Characteristics of coliphage ECP4 and potential use as a sanitizing agent for biocontrol of Escherichia coli O157:H7

Food Control 34 (2013) 255e260

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Characteristics of coliphage ECP4 and potential use as a sanitizing agent for biocontrol of Escherichia coli O157:H7 Young-Duck Lee 1, Jin-Young Kim 1, Jong-Hyun Park* Department of Food Science and Biotechnology, College of Engineering, Gachon University, Sungnam 461-701, South Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 December 2012 Received in revised form 15 April 2013 Accepted 27 April 2013

Escherichia coli O157:H7 is an important pathogenic bacterium to humans because it produces various toxins, such as shiga-toxin. Coliphage ECP4, which belongs to the Siphoviridae family, was isolated from bovine feces to test its utility as a potential agent for the biocontrol of E. coli O157:H7. The burst size of coliphage ECP4 was about 80 PFU/cell, after a latent period of 30e35 min. Coliphage ECP4 was susceptible to temperatures above 70
C; however, its stability was slightly reduced to 1e2 log PFU/ml after 30 min in 70% ethanol. In addition, the shiga toxin gene was not detected on coliphage ECP4. Coliphage ECP4 inhibited the growth of E. coli O157:H7 in vegetable juice, and was not detected in cabbage after 5 h. When coliphage ECP4 was applied to biofilm-formed E. coli O157:H7, E. coli O157:H7 was efficiently reduced. The newly identified coliphage ECP4 might effectively reduce E. coli O157:H7 or its biofilmedform. Therefore, the coliphage ECP4 might be an efficient sanitizer for fresh produce contaminated with E. coli O157:H7 in the biofilm environment. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: E. coli O157:H7 Coliphage Biocontrol Biofilm

1. Introduction Escherichia coli O157:H7 is a common strain of pathogenic E. coli, and is known to be a shiga-toxin producing food-borne pathogen. Infectious diseases caused by E. coli O157:H7 include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). E. coli O157:H7 is part of the normal microflora found in the digestive tract of ruminants, such as cattle, but is not found in humans (Gyles, 2007). E. coli O157:H7 infects humans through the ingestion of contaminated foods, direct contact with animals, and person-toperson transmission. Cattle serve as reservoirs for E. coli O157:H7, with the main animal sources of contamination including ground beef, ruminant-based foods, and unpasteurized milk. Other reservoirs include lettuce, sprouts, spinach, apple cider, and water (Fairbrother & Nadeau, 2006). E. coli O157:H7 forms biofilms to survive under harsh environmental conditions, which facilitate its attachment to soil or on food surfaces. With the potential to prevent the permeation of certain antimicrobial agents or antibiotics, the importance of biofilms in foods and medicines has been recognized. (Donlan & Costerton, 2002; Stewart & Costerton, 2001). Biofilms may also protect against a variety of environmental stresses, such as pH, osmotic shock, desiccation, and heat (O’Toole,

* Corresponding author. Tel.: þ82 31 750 5523; fax: þ82 31 750 5273. E-mail addresses: [email protected], [email protected] (J.-H. Park). 1 These authors contributed equally. 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.04.043

