E. coli O157:H7 on freshly cut iceberg lettuce and cantaloupe (Sharma et al., 2009). Lettuce pieces (9 cm) were inoculated using 3.76 log CFU/cm² of an E. coli O157:H7 mixture, then dried, and sprayed with PBS or 7.98 log PFU/cm² of ECP-100. After two days of storage at 4°C, populations of E. coli O157:H7 on lettuce treated with ECP-100 on days 0, 1, or 2 were 0.72, <0.22, and 0.58 log CFU/cm², which were significantly (p < 0.05) lower than the 2.64, 1.79, and 2.22 log CFU/cm² of the PBS group. In addition, cut pieces of cantaloupe inoculated with an E. coli O157:H7 mixture (4.55 log CFU/mL) were treated with PBS or ECP-100 (6.69 log PFU/mL) and then stored at 4 or 20°C for up to 7 days. E. coli populations on cut cantaloupes treated with ECP-100 on days 2, 5, and 7 were 0.77, 1.28, and 0.96 log CFU/mL, significantly lower than the PBS group which had 3.34, 3.23, and 4.09 log CFU/mL at 4°C (Sharma et al., 2009).

Vianzis et al. (2011) reported that treatment of experimentally contaminated baby spinach and baby romaine lettuce with a mixture of eight lytic bacteriophages (BEC8) significantly reduced (p < 0.05) the number of viable dried E. coli O157:H7 cells. Treating the contaminated leafy greens at three different MOI levels (1, 10, or 100) with BEC8 produced a reduction of at least one log CFU in the number of E. coli O157:H7 cells after 24 h at 8, 23, or 37°C. The highest levels of inactivation were observed with increased time, temperature, and MOIs (Viazis et al., 2011).

As an alternative to spraying, lytic bacteriophages have also been applied to vegetables by immersion (Ferguson et al., 2013). Immersion of cut lettuce pieces in a mixture of E. coli O157:H7-specific bacteriophage suspension (9.8 log PFU/mL) for 2 min significantly (p < 0.05) reduced the population of E. coli O157:H7 (compared with lettuce treated with PBS) when applied to fresh-cut lettuce stored at 4°C for 1, 3, and 7 days after inoculation. By days 6 and 7, E. coli O157:H7 populations were reduced to undetectable levels in bacteriophage-treated lettuce samples. The higher titer and longer contact time between the lettuce and bacteriophages (2 min) allowed more bacteriophage particles to adhere on the surface of the lettuce, leading to rapid and more effective reduction of E. coli O157:H7.

Bacteriophages were also effective (p ≤ 0.05) for reducing E. coli O157:H7 after one day of application by 1.19 log CFU/cm² on spinach, 3.21 log CFU/cm² on green leaf lettuce, and 3.25 log CFU/cm² on romaine lettuce stored at 4°C when compared with untreated inoculated control samples. Reduction was further improved when leafy greens were packaged under modified atmosphere packaging (MAP; high CO₂/low O₂) and stored at 4°C. Bacteriophages reduced E. coli O157:H7 populations by 2.18, 3.50, and 3.13 log CFU/cm² on spinach, green leaf lettuce, and romaine lettuce, respectively. Similar results were obtained when leafy greens were stored by MAP at 10°C for 15 days with a reduction of 3.08, 3.89, and 4.34 log units on spinach, green leaf, and romaine lettuce, respectively (Boyacioglu et al., 2013). Hong et al. (2014) also reported that the numbers of E. coli O157:H7 in spinach were reduced (p < 0.05) by 3.28, 2.88, and 2.77 log10 CFU/mL when stored at room temperature for 24, 48, and 72 h after treatment with a three-phage cocktail at an initial contamination of 10⁷ CFU of E. coli O157:H7 with an MOI of 1 (Hong et al., 2014).

