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Article

Efficacy of Escherichia coli O157:H7 Phage Φ241 in Model Food Systems

by
Zhongjing Lu
Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA 30144, USA
Appl. Microbiol. 2025, 5(3), 87; https://doi.org/10.3390/applmicrobiol5030087 (registering DOI)
Submission received: 2 August 2025 / Revised: 17 August 2025 / Accepted: 19 August 2025 / Published: 21 August 2025
(This article belongs to the Special Issue Exclusive Papers Collection of Editorial Board Members and Invited Scholars in Applied Microbiology (2025))
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Abstract

Escherichia coli O157:H7 has been an important foodborne pathogen causing severe disease in humans worldwide. It is challenging to control E. coli O157:H7 due to its intrinsic acid resistance, ability to survive in various environments, and the emergence of antimicrobial resistance. Recent research showed that phages are promising antibacterial agents. A phage (Φ241) infecting 48 E. coli O157:H7 strains from various sources was previously isolated from an industrial cucumber fermentation at pH 3.7 and 5% NaCl. The efficacy of phage Φ241 infection was evaluated in this study in four representative model food systems (beef broth, cucumber juice, cucumber juice supplemented with NaCl, and apple juice). Pronounced differences in phage effectiveness were found in the tested food systems, and impacted by pH, salinity, and multiplicity of infection. The potential of this phage is evident in beef broth and cucumber juice, where 4- to 6-log reduction in host concentration was achieved within 3 to 5 h. However, apple juice (pH 3.55) completely inhibited host growth and phage infection. Overall, the study shows the high potential of Φ241 as an antibacterial agent to improve food safety. Future research will incorporate a cocktail of phages targeting E. coli O157:H7 to mitigate phage resistance development.

1. Introduction

Food-borne pathogens are the leading causes of human illness and death in the United States and many other countries [1]. Escherichia coli O157:H7 has been one of the most important foodborne pathogens and a major public health concern in the United States. The pathogen can develop antibiotic resistance like many other members in the Enterobacteriaceae family [2]. E. coli O157:H7 produces multiple virulence factors, most notably Shiga toxin, which can cause severe hemorrhagic colitis and life-threatening hemolytic uremic syndrome (HUS) [3,4,5,6]. It is estimated that E. coli O157:H7 infections cause 73,000 illnesses, 2200 hospitalizations, and 60 deaths in the United States in 1997 [7,8]. The annual cost of illness due to E. coli O157:H7 infections in the United States was USD 405 million in 2003 [9]. E. coli O157:H7 has also been implicated in outbreaks in many other countries such as the United Kingdom, Canada, Japan, Australia, and Argentina [10,11]. E. coli O157:H7 has a very low infectious dose (as low as 10–100 cells) [12]. It can survive in low-pH environments such as gastrointestinal tracts and various acidic foods [13,14,15,16,17]. In addition, it can tolerate high concentrations of NaCl [18] and is multi-drug-resistant [19]. Multi-state outbreaks of E. coli O157:H7 infections frequently occur [20]. These outbreaks have been linked to many different types of contaminated foods and drinks, including meats (such as undercooked ground beef), dairy (raw milk and cheeses), vegetables and fruits (such as spinach, lettuce, sprouts, and unpasteurized apple juice) [21,22,23,24,25]. The combination of the ability to develop antibiotic resistance, the low infectious dose, and the ability to survive in acidic and high NaCl concentration environments, makes it very difficult to control E. coli O157:H7 in foods. Recent studies have revealed that using phages as biocontrol agents is a promising strategy to control E. coli O157:H7, thereby improving food safety [26]. Phages are host-specific, usually species-specific and even strain-specific [27]. The specificity toward bacterial hosts offers a unique advantage over antibiotic therapy because phages do not affect commensal flora [28,29]. In addition, phages do not produce any substances that can affect nutritional value and sensory attributes in foods. Furthermore, phages are ubiquitous in nature and can be isolated from a variety of environments.
An E. coli O157:H7 phage (designated as Φ241) was previously isolated from an industrial cucumber fermentation at high acidity (pH 3.7) and high salinity (5% NaCl) by using E. coli O157:H7 strain B0241 [30]. The tolerance to low pH and high salinity makes phage Φ241 potentially have a wider range of applications compared to other products currently available in the food industry. The previous study showed that the phage can infect all 48 tested E. coli O157:H7 isolates from various sources, but it was unable to infect the 18 tested non-O157 isolates such as E. coli O104:H7 [30]. The phage is a myovirus with a contractile tail. The phage genome does not contain antibiotic resistance genes or virulence factors, suggesting that Φ241 is safe to be used in foods to control E. coli O157:H7 [31]. The phage has a latent period of 15 min and an average burst size of 53 phage particles per infected cell. The phage infection at multiplicity of infections (MOIs) of 0.3 to 10 in tryptic soy broth caused a rapid cell lysis within 1 to 2 h, resulting in 3.5 to 4.5 log-unit reduction in host concentration [30]. This study was designed to evaluate the efficacy of Φ241 infection against E. coli O157:H7 in four model food systems: beef broth, cucumber juice, cucumber juice with added salt, and apple juice, representing meats, vegetables, salty foods, and fruits with low pH, respectively. The results from this study provided insight into the application of the phage in foods. Phage therapy offers an alternative way to target and kill antibiotic-resistant bacteria with high specificity [27,29]. This new phage may have potential to be used in combination with other phages to effectively control E. coli O157:H7 in foods.

