PHAGE COCKTAIL AGAINST E. COLI 0157

Abstract

The invention relates to the field of microbiology, specifically to an antimicrobial composition comprising a first and a second bacteriophage, wherein the composition has lytic activity against E. coli O157. The invention further relates to a use of the antimicrobial composition for controlling bacterial contamination in a food- or feed environment on or in food- or feed processing equipment or food- or feed containers or in a food- or feed product.

Claims

1-17. (canceled)

18. A method of controlling bacterial contamination in a food- or feed environment or in food- or feed processing equipment or food- or feed containers or in a food- or feed product comprising contacting a composition with the food- or feed processing equipment or the food- or feed containers or the food- or feed product, wherein the composition comprises: (a) a first bacteriophage, wherein the bacteriophage has a genome with at least 70% sequence identity with SEQ ID NO: 1 over the entire length of SEQ ID NO: 1, or has at least 70% sequence identity with the genome of bacteriophage EP75 over the entire length of the genome of bacteriophage EP75, deposited under number CBS 143858, or (b) a second bacteriophage, wherein the bacteriophage has a genome with at least 70% sequence identity with SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, or has at least 70% sequence identity with the genome of bacteriophage EP335 over the entire length of the genome of bacteriophage EP335, deposited under number CBS 143859, or (c) a first bacteriophage and a second bacteriophage, wherein: the first bacteriophage has a genome with at least 70% sequence identity with SEQ ID NO: 1 over the entire length of SEQ ID NO: 1, or has at least 70% sequence identity with the genome of bacteriophage EP75 over the entire length of the genome of bacteriophage EP75, deposited under number CBS 143858, and the second bacteriophage has a genome with at least 70% sequence identity with SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, or has at least 70% sequence identity with the genome of bacteriophage EP335 over the entire length of the genome of bacteriophage EP335, deposited under number CBS 143859.

19. The method according to claim 18, wherein the food product is a processed, non-processed, cured or uncured food product selected from the group consisting of meat, fish, shellfish, pastry, dairy products, vegetables, fruit and mixtures thereof.

20. The method according to claim 18, wherein the food product is selected from the group consisting of beef, pork, lamb, fruit, vegetables, including but not limited to lettuce, leafy greens, baby leafy greens, sprouts.

21. The method according to claim 18, wherein the pathogenic bacterium is a species of E. coli, preferably E. coli O157.

22. The method according to claim 18, wherein the composition is administered by spraying or misting the composition to the food product or by dipping or soaking the food product into the composition.

23. The method according to claim 18, wherein the first bacteriophage comprises: a tail spike protein that has at least 70% sequence identity with SEQ ID NO: 3 or has at least 70% sequence identity with SEQ ID NO: 4 over the entire length of SEQ ID NO: 4, and/or, a tail spike protein that has at least 70% sequence identity with SEQ ID NO: 5 over the entire length of SEQ ID NO: 5.

24. The method according claim 18, wherein the second bacteriophage comprises: a tail fiber protein that has at least 70% sequence identity with SEQ ID NO: 6 over the entire length of SEQ ID NO: 6 or has at least 70% sequence identity with SEQ ID NO: 7 over the entire length of SEQ ID NO: 7, and/or a tail fiber protein that has at least 70% sequence identity with SEQ ID NO: 8 over the entire length of SEQ ID NO: 8 or has at least 70% sequence identity with SEQ ID NO: 9 over the entire length of SEQ ID NO: 9.

25. The method according to claim 21, wherein the composition comprises progeny of: the first bacteriophage, and/or the second bacteriophage, wherein the progeny has the same phenotypic features and the same lytic activity against E. coli O157 as the respective first and second bacteriophage.

26. The method according to claim 18, wherein the composition is an aqueous liquid or a lyophilized aqueous liquid.

27. The method according to claim 18, wherein the composition comprises 110.sup.7 PFU/ml to 110.sup.13 PFU/ml of bacteriophage.

28. The method according to claim 18, wherein the composition further comprises an additional active ingredient selected from the group consisting of: a further bacteriophage, a bacteriostatic agent, a bactericide agent, an antibiotic, a surfactant and an enzyme.

29. A food product comprising at least 110.sup.3 PFU, or at least 110.sup.3 PFU equivalents, of a first bacteriophage, a second bacteriophage or a first and second bacteriophage as defined in claim 18 per average gram of food product.