Kaplan, & Kolter, 2000). Bacteria are able to adhere to surfaces and form sessile multi-cellular communities, known as biofilms. These biofilms are structural micro-colonies encased in a complex extracellular polysaccharide matrix (Kierek-Pearson & Karatan, 2002). Recently, bacteriophages (or phages) have been used for the removal or reduction of biofilms. Bacteriophages exist in all habitats on earth, with around 1031 bacteriophages being estimated, or approximately 10 million/cm3 in any environment (Hendrix, 2003). Since their discovery, bacteriophages have been intensively studied, and have been used for a variety of practical applications, such as a phage therapy, the biocontrol of food-borne pathogens, the detection of pathogenic bacteria-like phage-typing, fluorescent bacteriophage assay, and amplification technology (Dinnes et al., 2007; Greer, 2005; Hudson, Billington, Carey-Smith & Greening, 2005; Sulakvelidze, Alavidze & Morris, 2001; Withey, Cartmell, Avery & Stephenson, 2005). More recently, bacteriophages have been used in attempts to reduce bacterial biofilms (Cerca, Oliveira & Azeredo, 2007; Hughes, Sutherland & Jones, 1998; Lu & Collins, 2007; ). In addition, many researchers are investigating the potential use of bacteriophages as molecular tools, for host interactions and immunity (Boyd, Brigid & Bianca, 2001; Brabban, Hite, & Callaway, 2005; Campbell, 2003). Various studies on the biocontrol of E. coli O157:H7 by coliphages have been developed. For instance, Anany et al. reported that immobilized coliphages on cellulose membranes were able to inhibit the growth of E. coli O157:H7 in meats at different temperatures (Anany, Chen, Pelton & Griffiths, 2011). Furthermore, Viscardi et al. demonstrated

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that two coliphages were able to inhibit antibiotic resistant pathogenic E. coli (Viscardi et al., 2008). Many other investigators have also reported the use of various coliphages for the control of E. coli O157:H7 in animal and food products, including processed foods and fresh produce (Goodridge & Bisha, 2011; Hudson et al., 2005). There has been a recent increase in the consumption of fresh produce, such as vegetables and fruit, due to their nutritional value and functionality (Alegre, Abadias, Anguera, Oliverira & Viñas, 2010; Bari, Nei, Enomoto, Todoriki & Kawamoto, 2009). However, pathogenic E. coli might contaminate fresh produce, with these products presenting a very high risk factor to vulnerable persons such as children and the elderly. Dewaal et al. reported that 554 food-borne outbreaks were associated with vegetables, with 28,000 patients developing food-borne illnesses between 1990 and 2003 (Dewaal, Hicks, Barlow, Alderton & Vegosen, 2006). The CDC (Centers for Disease Control and Prevention) also demonstrated that various agricultural products (including salad, sprouts, lettuce, spinach, melons, onions, and apples) have been related to food-borne illnesses in the USA (CDC, 2011). In South Korea, 277 outbreaks were caused by pathogenic E. coli, which infected 17,200 patients between 2002 and 2011 (KFDA, 2012). According to the reports of the KFDA (Korea Food and Drug Administration), pathogenic E. coli has been a major food-borne pathogen in Korea. (KFDA, 2012). Some researchers reported that the contamination ratio of E. coli was 5e 18% in fresh produce, such as vegetables and fruits, and at least one virulence factor was detected in 19% (41/217) of animal products (Bae, Hong, Kang, Heu & Lee, 2011; Hong, Seo, Choi, Hwang & Kim, 2012; Kang et al., 2011). Fresh produce and related foods such as Sunsik (cereal powdered products) and green vegetable juice are especially widely used as breakfast substitutes and health care foods by the healthy and vulnerable individuals in Korea. The manufacturing processes of Sunsik and green vegetable juice are very simple; raw agricultural products are dried, pulverized, and mixed into food compositions similar to cereals, vegetables, fruits, and medicinal plants (Lee, Park & Chang, 2012). These foods might be contaminated by pathogenic E. coli; hence biocontrol management is required. Thus, a coliphage was isolated from bovine fecal samples, and analyzed for various characteristics in this study. The coliphage was also applied in the biocontrol of E. coli O157:H7 in foods, in addition to the reduction of biofilm-formed E. coli O157:H7. 2. Materials and methods 2.1. Isolation of coliphage from bovine feces The strain used as a target strain for bacteriophage was E. coli O157:H7 NCTC12079. E. coli O157:H7 was grown in LuriaeBertani broth (LBC) or agar (Difco Laboratory, Detroit, MI, US) supplemented with 10 mmol/l CaCl2 (Sigma Aldrich, St. Louis, MO, US) at 37
C overnight in a shaking incubator. To isolate coliphage for E. coli O157:H7, bovine fecal samples were analyzed by plaque assay. Five gram of bovine fecal sample was mixed with 7e8 log CFU/ml E. coli O157:H7 and incubated with shaking at 37
C for 18 h. Then, the culture was centrifuged, and the supernatant was filtered by a 0.22 um membrane filter. The filtrate was tested for coliphage by plaque assay using double overlay agar. The plaque was picked and coliphage eluted with SM buffer and re-picked. Coliphage was propagated and purified by Sambrook and Russel (2001). 2.2. Characterization of coliphage ECP4 2.2.1. One step growth E. coli O157:H7 was grown at 37
C in LBC broth until midexponential phase. One milliliter of the culture was harvested by