2. Materials and Methods

2.1. Bacterial Strain and Culture Conditions

E. coli O157:H7 strain B0241 was obtained from the culture collection of USDA Agricultural Research Service located at North Carolina State University. This strain was the original host used to isolate phage Φ241. A frozen stock of this bacterial strain was maintained at −80 °C in tryptic soy broth (TSB) supplemented with 20% glycerol (v/v). The working stock of this strain was maintained on tryptic soy agar (TSA) at 4 °C for a few weeks. An initial broth culture was prepared by inoculating 10 mL of TSB with a well-isolated colony from a TSA plate and then incubated at 37 °C statically for 12 h. Fresh overnight culture was prepared by inoculating 10 mL of TSB with 100 µL of initial broth culture or previous overnight culture and incubated statically at 37 °C for 12 h. Bacterial concentration was measured by the viable plate count method using TSA plates. All plates were incubated at 37 °C for 20 to 24 h before being examined.

2.2. Phage Isolation, Purification, and Propagation

Phage Φ241 was previously isolated from an industrial cucumber fermentation at pH 3.7 and 5% NaCl by using E. coli O157:H7 strain B024 [30]. The phage was purified and propagated on its original host (E. coli O157:H7 strain B0241) according to the method described by Lu and Breidt [30]. Briefly, brine samples were taken from seven industrial cucumber fermentation tanks. These samples were then centrifuged (5000× g for 10 min). The supernatants were filtered through syringe filters (0.45 μm pore size) to remove cellular materials and solid particles. The filtrates were used for phage enrichment in the presence of potential bacterial hosts. Spot tests were used to detect the presence of phages. An isolated single plaque from a positive spot-test plate was picked and propagated against its natural host in TSB at 37 °C. After two rounds of plaque purification, high-titer phage lysate (approximately 1 × 1010 PFU/mL) was prepared by incubation of the phage and the fresh overnight host culture in TSB at 37 °C for several hours. The phage lysate was then centrifuged at 4000× g for 20 min. The resulting supernatant was filtered through a bottle-top filter (0.45 µm pore size). The filtered phage solution was stored at 4 °C as a working stock until use. The phage stock containing 16% glycerol was maintained at −80 °C for long-term storage. The phage titer was measured by double agar overlay plaque assay. The soft TSA containing 0.6% agar was used as the top agar in the plaque assay. Right before use, the soft agar was supplemented with 100 µL of 0.3 M CaCl2. All plates were incubated overnight at 37 °C before the examination. The phage was further purified and concentrated according to the method described by Lu and Breidt [30]. Briefly, phage lysate was treated with DNase I and RNase A, and then concentrated by PEG precipitation. The concentrated phage was further purified by cesium chloride step density gradient ultracentrifugation at 600,000× g for 6 h at 4 °C followed by dialysis.