30. The food product according to claim 29, wherein the food product is a processed, non-processed, cured or uncured food product selected from the group consisting of meat, fish, shellfish, pastry, dairy products, vegetables, fruit and mixtures thereof.

31. The food product according to claim 29, wherein the food product is selected from the group consisting of beef, pork, lamb, fruit, vegetables, including but not limited to lettuce, leafy greens, baby leafy greens, sprouts.

Description

DESCRIPTION OF THE FIGURES

[0060] FIG. 1. Efficacy after 24 h of PhageGuard E on cold fresh beef contaminated with a single Stx() E. coli O157 isolate. Data presented is the average of three independent experiments of which each experiment consisted of a duplo sample. Error bars represent the standard deviation.

[0061] FIG. 2. Efficacy after 24 h of PhageGuard E on cold fresh beef contaminated with three different E. coli O157 mixes, each mix consisting of four different Stx(+) E. coli O157 isolate. Data presented is the average reduction of duplicate samples in a single experiment. Range bars indicate the minimum and maximum reduction obtained for each treatment on the respective E. coli O157 mix.

[0062] FIG. 3. E. coli O157 load on non-treated (control) and treated (310.sup.7 and 310.sup.8 PFU/cm2) romaine lettuce samples. The data presented is an average of three independent experiments containing two samples per treatment. Error bars represent the standard deviation. Asterisks indicate statistical significance according to two-way ANOVA (Tukey's multiple comparisons test) (***P<0.001, ****P<0.0001).

[0063] FIG. 4. Time trial with PhageGuard E on romaine lettuce (A), spinach (B), and zucchini (C). Samples of all vegetable types were contaminated with E. coli strain NCTC13127 and subsequently treated with either 310.sup.8 PFU/cm2 of PhageGuard E or tap water (Control). The data presented is an average of three independent experiments containing two samples per treatment and per time point. Error bars represent the standard error of the mean. Asterisks indicate statistical significance according to two-way ANOVA (Sidak's multiple comparisons test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

EXAMPLES

[0064] The inventors developed a composition named PhageGuard E which comprises two bacteriophages named EP75 and EP335. Both bacteriophages have a high specificity for E. coli O157 bacteria and a broad host range spectrum within the E. coli O157 group.

Example 1

[0065] Challenge Study I: PhageGuard E Efficacy on Meat Samples Inoculated with a Single Stx()) E. coli O157 Isolate.

Material and Methods

Bacterial Overnight Cultures

[0066] One colony of the respective E. coli O157 Stx() strain was inoculated in 5 ml LB broth and incubated overnight at 37 C. shaking.

Preparation of Samples

[0067] Beef sample pieces of 33(1) cm were prepared to achieve a 5 cm.sup.2 surface to be contaminated (Acon) and a surface of 9 cm.sup.2 to be treated with phages (Atreated). Samples were placed and stored in sterile petri dishes.

Artificial Contamination of Beef Samples

[0068] An appropriate dilution of the overnight culture is prepared in PBS buffer to allow the contamination of the samples with a final concentration of approximately 110.sup.5 cfu/cm.sup.2 (5 L liquid/cm.sup.2). In the laminar flow hood 5 l/cm.sup.2 of the dilution is transferred to each sample and rubbed in evenly with the pipette tip.

Treatment with PhageGuard E

[0069] To allow the treatment of the beef samples with a final concentration of 310.sup.7 or 310.sup.8 pfu/cm.sup.2, dilutions of PhageGuard E were prepared. In the fume hood, 10 l/cm.sup.2 was transferred onto the samples. The liquid was distributed with the pipette tip. The petri dishes were closed and incubated at 4 C. for 24 hours, before bacterial enumeration. Bacteria were retrieved by stomaching the beef samples with 20 ml of retrieval buffer for 180 seconds. Dilutions of retrieved sample were plated on LB agar plates. Bacteria were enumerated on two different beef samples per treatment at 0 hours and 24 hours after treatment.

Results

[0070] FIG. 1 shows the reduction levels achieved by two different phage concentrations on chilled beef samples after incubation of 24 hours at refrigeration temperature. Between 84% and 98% reduction can be achieved on all of the Stx() E. coli O157 cultures depending on concentration.

Example 2

[0071] Challenge study II: PhageGuard E efficacy on meat samples inoculated with three different E. coli O157 mixes, each mix consisting of four different Stx(+) E. coli O157 isolates

[0072] Next, the efficacy of PhageGuard E was tested on meat samples inoculated with three different E. coli mixes. Sample and bacterial culture preparation was performed as described in Example 1. Likewise contamination and treatment was performed as described in Example 1. Contamination of the samples took place with three different E. coli O157 mixes, each mix consisting of four different Stx(+) E. coli O157 isolates. The E. coli mixes used are described in table 1.