centrifugation (10,000 g, 5 min) and then mixed with 0.1 ml of coliphage lysate (M.O.I. of approximately 0.001). The phageebacteria complex was collected by centrifugation after 10 min and resuspended in 10 ml LBC to allow for adsorption of phage to the bacteria. During the subsequent incubation of the resuspension at 37
C, samples were taken at 5 min intervals for 50 min. Plaques were enumerated after 24 h of incubation at 37
C. 2.2.2. Morphology Phage particles (approximately 1010e11 PFU/ml) were negatively stained with 2% (w/v) aqueous uranyl acetate (pH 4.5) on a carboncoated grid and examined by transmission electron microscopy using a JEOL JEM-100S apparatus (Japan Electronics and Optics Laboratory, Tokyo, Japan) at an accelerating voltage of 80 kV. 2.2.3. Stability Resistance to heat and ethanol was determined as previously described (Capra, Binetti, Mercanti, Quiberoni, & Reinheimer, 2009). Three temperatures (55, 65 and 70
C) were selected to study the thermal tolerance of coliphage ECP4 in LBC. Resistance to biocides was determined using the common biocides of ethanol (30%, 50% and 70% v/v for each). The results were expressed as the concentration of viable particles (log 10) plotted against time. The surviving phages were diluted and counted immediately. 2.2.4. Restriction fragmentation analysis Phage DNA was extracted using a method modified from that of Lee, Park, et al. (2012); Lee, Kim, Park and Chang (2012) DNAse I (SigmaeAldrich) and RNase A (SigmaeAldrich) were added to each 5 ml volume of phage lysate to produce a final concentration of 10 and 20 mg/ml, respectively. After incubation at room temperature for 15 min, 0.8 ml of 0.5 M EDTA (SigmaeAldrich) (pH 8) and proteinase K (Boehringer Mannheim, Mannheim, Germany; final concentration of 50 mg/ml) were added, followed by incubation at 65
C for 30 min. After incubation, sodium acetate was added, and the nucleic acid was extracted with phenol-chloroform-isoamyl alcohol (SigmaeAldrich). The nucleic acid was precipitated with isopropanol (SigmaeAldrich), and dissolved in sterile distilled water. Phage DNA was stored at 80
C. The DNA was digested with restriction enzymes according to the manufacture’s recommendations (Roche, Mannheim, Germany). 2.2.5. PCR To confirm the shiga toxin-carrying bacteriophage, PCR was performed for detection of shiga toxin encoding gene in coliphage ECP4. The primers used to detect stx1 and stx2 genes were based on published sequences (Dumke, Schröter-Bobsin, Jacobs & Röske, 2006). PCR amplifications were performed with a MJ cycler (BioRad, Hercules, USA). The PCR products were analyzed using a 1% TAE buffer with 0.5 mg/ml of ethidium bromide, and the gel visualized and photographed under a UV transilluminator (Seolin, Suwon, Korea) after electrophoresis at 5 V/cm. 2.3. Growth inhibition of E. coli O157:H7 using coliphage ECP4 in cabbage and vegetable juice Vegetable juice and cabbage were purchased from a local market. Exponentially growing E. coli O157:H7 were diluted with 0.85% saline and added to the vegetable juice and cabbage to obtain a final concentration of w104 CFU/ml. The coliphage ECP4 was added until a final concentration of w108 PFU/ml was obtained, and the mixtures were incubated at 37
C with shaking. At designated time points, 1 ml was removed, diluted with 0.85% saline, and plated onto EMB agar (Oxoid, Hampshire, England).