2.3. Model Food Systems

Beef broth (BB), cucumber juice (CJ), and apple juice (AJ) were used as model food systems to represent meats, vegetables, and fruits with low pH, respectively. Beef broth and apple juice were purchased from a local grocery store and then centrifuged at 10,000× g and 4 °C for 30 to 60 min. Each resulting supernatant was filtered through a bottle-top filter (0.45 µm pore size). Cucumber juice was prepared from fresh cucumbers according to the methods described by Lu et al. [32]. Fresh cucumbers (size 3A, 44–51 mm in diameter) were washed, cut into pieces, and blended at maximum speed for 1 min (Waring Co., Torrington, CT, USA). The resulting cucumber slurry was frozen overnight. After thawing, the slurry was centrifuged at 10,000× g and 4 °C for 30 min. The supernatant was then filtered using a bottle-top filter with a pore size of 0.45 μm. Each filtered food was aliquoted and stored at −80 °C until used as a model food system. The pHs in each model food system and in TSB were measured with a Fisher Science Education pH meter (Fisher Scientific, Hampton, NH, USA). In addition, cucumber juices supplemented with 1 to 2% NaCl were also prepared as a model food system to represent certain salty foods. A 20% NaCl stock solution was prepared and then filtered (0.45 µm pore size). A proper amount of the stock solution was added to CJ to increase the salt concentration by 1%, 1.5%, or 2% (higher than the natural salt concentration in CJ. The salt concentrations in CJ with and without supplemented salt were measured with a salinity meter (Traceable Salinity Meter, Fisher Scientific, Hampton, NH, USA).

2.4. Efficacy of Phage Infection Against E. coli O157:H7 in Three Model Food Systems

The lytic activity of phage Φ241 against E. coli O157:H7 B0241 was evaluated at three different MOIs in three model food systems (CJ, BB, and AJ) using the methods described by Lu et al. [32] with some modifications. For the phage infection in CJ, the frozen CJ was thawed at 4 °C and then equilibrated to room temperature before use. An overnight host culture grown in TSB was diluted with CJ to reach the host concentration of approximately 106 CFU/mL. A 9.9 mL aliquot of the CJ-diluted host culture was added to each of the four 15-mL tubes. To one of the three tubes, 100 µL of phage solution at an appropriate titer (106, 107, or 108 PFU/mL) was added to achieve an initial MOI of 1, 10, or 100. No phage was added to the fourth tube, but 100 µL of saline was added. This phage-free tube served as a control. After briefly vortexed, all four tubes were incubated statically in a water bath at 37 °C. Hourly samples were taken from each tube for 8-h period. Each sample was processed immediately to measure host concentration using the plate count method as described above. The last samples from three infection tubes were also subject to plaque assay to measure the final phage titer. The same procedure was used for the phage infection in BB or AJ. Each experiment was independently conducted three or more times.

2.5. Phage Infection at MOI of 10 in Cucumber Juice Supplemented with NaCl

The lytic activity of the phage was further evaluated in CJ supplemented with 1%, 1.5%, or 2% salt (NaCl). Two controls were used: CJ without added salt and CJ with 2% added salt. Neither control contained phages. The phage infection started at MOI 10 and continued for 8 h at 37 °C. Hourly samples were taken and analyzed immediately. Each experiment was independently conducted three or more times.

2.6. Statistical Analysis

All experiments were carried out in triplicate and each sample was plated in duplicate. One-way analysis of variance (ANOVA) was performed using Statistica 10 for Windows (StatSoft, Tulsa, OK, USA). Tukey’s Honest Significant Difference test was used to compare the mean values of data for significant difference (p ≤ 0.05).