TABLE-US-00002 TABLE 1 Overview of E. coli O157 strains used in presented challenge studies on roast beef Mix# Database ID number Isolation source E. coli O157 USDA* 38 Human (1991 cider outbreak) mix 1 USDA* 39 Human (Salami outbreak) Unknown 40 Human Unknown 45 Human E. coli O157 OARDC** EC 260 Bovine (dairy cattle, Creston, mix 2 Ohio) OARDC** EC 274 Bovine (dairy cattle, Wooster, Ohio) OARDC** EC 280 Bovine (dairy cattle, Minerva, Ohio) OARDC** EC 302 Bovine (dairy cattle, Millersburg, Ohio) E. coli O157 OARDC** EC 1787 Bovine (Beef feedlot, Kansas) mix 3 OARDC** EC 1805 Bovine (Beef feedlot, Kansas) OARDC** EC 1828 Bovine (Beef feedlot, Kansas) OARDC** EC 1890 Bovine (Beef feedlot, Missouri) NENT*** 999/1 unknown NENT*** 396 unknown NENT*** 419 unknown NENT*** 1286 unknown *United States Department of Agriculture **Ohio Agricultural Research and Development Center ***National Reference Centre for Enteropathogenic Bacteria and Listeria

Results

[0073] FIG. 2 shows the reduction levels achieved by two different phage concentrations on chilled beef samples after incubation of 24 hours at refrigeration temperature. Between 84% and 96% reduction can be achieved on all mixes of Stx(+) E. coli O157 cultures depending on concentration.

Example 3

Host Range Analysis

Materials and Methods

[0074] Host range analysis of PhageGuard E was performed using the spot-on-the-lawn methods. In brief, dry LB agar plates (1.5% agar) were flooded with 4 ml of soft agar (0.4% agar) containing 100 uL of an overnight E. coli culture. The soft agar containing the E. coli cells was allowed to solidify after which 5-10 l of phage dilutions 10-2, 10-4, 10-6, and 10-7 of the respective phage stocks (1*10.sup.10 to 1*10.sup.11 PFU/ml) were spotted onto the plates. After the spot plates were incubated overnight at 20 C., all the spots were scored for formation of a confluent lysis zone or single plaques.

Results

[0075] PhageGuard E infection was tested on 88 E. coli O157 clinical isolates and on 56 other E. coli strains (table 2). PhageGuard E is able to lyse all but 5 of the clinical E. coli O157 isolates when spotted. The other E. coli strains were not found to be sensitive for PhageGuard E. Thus, PhageGuard E has been proven to be very specifically active against E. coli O157.