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2.4. Reduction of biofilm-formed E. coli O157:H7 with coliphage ECP4 The biofilms of E. coli O157:H7 were analyzed by microtiter plate assay, following the method of Coenye and Nelis (2010). Biofilm formation was carried out in 96-well microplates containing 300 ml of LBC and 10 ml of E. coli O157:H7 with an Abs. 600 nm of 1.0. Biofilm was formed for 48 h and the microplates were incubated at 37
C. After biofilm formation of E. coli O157:H7, 108 PFU of the coliphage ECP4 and 0.85% saline were added to microplate wells, respectively. The microplate plates were incubated at 37
C. For confirmation of the biofilm reduction, microplate wells were washed twice in 0.85% saline, and then biofilms were fixed with 1 ml of methanol for 15 min. After this time, methanol was removed and 1 ml of 1% crystal violet was added for 5 min. The wells were then washed with water, and 1 ml of 33% acetic acid was added to dissolve the stain. Then, absorbance of the 96-well microplates was read by an ELISA reader at 600 nm. 3. Results and discussions To isolate the coliphage of E. coli O157:H7, bovine fecal samples were analyzed using the plaque assay. Out of 6 tested coliphages, coliphage ECP4 exhibited a broad host range spectrum, which appeared the characteristic to impede E. coli O157:H7 growth, infecting 27 out of 28 E. coli O157:H7. The morphology of coliphage ECP4 included a long non-contractile tail and an icosahedral head (Fig. 1), and showed high similarity with somatic coliphage T1 belonging to Siphoviridae family (Kropinski et al., 2012). The results of restriction fragmentation analysis by HaeIII and XbaI, which cut double-stranded DNA, indicated various patterns (Fig. 2). Coliphages are a group of phages that infect in E. coli and divided into two types such as somatic and male-specific (Fþ) coliphages. Male-specific coliphages are most RNA viruses that infect via the F-pilli of male strains of E. coli, while somatic coliphages are infected through receptors on the E. coli. Two classes of coliphages show different morphology, nucleic acid (DNA or RNA) and infection types. Multiplication parameters of the lytic cycle of coliphage ECP4 were determined from the one-step growth curve (Fig. 3). The latent

Fig. 2. Restriction enzyme digestion patterns of coliphage ECP4.

period was 30e35 min, the burst period was about 45 min, and the burst size was estimated at about 80 PFU/infected cells. In case of coliphage T1 multiplies rapidly, with a latent period of about 13 min and a burst size of about 100 PFU/infected cells. Stability tests indicated a loss in viability when coliphage ECP4 was treated at 70
C (Fig. 4(A)). At 70
C, coliphage ECP4 was reduced by 6 log PFU/ml after 10 min, and was not detected after 20 min. Coliphage ECP4 showed heat resistance at temperatures below 65
C (data not shown). Li et al. reported that coliphage EEP was reduced by 3 log PFU/ml after 30 min at 72
C. Coffey et al. demonstrated that coliphages e11/2 and e4/1c were significantly reduced following exposure at 70
C (Li et al., 2010). In most dairy bacteriophages, high temperatures (>75
C) allowed efficient inactivation in a short time; however, some bacteriophages were resistant to very high temperature (>90
C) (Guglielmotti, Mercanti, Reinheimer & Quiberoni Adel, 2011). When coliphage ECP4 suspensions were treated with ethanol, the viral particles were mostly unaffected in the presence of 30% (data not shown), 50% (data not shown), and 70% ethanol (Fig. 4(B)). Li et al. also confirmed resistance to 25e100% ethanol. Some investigators reported that most bacteriophages are resistant to low ethanol concentration (<50%) (Binetti and Reinheimer, 2000; Ebrecht, Guglielmotti, Tremmel, Reinheimer & Suárez, 2010; Quiberoni, Suárez & Reinheimer, 1999). Thus, stability might vary according to the characteristics of each bacteriophage. PCR was performed to confirm whether it was a shiga toxin-carrying bacteriophage. Stx1

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Fig. 3. One step growth curve of coliphage ECP4 for E. coli O157:H7.