3. Results and Discussion

3.1. pH in Model Food Systems

The pH in the three model food systems and in TSB was measured. Cucumber juice and TSB had pHs of 7.0 and 7.3, respectively, which was at or near neutral pH. Beef broth had a pH of 5.15, which was moderately acidic. The pH of beef was reported to be in the range of 5.4 to 5.7 [33,34]. The pH of beef broth was about 6.37 [35]. The pH of apple juice was 3.55 which is in the pH range of 3.3 to 4.4 for apple juice [34,36].

3.2. Efficacy of Phage Infection Against E. coli O157:H7 in Beef Broth

Beef broth is often chosen as a model food system for studying microorganisms due to its rich nutritional composition supporting diverse microbial growth. It is valuable for understanding microbial behavior in meat products in a controlled laboratory setting. Most illnesses caused by E. coli O157:H7 have been associated with the consumption of undercooked ground beef. Thus, beef broth (BB) was used as a model food system in this study simulating meats to evaluate the efficacy of phage Φ241 infection against E. coli O157:H7 at three different MOIs (1, 10, and 100).
Figure 1 showed the host concentration changes in beef broth over the 8-h incubation at 37 °C. In the absence of phage Φ241 (the control), the host concentration did not change during the first hour, but increased exponentially by two log units during the following 4 h, and then increased slowly between the fifth and sixth hour. At the end of the sixth hour, the host concentration reached 2.3 × 108 CFU/mL and then remained unchanged afterwards. In contrast, in the presence of phage Φ241 (in the infection tubes), the host concentrations decreased as soon as the infection started. At the MOI of 1, the host concentration decreased slowly during the first two hours but rapidly during the third hour, and then slowly during the following 2 h. Within 5 h, the host concentration decreased from 106 CFU/mL to 4.6 × 102 CFU/mL which was 5.3-log units lower than that in the control. At the MOI of 10, the host concentration decreased rapidly during the first three hours, then slowly during the fourth hour and increased slightly during fifth hour, resulting in 5.6-log units lower in host concentration than that in the control. The slight increase in host concentration during the fifth hour might be due to the development of phage resistance. At the MOI of 100, the host concentration decreased much more rapidly during the first hour, but much more slowly during the following 4 h than those at lower MOIs (1 and 10). As a result, the host concentration was 4.3-log units lower than that in the control at the fifth hour, indicating that the infection at MOI 100 did not achieve the same level of reduction in host concentration as that at MOI 1 or 10. This seemingly odd result might be explained by the fact that phage infection at higher initial MOI (100) quickly killed more host cells during the first hour, resulting in fewer host cells available for phage replication; thus, the host concentration no longer decreased rapidly. We measured the final phage concentrations after 8-h infection in all three infection tubes. The phage concentration in the infection tube with initial MOI of 100 was the lowest among the three infection tubes, which supported the above discussions. Nevertheless, regardless of initial MOI used, 5-h Φ241 infection resulted in at least 4.3-log reduction in host concentration compared to the control. The significant differences between treatments and control indicated that Φ241 infections at all three MOIs are highly effective to inhibit the growth of E. coli O157:H7 and to kill it in beef broth. Lu et al. [32] evaluated the efficacy of Salmonella phage ΦEnt in the same model food system (BB) under the similar condition (initial MOIs of 1, 10, and 100 at 37 °C). They found that regardless of initial MOIs, the reduction in host concentration occurred only after 3.5 h of phage infection. The delayed decline in host concentration indicated that the Salmonella phage has a much longer lysis onset time than phage Φ241 in beef broth.
It was noticed that after the fifth or sixth hour (Figure 1), the host concentration in all infection tubes started to increase, which might indicate the emergence of the phage-resistant mutants. Further study is needed to better understand the process. Notably, despite the increase, the host concentrations in all three infection tubes at the eighth hour were still about 4 log units lower than that in the control, and about 2 log units lower than the initial host concentration at time 0. Evolution of phage resistance in the host during phage infection is very common. When exposed to phages, bacteria can rapidly evolve phage resistance through different mechanisms such as spontaneous mutations, restriction modification, and CRISPR-Cas adaptive immune [28,37,38,39]. In addition, many bacteria can modify the receptors on the cell to prevent phage adsorption [39,40,41]. Some studies showed that using phage cocktails can suppress the emergence of phage resistance [42,43,44]. Commercial phage cocktail products have been developed as food-processing aids to control E. coli O157:H7. One of such phage cocktail products is EcoShield™ PX (Intralytix, Inc., Baltimore, MD, USA). This product is effective against E. coli O157:H7 and six other Shiga-toxin-producing E. coli serotypes [45]. The Food and Drug Administration (FDA) has affirmed EcoShield™ PX as generally recognized as safe (GRAS) for use in various food applications (GRN 000834) [45].