TABLE-US-00003 TABLE 2 Host range analysis PhageGuard E PhageGuard (EP75 + EP335) Lysis in Single Source Organism Serogroup STRAIN # spots plaques 4 E. coli K12 C600 5 E. coli O157 PARC 37 ++ + 5 E. coli O157 PARC 38 ++ + 5 E. coli O157 PARC 39 ++ + 5 E. coli O157 PARC 40 ++ + 5 E. coli O157 PARC 43 ++ + 5 E. coli O157 PARC 44 ++ + 5 E. coli O157 PARC 45 ++ + 5 E. coli O157 PARC 46 ++ + 5 E. coli O157 PARC 51 ++ + 5 E. coli O157 PARC 54 ++ + 5 E. coli O157 PARC 66 ++ + 5 E. coli O157 PARC 273 ++ + 5 E. coli O157 PARC 275 ++ + 5 E. coli O157 PARC 276 ++ + 5 E. coli O157 PARC 277 5 E. coli O157 PARC 443 ++ + 5 E. coli O157 EC 66 ++ + 5 E. coli O157 EC 260 ++ + 5 E. coli O157 EC 274 ++ + 5 E. coli O157 EC 280 ++ + 5 E. coli O157 EC 285 ++ + 5 E. coli O157 EC 302 ++ + 5 E. coli O157 EC 306 ++ + 5 E. coli O157 EC 317 ++ + 5 E. coli O157 EC 338 ++ + 5 E. coli O157 EC 565 ++ + 5 E. coli O157 EC 617 ++ + 5 E. coli O157 EC 1082 ++ + 5 E. coli O157 EC 1486 ++ + 5 E. coli O157 EC 1649 ++ + 5 E. coli O157 EC 1784 5 E. coli O157 EC 1785 ++ + 5 E. coli O157 EC 1787 ++ + 5 E. coli O157 EC 1794 ++ + 5 E. coli O157 EC 1805 ++ + 5 E. coli O157 EC 1818 ++ + 5 E. coli O157 EC 1828 ++ + 5 E. coli O157 EC 1839 ++ + 5 E. coli O157 EC 1890 ++ + 5 E. coli O157 EC 1894 ++ + 5 E. coli O157 EC 1912 ++ + 5 E. coli O157 EC 1949 ++ + 5 E. coli O157 EC 1969 ++ + 5 E. coli O157 EC 1971 ++ + 5 E. coli O157 EC 2004 ++ + 5 E. coli O157 EC 2063 ++ + 5 E. coli O157 EC 2064 ++ + 5 E. coli O157 EC 2065 ++ + 5 E. coli O157 EC 2067 ++ + 5 E. coli O157 EC 2068 ++ + 5 E. coli O157 EC 2070 ++ + 5 E. coli O157 EC 2071 ++ + 5 E. coli O157 EC 2074 ++ + 5 E. coli O157 EC 2079 ++ + 5 E. coli O157 EC 2080 ++ + 5 E. coli O157 EC 2081 ++ + 2 E. coli O157 NC13128 ++ + 2 E. coli O157 NC13125 ++ + 2 E. coli O157 NC13126 ++ + 2 E. coli O157 NC13127 ++ + 3 E. coli O157 777/1 3 E. coli O157 2905 + 3 E. coli O157 264 + 3 E. coli O157 332 ++ + 3 E. coli O157 584 + 3 E. coli O157 877 ++ + 3 E. coli O157 2929 ++ + 3 E. coli O157 419 ++ + 3 E. coli O157 922 ++ + 3 E. coli O157 396 ++ + 3 E. coli O157 999/1 ++ + 3 E. coli O157 261 ++ + 3 E. coli O157 740/1 ++ + 1 E. coli O157 TW07496 ++ + 1 E. coli O157 TW07793 ++ + 1 E. coli O157 TW07794 ++ + 1 E. coli O157 TW07796 + 1 E. coli O157 TW07797 ++ + 1 E. coli O157 TWO7798 + 1 E. coli O157 TW04583 1 E. coli O157 TW07492 ++ + 1 E. coli O157 TW07493 ++ + 1 E. coli O157 TW07494 ++ + 1 E. coli O157 TW07495 ++ + 1 E. coli O157 TW00018 ++ + 1 E. coli O157 TW01286 ++ + 1 E. coli O157 TW01289 ++ + 1 E. coli O157 TW01292 ++ + 1 E. coli ECOR-01 1 E. coli ECOR-02 1 E. coli 1 ECOR-03 1 E. coli ECOR-04 1 E. coli 79 ECOR-05 1 E. coli ECOR-06 1 E. coli 85 ECOR-07 1 E. coli 6 ECOR-10 + 1 E. coli 6 ECOR-11 1 E. coli 7 ECOR-12 1 E. coli ECOR-13 1 E. coli 25 ECOR-15 1 E. coli ECOR-16 1 E. coli 106 ECOR-17 1 E. coli 5 ECOR-19 + 1 E. coli 89 ECOR-20 1 E. coli 121 ECOR-21 1 E. coli ECOR-22 1 E. coli 15 ECOR-24 1 E. coli ECOR-25 1 E. coli 104 ECOR-26 1 E. coli 150 ECOR-29 1 E. coli 113 ECOR-30 1 E. coli 79 ECOR-31 1 E. coli 7 ECOR-32 1 E. coli 7 ECOR-33 + 1 E. coli 88 ECOR-34 ++ + 1 E. coli 1 ECOR-35 1 E. coli 79 ECOR-36 1 E. coli ECOR-37 1 E. coli 7 ECOR-38 1 E. coli 7 ECOR-39 1 E. coli 7 ECOR-40 + 1 E. coli 7 ECOR-41 1 E. coli ECOR-42 1 E. coli ECOR-43 1 E. coli ECOR-47 1 E. coli 2 ECOR-49 1 E. coli 2 ECOR-50 + 1 E. coli 25 ECOR-51 + 1 E. coli 25 ECOR-52 1 E. coli 25 ECOR-54 1 E. coli 25 ECOR-55 1 E. coli 6 ECOR-56 1 E. coli ECOR-57 1 E. coli 4 ECOR-59 ++ + 1 E. coli 4 ECOR-60 ++ + 1 E. coli 2 ECOR-62 1 E. coli ECOR-63 1 E. coli ECOR-65 ++ 1 E. coli 4 ECOR-66 1 E. coli ECOR-68 1 E. coli ECOR-69 ++ 1 E. coli 78 ECOR-71 1 E. coli 144 ECOR-72 Source 1 STEC center at Michigan state University (www.shigatox.net) Source 2 Public Health of England (PHE) Source 3 National Reference Centre for Enteropathogenic Bacteria and Listeria (NENT) Source 4 Prof. Dr. Richard Calendar (University of California, Berkeley) Source 5 Ohio Agricultural Research and Development Center (OARDC)/Food Animal Health Research Program (FAHRP)