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and stx2 genes were not detected in coliphage ECP4 based on the PCR results (data not shown). E. coli O157:H7 is known to be a shiga toxin-producing pathogen, with shiga toxins occurring in two forms called stx1 and stx2. In general, shiga toxin genes are encoded by temperate bacteriophages, and are involved in toxicity and pathogenesis during the induction and regulation of several bacteria (Herold, Karch & Schmidt, 2004). Recently, shiga toxincarrying bacteriophages have been reported in various environments (Dumke et al., 2006; Gamage, Patton, Hanson, & Weiss, 2004; García-Aljaro, Muniesa, Jofre & Blanch, 2009; Imamovic, Jofre, Schmidt, Serra-Moreno & Muniesa, 2009; Lee et al., 2007). The correlation between the pathogenic E. coli and the shiga toxincarrying coliphage was analyzed with respect to gene transfer and epidemiological properties by molecular characteristics (Creuzburg et al., 2005; García-Aljaro, Muniesa, Jofre & Blanch, 2006; Rode et al., 2011; Shimizu, Ohta & Noda, 2009). Coliphage ECP4 might serve as a possible form of biocontrol for E. coli O157:H7. However, further study of coliphage ECP4 is required to isolate the whole genome sequence. Coliphages have been detected in many foods and environmental samples, and might also serve as indicators for the microbial water quality. Hence, various coliphages for E. coli O157:H7 might be easily isolated and applied as biocontrol agents to reduce the levels of E. coli O157:H7 in foods, or might be used as therapeutic agents to cure antibiotic resistant E. coli O157:H7. In addition, toxin gene deficient coliphages might be used for the biocontrol of E. coli O157:H7, because of the transfer to nonpathogenic E. coli from shiga toxin gene-carrying coliphage. To confirm the inhibition of E. coli O157:H7 growth in foods by coliphage ECP4, cabbage and vegetable juice was contaminated with E. coli O157:H7. Then, coliphage ECP4 was applied to the contaminated cabbage and vegetable juice. Fig. 5 showed the

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Fig. 5. Viability of E. coli O157:H7 with coliphage ECP4 in (A) cabbage and (B) vegetable juice. [A: Non-treatment coliphage ECP4, -: treatment coliphage ECP4].

reduction in E. coli O157:H7 viable counts after cabbage and vegetable juice were treated with 8 log PFU/ml coliphage ECP4. E. coli O157:H7 grew to 7e8 log CFU/g on cabbage, but was not detected at 3 h after treatment with coliphage ECP4. Furthermore, at 5 h after treatment with coliphage ECP4, E. coli O157:H7 decreased approximately 3 log CFU/ml in vegetable juice. Coliphage ECP4 could be effectively reduced the population of E. coli O157:H7 to cabbage than vegetable juice. These results might be caused by different conditions such as food type, nutrient component, and surface type during infection of coliphage ECP4 to E. coli O157:H7. Phages recognize and bind the receptors on bacteria and the phage DNA is inserted into bacteria. But, if phage adsorption is inhibited due to various conditions such as pH, phage could be impeded attachment to receptor on host, and might be difficult to decrease population of host. In vegetable juice was mixed with carrot, lemon and tomato, etc., and showed low pH (about 3.8). Marcó, Reinheimer, and Quiberoni (2010) reported that infectivity of phages for lactobacilli was reduced at low pH (3.0 and 4.0). Thus, our results indicate that coliphage ECP4 might be used to reduce E. coli O157:H7 in fresh vegetable juice, which contains various compounds. The biofilm of E. coli O157:H7 was analyzed by microtiter plate assay. Coliphage ECP4 was applied to biofilms of E. coli O157:H7, and then the absorbance of 96-well microplates was determined by an ELISA reader at 600 nm. Fig. 6 showed that 8 log PFU/ml coliphage ECP4 treatment efficiently reduced biofilm-formed E. coli O157:H7 at OD600 compared to the control. Recently, many researchers have reported that various bacteriophages are able to infect and remove the host cells of various pathogenic bacteria biofilms, including E. coli, Staphylococcaltaphylococcus aureus, Listeria monocytogenes, Bacillus -