3.3. Efficacy of Phage Infection Against E. coli O157:H7 in Cucumber Juice

Many E. coli O157:H7 outbreaks have been associated with the consumption of raw vegetables. Cucumbers are one of the most popular vegetables. Like whole cucumbers, cucumber juice (CJ) contains essential nutrients required for microbial growth and provides a natural environment for studying microorganisms, similar to the conditions found in many other plant-based food products. Because of these reasons, cucumber juice has been used as a valuable model food system in microbiology research, allowing for focused and relevant studies [46,47,48,49,50,51,52]. In this study, CJ was used as a model system simulating vegetables to evaluate the efficacy of phage Φ241 infection against E. coli O157:H7 at three different MOIs (1, 10, and 100).
Figure 2 showed the host concentration changes in cucumber juice over the 8-h incubation at 37 °C. In the absence of phage (the control), E. coli O15:H7 grew exponentially in CJ after 1-h incubation. The host concentration increased from 1 × 106 CFU/mL to 1 × 108 CFU/mL within 4 h. The growth continued, but less rapidly, until the sixth hour. The host concentration reached 3.4 × 108 CFU/mL and then remained constant thereafter, indicating that the bacterial population entered a stationary phase. In the presence of phage Φ241, the host concentration decreased rapidly during the first three h. The higher the initial MOI, the faster the decrease in host concentration. The phage infection at the initial MOIs of 1, 10, and 100 caused 5.3-, 5.5-, and 6-log reduction in host concentration, respectively, compared to the control at the end of the third h. That is, regardless of initial MOI, Φ241 infection caused at least 5.3-log reduction in host concentration within 3 h. The significant differences between treatments and control indicated that Φ241 infection is highly effective against E. coli O157:H7 in cucumber juice. After 3 h, the host concentrations in all three infection tubes started to increase, which might indicate the emergence of phage-resistant mutants. Such an increase continued at a similar rate, regardless of initial MOIs. But even so, the host concentrations in those tubes were still about 2.6 to 3.3 log units lower than that in the control at the end of the eighth hour. Lu et al. [32] used the same model food system (CJ) to evaluate the efficacy of Salmonella phage ΦEnt-infecting Salmonella Thompson under similar conditions. They found that during the first 3 h, Salmonella phage infection at initial MOI of 1 or 10 did not affect host growth at all while the phage infection at an MOI of 100 completely prevented host growth but did not kill the host. The rapid decline in host concentration caused by Salmonella phage ΦEnt infection occurred only after 3 h regardless of initial MOI, and a 5-log reduction in Salmonella concentration was achieved at the end of the fourth hour. The different results from E. coli O157:H7 phage and Salmonella phage may be attributed to the differences in phage-host interactions.
It was noticed that during the first three hours, Φ241 infection caused a much more rapid decrease in E. coli O157:H7 concentration, and to a greater extent in cucumber juice than in beef broth (Figure 1). A similar observation was obtained when Salmonella phage ΦEnt infected its host in CJ and BB [34]. Such differences in CJ and BB may be explained by the fact that BB contains a higher concentration of organic matter (e.g., proteins and lipids) than CJ. Organic matter could act as a physical barrier to block cell surface receptors and/or receptor-binding proteins, thereby interfering with phage adsorption to host cells. It is also noteworthy that the host started growing, which might indicate the emergence of resistant mutants in cucumber juice (Figure 2) at least 1 h earlier than that in beef broth (Figure 1), which may be explained by the fact that the rapid phage infection occurred 1 h earlier in CJ than in BB. Phages and their bacterial hosts usually coevolve. As phage rapidly propagates early, the bacteria evolve defenses against the phage early, leading to the early emergence of phage-resistant mutants. Consequently, phage also evolves to combat such resistance. Phage–host coevolution results in an increase in the genomic diversity of both partners to ensure population survival [53,54]. To address the issue of resistance development, incorporating a cocktail of phages targeting E. coli O157:H7 will be necessary in future studies.