In the investigations of the effectiveness of lytic bacteriophage cocktails against E. coli O157:H7 on broccoli, cantaloupe, and strawberries, significant reductions of E. coli O157:H7 were only detected on two samples each of cantaloupe and broccoli (p < 0.05) and were not observed on any strawberry samples (Magnone et al., 2013). It was suggested that the use of bacteriophages together with combinations of produce washes or other treatment methods may help mitigate the effects of exposure to food pathogens in fresh fruits and vegetables. Carter et al. (2012) found that the application of bacteriophages at a typical concentration of 1–5 × 10⁶ PFU/g for lettuce significantly (p < 0.05) reduced E. coli O157:H7 contamination by nearly 87% with 5 min of contact time. That reduction was maintained for at least 7 days of refrigeration, mimicking typical food storage conditions (Carter et al., 2012).

Such successful studies suggest that naturally occurring bacteriophages may be useful for reducing contamination of various fruits and vegetables by E. coli O157:H7. It is concluded that phages can be applied as an alternative antimicrobial agent to control bacterial contamination of agricultural produce in vitro.

Commercial Use of Bacteriophages in the Food Industry

Multidrug-resistant bacteria pose a major threat to human health and the long-term usefulness of conventional antibiotics (Cars et al., 2008). The reduction in investment and the cautious attitude of big pharmaceutical companies toward the development of new antibiotics have prompted increasing interests in phage therapies (Ryan et al., 2011). At present, many pharmaceutical companies are engaged in phage therapy research and carrying out clinical trials. Phage prepared products have already been launched into the market by some companies.

In 2007, the U.S. Department of Agriculture (USDA) issued a no objection letter for the use of E. coli O157:H7-targeted bacteriophages developed by Omnilytics™ (Salt Lake City, UT) for use as hide sprays on cattle before slaughter (Anonymous, 2007). Elanco (Greenfield, IN) in conjunction with Omnilytics has produced a product called Finalyse to reduce E. coli O157:H7 on cattle hides to decrease transfer of these pathogens from hide to meat (Omnilytics, 2016). In 2011, Intralytix Incorporated received regulatory clearance in the form of a Food Contact Notification (FCN) from the Food and Drug Administration (FDA) for its phage-based EcoShield™ food safety product, which significantly reduced E. coli O157:H7 in ground meat by 95% to 100%. The FCN will allow the use of EcoShield on trimmed red meat parts intended for grinding (Intralytix, 2016). Further approval of phage-based products may become easier to achieve with the FDA and European Food Safety Authority Biohazards Panel endorsement of the use of phages as a treatment to control E. coli O157:H7 from foods of animal origin, including carcasses and meat.

Problems in the Applications of Bacteriophages

Despite the positive results obtained with phage therapy, there are still some disadvantages postponing the use of phages in clinical practice. The fast development of bacterial resistance to phages, the immune response induced by phages in the animal body, and the biosafety regulations regarding phage preparations, as well as the limited spectrum of activity, are just some of the major problems of phage therapy limiting its broad application. When compared with antibiotic resistance developed by pathogenic bacteria, phage therapy seems to face the same problem of resistance. However, the use of phage cocktails provides an effective way to improve the effect of phage therapy, and the cocktail treatment may also counteract the limitation of the narrow host range of phages (Sarker et al., 2012).

Conclusions

Despite the fact that most current research is mainly in the experimental stage, the increasing number of publications and emerging companies in this area are paving the way for the future of phage-based antimicrobial technology. With the use of biotechnology, the aim to amplify the host range and overcome bacterial resistance to phages could be attained through gene modification. It is hoped that further studies will be conducted on bacteriophages, demonstrating their successful applications in human therapy, veterinary prophylaxis, food safety, and environmental protection.

Footnotes

Acknowledgments

This work was supported by the Cultivation Plan for Youth Agricultural Science and Technology Innovative Talents of Liaoning Province (2015011), the 48th Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry for the collection of data, and the National Public Science and Technology Research Funds Projects of Ocean (201405003) for the analysis and interpretation of data. The authors would like to thank Professor Phil Thacker for help in revising the manuscript.

Authors' Contribution

L.W. drafted the manuscript. Z.C., Z.L., Y.S., and L.W. collected the information of phage-related research to E. coli O157:H7. K.Q. categorized the data and sorted the tables. X.W. and X.L. contributed as our advisors. Y.X. conceived and supervised the manuscript. All authors read and approved the final manuscript.

Disclosure Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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