Example 4

Host Range Analysis Single Phages.

Materials and Methods

[0076] Host range analysis of phages EP75 and EP335 was performed using the spot-on-the-lawn methods. In brief, dry LB agar plates (1.5% agar) were flooded with 4 ml of soft agar (0.4% agar) containing 100 uL of an overnight E. coli culture. The soft agar containing the E. coli cells was allowed to solidify after which 5-10 l of phage dilutions 10-2, 10-4, 10-6, and 10-7 of the respective phage stocks (1*10.sup.10 to 1*10.sup.11 PFU/ml) were spotted onto the plates. After the spot plates were incubated overnight at 20 C., all the spots were scored for formation of a confluent lysis zone or single plaques.

Results

[0077] EP75 and EP335 infection was tested on 88 E. coli O157 clinical isolates and on 56 other E. coli strains (table 3).

TABLE-US-00004 TABLE 3 Host range analysis EP75 and EP335 EP75 EP335 Lysis in Single Lysis in Single Source Organism Serogroup spots plaques spots plaques 4 E. coli 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 5 E. coli O157 ++ + ++ + 2 E. coli O157 ++ + ++ + 2 E. coli O157 ++ + ++ + 2 E. coli O157 ++ + ++ + 2 E. coli O157 ++ + ++ + 3 E. coli O157 + 3 E. coli O157 + + 3 E. coli O157 + 3 E. coli O157 ++ + 3 E. coli O157 + + 3 E. coli O157 ++ + ++ + 3 E. coli O157 ++ + ++ + 3 E. coli O157 ++ + ++ + 3 E. coli O157 ++ + + 3 E. coli O157 ++ + 3 E. coli O157 ++ + + 3 E. coli O157 ++ + ++ + 3 E. coli O157 ++ ++ + 1 E. coli O157 ++ + ++ + 1 E. coli O157 ++ + ++ + 1 E. coli O157 ++ + ++ + 1 E. coli O157 + + 1 E. coli O157 + ++ + 1 E. coli O157 + + 1 E. coli O157 1 E. coli O157 ++ + ++ + 1 E. coli O157 ++ + ++ + 1 E. coli O157 ++ + ++ + 1 E. coli O157 ++ + ++ + 1 E. coli O157 ++ + ++ + 1 E. coli O157 ++ + ++ + 1 E. coli O157 ++ + ++ + 1 E. coli O157 ++ + ++ + 1 E. coli 1 E. coli 1 E. coli 1 1 E. coli 1 E. coli 79 1 E. coli 1 E. coli 85 1 E. coli 6 + 1 E. coli 6 1 E. coli 7 1 E. coli 1 E. coli 25 1 E. coli 1 E. coli 106 1 E. coli 5 + 1 E. coli 89 1 E. coli 121 1 E. coli 1 E. coli 15 1 E. coli 1 E. coli 104 1 E. coli 150 1 E. coli 113 1 E. coli 79 1 E. coli 7 1 E. coli 7 + 1 E. coli 88 ++ + 1 E. coli 1 1 E. coli 79 1 E. coli 1 E. coli 7 1 E. coli 7 1 E. coli 7 + 1 E. coli 7 1 E. coli 1 E. coli 1 E. coli 1 E. coli 2 1 E. coli 2 + 1 E. coli 25 + 1 E. coli 25 1 E. coli 25 1 E. coli 25 1 E. coli 6 1 E. coli 1 E. coli 4 ++ + 1 E. coli 4 ++ + 1 E. coli 2 1 E. coli 1 E. coli ++ 1 E. coli 4 1 E. coli 1 E. coli ++ 1 E. coli 78 1 E. coli 144 Source 1 STEC center at Michigan state University (www.shigatox.net) Source 2 Public Health of England (PHE) Source 3 National Reference Centre for Enteropathogenic Bacteria and Listeria (NENT) Source 4 Prof. Dr. Richard Calendar (University of California, Berkeley) Source 5 Ohio Agricultural Research and Development Center (OARDC)/Food Animal Health Research Program (FAHRP)

Example 5

Effectivity of PhageGuard E on Vegetables

Introduction

[0078] Efficacy of the bacteriophage product PhageGuard E was performed on vegetable samples in the laboratory.

