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Bacteriophage Cocktail Significantly Reduces Escherichia Coli O157:H7 Contamination of Lettuce and Beef, but Does Not Protect Against Recontamination

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Bacteriophage Cocktail Significantly Reduces Escherichia Coli O157:H7 Contamination of Lettuce and Beef, but Does Not Protect Against Recontamination RESEARCH PAPER Bacteriophage 2:3, 178–185; July/August/September 2012; © 2012 Landes Bioscience Bacteriophage cocktail significantly reduces Escherichia coli O157:H7 contamination of lettuce and beef, but does not protect against recontamination Chandi D. Carter,1,* Adam Parks,1 Tamar Abuladze,1,† Manrong Li,1,† Joelle Woolston,1 Joshua Magnone,2 Andre Senecal,2 Andrew M. Kropinski3 and Alexander Sulakvelidze1 1Intralytix, Inc.; Baltimore, MD USA; 2United States Army Natick Soldier Research Development and Engineering Center; Combat Feeding Directorate; Natick, MA USA; 3Public Health Agency of Canada; Laboratory for Foodborne Zoonoses; and Department of Molecular & Cellular Biology; University of Guelph; Guelph, ON Canada †These authors contributed equally to this work. Keywords: Escherichia coli O157:H7, bacteriophage, phage, EcoShield™, food safety, beef, ground beef, lettuce, genomics, phylogeny Abbreviations: SEM, Standard Error of the Mean; STEC, Shiga-like toxin producing E. coli; ca., approximately Foods contaminated with Escherichia coli O157:H7 cause more than 63,000 foodborne illnesses in the United States every year, resulting in a significant economic impact on medical costs and product liabilities. Efforts to reduce contamination with E. coli O157:H7 have largely focused on washing, application of various antibacterial chemicals, and gamma- irradiation, each of which has practical and environmental drawbacks. A relatively recent, environmentally-friendly approach proposed for eliminating or significantly reducing E. coli O157:H7 contamination of foods is the use of lytic bacteriophages as biocontrol agents. We found that EcoShield™, a commercially available preparation composed of three lytic bacteriophages specific for E. coli O157:H7, significantly (p < 0.05) reduced the levels of the bacterium in experimentally contaminated beef by ≥ 94% and in lettuce by 87% after a 5 min contact time. The reduced levels of bacteria were maintained for at least one week at refrigerated temperatures. However, the one-time application of EcoShield™ did not protect the foods from recontamination with E. coli O157:H7. Our results demonstrate that EcoShield™ is effective in significantly reducing contamination of beef and lettuce with E. coli O157:H7, but does not protect against potential later contamination due to, for example, unsanitary handling of the foods post processing. Introduction to $405 million in medical costs and lost productivity annually.6 Furthermore, substantive costs to manufacturers and growers may Escherichia coli (E. coli) O157:H7 is a Shiga toxin producing be incurred in the form of product loss and brand-damaging pub- member of enterohemorrhagic E. coli (STEC). It is a major food- licity associated with recalling products contaminated with this borne bacterial pathogen, which was first associated with human bacterium. These costs significantly increase (due to additional illness during an outbreak of hemorrhagic colitis in 1982, and has legal fees and settlement agreements) if the consumption of those been listed by the Centers for Disease Control and Prevention foods results in human illness or mortality, and may force the as a national notifiable disease since 1994.1,2 This bacterium company out of business.7–9 For example, a single E. coli O157:H7 has been estimated to cause > 63,000 foodborne illnesses and outbreak associated with contaminated spinach in 2006 cost the approximately 61 deaths annually in the United States.2,3 E. coli spinach industry between $37 and $74 million.10 Thus, there are O157:H7 infections are of particular concern in young children very strong public health and economic incentives to develop novel, and elderly persons because it is associated with hemolytic uremic environmentally-friendly, safe and effective approaches for manag- syndrome which may permanently damage the kidneys.4,5 ing E. coli O157:H7 contamination of a broad range of foods. In addition to being of significant importance to public health, A variety of treatment strategies are currently employed to the economic impact of E. coli O157:H7 contamination of foods is eliminate or significantly reduceE. coli O157:H7 contamination, substantial. It has been estimated that hospitalizations and deaths ranging from simple washing of foods to chemical or physical due to E. coli O157:H7 infections in the United States may lead decontamination of foods. These methods vary with regard to *Correspondence to: Chandi D. Carter; Email: [email protected] Submitted: 07/18/12; Revised: 11/07/12; Accepted: 11/07/12 http://dx.doi.org/10.4161/bact.22825 178 Bacteriophage Volume 2 Issue 3 RESEARCH PAPER RESEARCH PAPER approved for direct food applications in the United States and Europe; e.g., ListShield™, Listex P-100™ and EcoShield™.24 One of those FDA-cleared preparations, EcoShield™, is a bac- teriophage cocktail composed of three E. coli O157:H7-specific lytic bacteriophages, which