3.4. Efficacy of Phage Infection Against E. coli O157:H7 in Cucumber Juice Supplemented with NaCl

Cucumber juice naturally contains approximately 0.3% NaCl. Some studies showed that elevated NaCl concentration generally inhibits E. coli growth [55], and NaCl concentration at ≥2% significantly inhibits its growth [56,57]. Since phage needs a living host cell to reproduce, an elevated NaCl concentration may also inhibit phage infection. In this study, cucumber juice supplemented with 1%, 1.5%, or 2% NaCl was used to evaluate the effects of elevated NaCl concentration on the growth of E. coli O157:H7 and on phage Φ241 infection at MOI of 10.
In the absence of phage, E. coli O157:H7 in CJ without added NaCl grew exponentially after a short (<1 h) lag phase (Figure 3). The host concentration increased from 1 × 106 CFU/mL to 1 × 108 CFU/mL within 4 h. After 6 h, the host concentration reached 4 × 108 CFU/mL and remained unchanged thereafter. In CJ supplemented with 2% NaCl without phage, the growth was significantly less rapid, but continued until it reached the same final host concentration (4 × 108 CFU/mL) as in CJ without added NaCl. These results indicated that 2% added NaCl inhibited the growth of E. coli O157:H7, but it did not stop the growth and did not affect the final host concentration, indicating that E. coli O157:H7 tolerates such elevated NaCl concentration. Similar results were reported in the literature when E. coli O157:H7 grew under the same condition (CJ with the addition of 2% NaCl) [47] and in TSB containing 2 to 2.5% NaCl [16]. The inhibitory effect of higher NaCl concentration on bacterial growth is mainly due to the reduced water activity caused by NaCl [58,59]. A very high NaCl concentration could significantly reduce bacterial motility [54], which in turn directly affects chemotactic activity, thereby affecting bacterial growth.
In the presence of phage Φ241 at MOI of 10, the host concentration decreased rapidly during the first two hours and less rapidly during the third hour regardless of salt concentration in CJ (Figure 3). The decrease in host concentration was slightly less rapid in the CJ supplemented with 1.5% or 2% NaCl than that in CJ supplemented with 0 or 1% NaCl, suggesting that higher NaCl concentration slowed down phage infection. After the third hour, the host concentration in CJ with 0% or 1% added NaCl started to increase, indicating that phage-resistant mutants might emerge. In contrast, the host concentration in CJ with 1.5 or 2% added salt continued to decrease, although slowly, indicating that the high NaCl concentrations did not stop phage infection for 4 h. Interestingly, at the end of the fourth hour, regardless of the NaCl concentration in CJ, the host concentrations in all phage-containing CJs were about the same (around 1 × 102 CFU/mL) which was 4 log units lower than the initial host concentration (at time 0) and 6 or 5.3 log units lower than that in phage-free CJs (with or without 2% added NaCl) at the same time point (the fourth hour). These significant differences indicated that the phage infection was highly effective to kill host cells during these hours. After the fourth hour, host concentrations in all phage-containing CJs started to increase, which might mark the emergence of phage-resistant strain. It was noticed that the host concentration increased more rapidly in CJ without added NaCl than that in CJs with added NaCl. As NaCl concentration in CJ increased, the bacterial growth rate decreased (although slightly), suggesting that higher NaCl concentration is also inhibitory to the phage-resistant mutants. It is noteworthy that even though phage-resistant mutants emerged after fourth hour in all phage-containing CJs, the final host concentrations were still significantly lower (3.2 to 4.5 log units lower) than those in phage-free CJs at the eighth hour. It was reported that although E. coli growth was inhibited by increased salinity, E. coli is capable of surviving at salinity levels greater than 4% NaCl, which is the salinity of the ocean [60]. Since phage Φ241 was isolated from an industrial cucumber fermentation with high salinity (5% NaCl), it would be interesting to explore the host survival and the lytic effect of the phage in CJ supplemented with higher (>2%) NaCl concentrations in the future study.