Material and Methods

Bacterial Overnight Cultures

[0079] One colony of each E. coli O157 strain (see Table 5) was inoculated in LB broth and incubated overnight at 37 C. shaking.

Preparation of Samples

[0080] Vegetable sample pieces of 63 cm were prepared to achieve a 10 cm.sup.2 surface to be contaminated (A.sub.con) and a surface of 18 cm.sup.2 to be treated with phages (A.sub.treated). Samples were placed and stored in sterile petri dishes.

Artificial Contamination of Vegetable Samples

[0081] An appropriate dilution of the overnight culture were prepared in PBS buffer to allow the contamination of the samples with a final concentration of approximately 510.sup.5 cfu/cm.sup.2 E. coli O157 (2.5 L liquid/cm.sup.2). In a laminar flow hood, 2.5 l/cm.sup.2 of the dilution was transferred to each sample and rubbed in evenly with the pipette tip.

Treatment with PhageGuard E

[0082] To allow treatment of the vegetable samples with a final concentration of 310.sup.7 or 310.sup.8 pfu/cm.sup.2, dilutions of PhageGuard E were prepared. Vegetable samples were treated by hand spraying the respective PhageGuard E onto the sample surface to achieve 5 l/cm.sup.2. The petri dishes were closed and incubated at 4 C. for the indicated time periods, before bacterial enumeration. Bacteria were retrieved by stomaching the vegetable samples with 40 ml of retrieval buffer for 180 seconds. Dilutions of retrieved sample were plated on LB agar plates, which were supplemented with 500 pg/mL Streptomycin for the time trial experiments. Bacteria were enumerated on two different vegetable samples per treatment at the indicated time points after phage treatment.

TABLE-US-00005 TABLE 5 Overview of E. coli O157 strains used in presented challenge studies on vegetables Database ID number Isolation source NENT* 999/1 unknown NENT* 396 unknown NENT* 1286 unknown PHE** NCTC13127 Human diarrhea - stool *National Reference Centre for Enteropathogenic Bacteria and Listeria **Public Health of England

Results

[0083] Challenge Study 1: PhageGuard E Efficacy on Shiga Toxin Negative (Stx()) E. coli O157 Inoculated Romaine Lettuce 24 Hours Post Phage Treatment

[0084] It is evident from FIG. 3 that PhageGuard E reduces E. coli O157 on refrigerated romaine lettuce. Romaine lettuce samples contaminated with E. coli strains 1286, 396, or 999/1 were treated with either 310.sup.7 or 310.sup.8 PFU/cm.sup.2 of PhageGuard E or with tap water (Control). After storage of the samples at 4 C. for 24 hours, microbial loads were determined. The two different phage treatments both achieved significant (P<0.001) E. coli O157 reductions of 2.1 to 3.4 log CFU/cm.sup.2, respectively, after incubation of 24 hours at refrigeration temperature.

Challenge Study 2: PhageGuard E Efficacy Time Trial on Shiga Toxin Negative (Stx()) E. coli O157 Inoculated Romaine Lettuce, Spinach, and Zucchini

[0085] It is evident from FIG. 4 that PhageGuard E reduces E. coli O157 on refrigerated romaine lettuce, spinach, and zucchini over time. Romaine lettuce, spinach, and zucchini samples were artificially contaminated with the streptomycin resistant E. coli O157 strain NC13127, after which half of the samples were treated with tap water (control), while the other half were treated with 310.sup.8 PFU/cm.sup.2 of PhageGuard E. Subsequently, the E. coli O157 loads were determined at 2, 6, 24, 30, 54, and 72 hours post treatment for both the control and phage treated samples. For romaine lettuce, spinach, and zucchini reductions of at least 1.8 log CFU/cm.sup.2 were observed at all time points (P<0.05), with no marked difference in microbial reductions between the time points, as can be observed in FIG. 4.