has been previously reported to sig- nificantly reduce theE. coli O157:H7 contamination on surfaces and various foods.20 The studies presented in the current com- munication were performed to determine whether treatment with EcoShield™ can (1) safely and significantly reduce E. coli O157:H7 levels in lettuce and beef under conditions that mimic their usual storage conditions, and (2) protect the phage-treated foods from recontamination with E. coli O157: H7. Results and Discussion Figure 1. Effect of phage contact time on E. coli O157:H7 levels in beef. Effect of phage contact time on E. coli O157:H7 levels. In an White bars indicate PBS controls lacking EcoShield™, gray bars indicate earlier study in which the efficacy of EcoShield™ on the E. coli EcoShield™ treated test groups. Error bars represent the standard error of the mean (SEM) (n = 3). ** p < 0.05. O157:H7 levels on various foods and hard surfaces was evaluated, the standard incubation time between phage application and test- ing for the residual bacterial contamination levels (i.e., “contact their efficacy, cost and impact on the flavor and aesthetic integ- time”) was 5 min.20 To determine if longer contact times would rity of food. For example, gamma-irradiation is considered to be increase the efficacy of EcoShield™, a time-course experiment one of the most effective treatments, capable of reducing E. coli was performed in which artificially contaminated beef steaks 11 O157:H7, and various other bacteria, by 5 log10. However, were treated with EcoShield™ (or PBS control) and held for 5, the process is very expensive and more effective (high) levels of 15, 30, 60, 120 and 1440 min at 4°C. The levels of viable E. coli gamma irradiation may adversely affect the organoleptic quali- O157:H7 in the meat were measured immediately after sampling. ties of foods, including taste and appearance.12 Other strategies EcoShield™ application significantly (p < 0.05, unpaired t-test) involve the application of various antibacterial chemicals, such as reduced the concentration of viable E. coli O157:H7 on beef sam- calcium hypochlorite, which has been reported to reduce E. coli ples at all contact times examined (Fig. 1) compared with the contamination by 1.5 – 2.5 logs,13 but many of those chemicals PBS-treated control samples. The reductions were similar for all have a negative environmental impact. In addition to targeting of the contact times examined, ranging from 94% to 98%. The pathogenic bacteria, both gamma-irradiation and chemical anti- 5 min contact time is considerably shorter than the time required bacterials target beneficial bacteria, thus negatively impacting the for a full replication cycle of lytic bacteriophage (e.g., the rep- availability of beneficial bacteria in foods.14 A relatively new inter- lication cycle of a typical lytic phage such as T4 takes approxi- vention strategy that has the potential to alleviate those problems mately 20–40 min25), indicating our results support the idea that involves using lytic bacteriophages to target specific foodborne E. coli cells were infected within the first 5 min and there was bacterial pathogens in various foods. no additional significant killing during the storage conditions we Bacteriophages (or viruses that lyse bacteria) are the most examined (Fig. 1). This observation may have some important ubiquitous life form on Earth and they are part of the normal practical implications for designing proper treatment strategies microflora of all fresh foods.15,16 The concept of using lytic bac- with bacteriophages in industrial food processing facilities. teriophages to improve food safety relies on application of an Impact of food storage conditions on E. coli O157:H7 appropriate lytic phage preparation onto foods that may be con- post phage treatment. Most foods that are at high risk of con- taminated with foodborne bacterial pathogens that are suscep- tamination with E. coli O157:H7 (including ground beef) are tible to those phages. If the foods happen to be contaminated typically stored and transported refrigerated or frozen. E. coli with the targeted bacterial pathogen, the phages will eliminate O157:H7 typically does not grow or grows very slowly at refriger- or significantly reduce the contamination, thus making the foods ated (2–8°C) temperatures,26 and growth may also occur dur- safe to consume without deleterious effect on their normal, ben- ing cooling from room to refrigeration temperatures. Thus, we eficial microflora. This strategy is referred to as bacteriophage- were interested in determining if the decrease in bacterial load mediated biocontrol. as the result of EcoShield™ treatment was consistent through- Interest in bacteriophage-mediated biocontrol has recently out a storage period of up to one week at refrigerated tempera- gained increased momentum, as a variety of laboratories are tures. This time period is consistent with the time it takes for pursuing the development of applications that utilize bacterio- beef processing, transport and sales. Artificially contaminated phages for pathogen control.17–19 Several reports have detailed beef steaks were treated with EcoShield™ (or PBS control) then the successful use of bacteriophages in significantly reducing the ground after 24 h at 4°C.
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