3.5. Efficacy of Phage Infection Against E. coli O157:H7 in Apple Juice

Fruit is an essential part of a healthy diet. In the United States, apples held the top spot for total fruit available for consumption in 2021 with loss-adjusted apple juice availability at 14.7 pounds (1.7 gallons) per person [61]. Apple juice contains sugars and other nutrients that can support the growth of various microorganisms. Apple juice is naturally acidic. Its pH range is usually between 3.5 and 4.4 [34]. Most “100% apple juices” have a pH level between 3.3 and 4 [34]. Based on pH, apple juice is an acid food, a food with a natural pH of ≤4.6 [62]. The available nutrients and low pH in apple juice make it suitable for studying acid-tolerant microorganisms including E. coli O157:H7. In this study, apple juice was used as a model food system representing fruit juices with low pH and other acid foods to evaluate the efficacy of phage Φ241 infection against E. coli O157:H7 at three different MOIs (1, 10, and 100).
In the absence of phage (the control), host concentration in AJ did not increase over the 8-h incubation (Figure 4A), indicating that bacterial growth was completely inhibited in AJ. It is known that E. coli grows in a broad pH range of 4.4–10.0 with an optimum pH of 6–7 [63]. Since the pH (3.55) in AJ was lower than 4.4, the bacteria did not grow. This result is consistent with the previous report that fruit juices with a pH value less than 4.0, in general, do not support the growth of E. coli O157:H7 [64]. While low-pH stress is a primary cause of the inhibition of bacterial growth, the intracellular accumulation of anions from organic acids is also a contributor to the inhibition [65]. It is noticed that the host concentration remained constant without significant decrease over the 8 h incubation, indicating that E. coli O157:H7 survived very well in AJ at pH 3.55. It is well known that E. coli O157:H7 is more acid-resistant than other E. coli strains [66]. The organism has evolved multiple mechanisms to survive in low-pH environments [13,14,15,66,67,68,69,70] such as gastrointestinal tracts and various acid foods [15,16,17]. A study showed that at pH 3 and 30 °C, O157:H7 strains survived for up to 3 days [14]. The remarkable acid resistance of E. coli O157:H7 increases the chance of bacteria colonizing the intestines and causing infection, and thus contributes to its low infectious dose [15,71]. Conner and Kotrola reported that E. coli O157:H7 is able to survive in acidic conditions (pH ≥ 4.0) for up to 56 days [72], but survival is affected by other factors such as type of acidulant, temperature, and growth phase [72,73,74,75].
In the presence of phage Φ241, regardless of the initial MOI, the host concentration profile was identical to that in the control (Figure 4A), indicating that phage infection was also completely inhibited. As mentioned earlier, phage Φ241 was previously isolated from cucumber fermentation, where the pH was 3.7 which is higher than the pH (3.55) in apple juice. In order to know if the phage survived in AJ, the phage titers were also measured. Figure 4B showed that phage titers, regardless of initial MOIs, declined rapidly in exponential fashion and at about the same rate during the first 45 min. Within 60 to 90 min (depending on the MOI), the phage titers decreased to below the detection limit (10 CFU/mL), indicating that the phage died off. The instability of the phage in AJ explained why the host concentrations in all infection tubes did not decrease and maintained the same as that in the control tube over the 8-h period. The instability of phage Φ241 in AJ is mainly due to the lower pH (3.55). As acid anions from organic acids can inhibit bacterial growth, it is not clear how different acids affect phage stability. Malic acid is the major acid present in apple juice [76] while lactic acid is the major acid produced in cucumber fermentation [77,78]. It would be interesting to study the phage stability in buffered AJ and in malic acid and lactic acid at the same pH.
Most of the juice sold in the United States is pasteurized to kill harmful bacteria [79]. The Food and Drug Administration requires all fruit juices to pass a mandatory HACCP plan to assure at least a 5-log reduction in a pertinent microorganism [80]. Since phage Φ241 is not stable in apple juice, it cannot be used to achieve such a level of reduction in E. coli O157:H7 concentration in apple juice and other acid foods.