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cereus, and Pseudomonas aeruginosa. Jassim, Abdulamir and Abu Bakar (2012) demonstrated that a bacteriophage reduced E. coli in biofilm by > 3 log. Furthermore, Sharma, Ryu and Beuchat (2005) reported the inactivation of E. coli O157:H7 in biofilms growing on stainless steel by bacteriophages. Thus, coliphage ECP4 might be used for the control of biofilm-formed E. coli O157:H7. Coliphage ECP4 was isolated from bovine fecal samples for the control of E. coli O157:H7. The morphology, stability, and molecular characterization of coliphage ECP4 were experimentally confirmed. Coliphage ECP4 might serve as an effective form of biocontrol to reduce E. coli O157:H7 in foods and remove biofilm-formed E. coli O157:H7 at the same time. Acknowledgment This research was supported by Korea Institute of Planning and Evolution for Biotechnology of Food, Agriculture, Forestry and Fisheries funded by Korean Government (311039-03-2-HD110). Reference Alegre, I., Abadias, M., Anguera, M., Oliverira, M., & Viñas, I. (2010). Factors affecting growth of foodborne pathogens on minimally processed apples. Food Microbiology, 27, 70e76. Anany, H., Chen, W., Pelton, R., & Griffiths, M. W. (2011). Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in meat by using phages immobilized on modified cellulose membranes. Applied and Environmental Microbiology, 77, 6379e6387. Bae, Y.,M., Hong, Y. J., Kang, D. H., Heu, S., & Lee, S. Y. (2011). Microbial and pathogenic contamination of ready-to-eat fresh vegetables in Korea. Korean Journal of Food Science and Technology, 43, 161e168. Bari, M. L., Nei, D., Enomoto, K., Todoriki, S., & Kawamoto, S. (2009). Combination treatments for killing Escherichia coli 0157:H7 on alfalfa, radish, broccoli, and mung bean seeds. Journal of Food Protection, 72, 631e636. Binetti, A. G., & Reinheimer, J. A. (2000). Thermal and chemical inactivation of indigenous Streptococcus thermophilus bacteriophages isolated from Argentinian dairy plants. Journal of Food Protection, 63, 509e515. Boyd, E. F., Brigid, M. D., & Bianca, H. (2001). Bacteriophageebacteriophage interactions in the evolution of pathogenic bacteria. Trends in Microbiology, 9, 137e144. Brabban, A. D., Hite, E., & Callaway, T. R. (2005). Evolution of foodborne pathogens via temperate bacteriophage-mediated gene transfer. Foodborne Pathogens and Disease, 2, 287e303. Campbell, A. (2003). The future of bacteriophage biology. Nature Reviews. Genetics, 4, 471e477. Capra, M. L., Binetti, A. G., Mercanti, D. J., Quiberoni, A., & Reinheimer, J. A. (2009). Diversity among Lactobacillus paracasei phages isolated from a probiotic dairy product plant. Journal of Applied Microbiology, 107, 1350e1357. Centers for Disease Control and Prevention. (23 March, 2011). Investigation announcement: Multistate outbreak of E. coli O157:H7 infections associated with Lebanon Bologna. Available at: http://www.cdc.gov/ecoli/2011/O157_0311/ index.html. Cerca, N., Oliveira, R., & Azeredo, J. (2007). Susceptibility of Staphylococcus epidermidis planktonic cells and biofilms to the lytic action of Staphylococcus bacteriophage K. Letters in Applied Microbiology, 45, 313e317. Coenye, T., & Nelis, H. J. (2010). In vitro and in vivo model systems to study microbial biofilm formation. Journal of Microbiological Methods, 83, 89e105.

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