4. Conclusions

This study evaluated the efficacy of phage Φ241 infection against E. coli O157:H7 in four model food systems: beef broth, cucumber juice, cucumber juice with added salt, and apple juice, representing meats, vegetables, salty foods, and fruits with low pH, respectively. Pronounced differences in phage effectiveness were found in the tested food systems, impacted by pH, salinity, and the different MOIs. The potential of this phage is evident in beef broth and cucumber juice, though increasing salt concentration in cucumber juice slowed down its action. It was noticed that the host started growing in beef broth and cucumber juices a few hours after phage infection, which might indicate the emergence of phage-resistant mutants. But even so, the host concentrations were still 3 to 4 log units lower than those in the controls. In contrast, apple juice (pH 3.55) completely inhibited phage infection as well as host growth. Overall, the study shows the high potential of Φ241 as an antibacterial agent to improve food safety. Future research will incorporate a cocktail of phages targeting E. coli O157:H7 to mitigate the resistance development in beef broth, cucumber juice, and other model food systems.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the article.

Acknowledgments

The author would like to thank Havish Deepnarain for his assistance in the experiments and data analysis.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Efficacy of phage infection by Φ241 against E. coli O157:H7 in beef broth. Each point represents the mean ± standard error based on three measurements.
Figure 1. Efficacy of phage infection by Φ241 against E. coli O157:H7 in beef broth. Each point represents the mean ± standard error based on three measurements.
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Figure 2. Efficacy of phage infection by Φ241 against E. coli O157:H7 in cucumber juice. Each point represents the mean ± standard error based on three measurements.
Figure 2. Efficacy of phage infection by Φ241 against E. coli O157:H7 in cucumber juice. Each point represents the mean ± standard error based on three measurements.
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Figure 3. Efficacy of phage infection by Φ241 against E. coli O157:H7 in cucumber juice supplemented with 1%, 1.5% or 2% NaCl. Each point represents the mean ± standard error based on three measurements.
Figure 3. Efficacy of phage infection by Φ241 against E. coli O157:H7 in cucumber juice supplemented with 1%, 1.5% or 2% NaCl. Each point represents the mean ± standard error based on three measurements.
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Figure 4. (A) Efficacy of phage infection by Φ241 against E. coli O157:H7 in apple juice. Concentration-time profile of E. coli O157:H7. (B). Efficacy of phage infection by Φ241 against E. coli O157:H7 in apple juice. Titer-time profile of phage Φ241. Each point represents the mean ± standard error based on three measurements.
Figure 4. (A) Efficacy of phage infection by Φ241 against E. coli O157:H7 in apple juice. Concentration-time profile of E. coli O157:H7. (B). Efficacy of phage infection by Φ241 against E. coli O157:H7 in apple juice. Titer-time profile of phage Φ241. Each point represents the mean ± standard error based on three measurements.
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Lu, Z. Efficacy of Escherichia coli O157:H7 Phage Φ241 in Model Food Systems. Appl. Microbiol. 2025, 5, 87. https://doi.org/10.3390/applmicrobiol5030087

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Lu Z. Efficacy of Escherichia coli O157:H7 Phage Φ241 in Model Food Systems. Applied Microbiology. 2025; 5(3):87. https://doi.org/10.3390/applmicrobiol5030087

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Lu, Zhongjing. 2025. "Efficacy of Escherichia coli O157:H7 Phage Φ241 in Model Food Systems" Applied Microbiology 5, no. 3: 87. https://doi.org/10.3390/applmicrobiol5030087

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Lu, Z. (2025). Efficacy of Escherichia coli O157:H7 Phage Φ241 in Model Food Systems. Applied Microbiology, 5(3), 87. https://doi.org/10.3390/applmicrobiol5030087

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