Field of the invention

The invention relates to the field of microbiology, specifically to a bacteriophage or a combination of bacteriophages, to a composition comprising said bacteriophage or said combination of bacteriophages preferably for preventing, treating or controlling contamination with and/or growth of Escherichia coli 0157. The invention further relates to an antimicrobial agent, preferably a food preservative or a disinfectant for controlling bacterial contamination in a food- or feed environment on or in food- or feed processing equipment or food- or feed containers, wherein said antimicrobial agent comprises a bacteriophage or a combination of bacteriophages according to the invention.

Background art

Escherichia coli (E. coli) bacteria are short Gram-negative rods that are part of the normal flora of the intestines of most warm-blooded animals. These organisms are the most common facultative anaerobe in the large bowel and provide protection against colonization by other harmful microbes. However, shiga toxin-producing strains of E. coli (STECs), such as E.coli 0157 are responsible for most severe cases of food poisoning. The toxin produced by E. coli 0157 in the intestines can cause anything from a mild diarrhea to severe hemorrhagic colitis, where the cells of the intestinal lining are damaged, allowing blood to pass into stool.

E. coli 0157 is found regularly in the feces of healthy cattle, and is transmitted to humans through contaminated food, water, and direct contact with infected people or animals. During the slaughter process, intestinal fluid or feces of infected cattle can drip onto the surface of the meat, contaminating it. Other foods that are often found to be contaminated with E. coli bacteria include unpasteurized milk and cheese, unpasteurized juices, alfalfa and radish sprouts, lettuce, spinach, and water. However, any food is at risk of becoming contaminated with E. coli through crosscontamination.

A variety of treatment strategies are currently employed to eliminate or significantly reduce E. coli 0157 contamination, ranging from simple washing of foods to chemical or physical decontamination of foods. Efforts to reduce or prevent E.coli outbreaks and control the growth of E.coli on foodstuff have been made. For example, Seo et al (Korean J. Food Sci. An. Vol. 36, No. 2, pp. 186-193 2016) describes inhibiting the growth of E.coli 0157 in meat using bacteriophage BPEC019. Carter et al (Bacteriophage 2:3, 178-185; 2012) describe that a bacterial phage cocktail containing ECML-4, ECML-117, and ECML-134 could be used to reduce levels of E. coli 0157:H7 in lettuce and beef but that this method could not protect against recontamination. US7,625,741 US7,635,584, and US7,625, 556 describes bacteriophages using ECML-4, ECTA-47, ECML-84, ECML-117, ECML-119, ECML-122 and ECML-134 for the treatment of foodstuff and food equipment and containers to prevent or control the growth of E.coli 0157. However despite the efforts described above there remains a need for novel bacteriophages to treat food products and food processing facilities or containers to reduce, eliminate or prevent colonization with pathogens such as E. coli 0157. In particular, there remains a need to develop antimicrobial agents capable of specifically targeting a broad range of E. coli 0157 (clinical) isolates without affecting other E. coli strains.

Summary of the invention

In one aspect, the invention provides for a composition comprising 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,

wherein the composition has lytic activity against E. coli 0157.

The invention further provides for a composition that comprises the progeny of the first bacteriophage as described herein, and/or the second bacteriophage as described herein.

In another aspect, the invention provides the use of a composition according to the invention, preferably as an antimicrobial agent, more preferably as a food preservative or disinfectant, preferably for controlling a bacterium, preferably by lysing said bacterium, preferably a bacterium of the species E. coli, more preferably E. coli 0157.

In yet another aspect, the invention relates to a method for controlling bacterial contamination in a food- or feed environment, on and/or in food- or feed processing equipment, on and/or in food- or feed containers comprising contacting a composition according to the invention with the food- or feed processing equipment and/or the food- or feed containers.

In a further aspect, the invention provides a method of controlling bacterial contamination in a food- or feed product comprising administering to the food product an effective amount of a composition according to the invention to reduce the number of a pathogenic bacterium in said food product. The invention also provides for a food product obtainable the methods according to the invention.

In an another aspect, the invention provides for a composition comprising a 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, and wherein the composition has lytic activity against E. coli 0157.

In yet another aspect, the invention provides for a composition comprising a 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, and wherein the composition has lytic activity against E. coli 0157.

Description of the invention

Provided here are newly isolated bacteriophages named EP75 and EP335 and compositions comprising EP75 and/or EP335. The inventors surprisingly found that compositions comprising EP75 and/or EP335 are very specifically active against a broad range of E. coli 0157 strains and clinical isolates.

Sequence analysis revealed that EP75 contains two open reading frames (ORF) 169.1 and ORF167 that have little to no homology with any of the known Escherichia phages.

ORF167 is a tailspike protein of 754 Amino acids in length. ORF167 is homologous to the N-terminal region spanning approximately the first 180 amino acids in several Escherichia phages (Phaxl and ECML-4), yet no homology is found against the C-terminal region of about 570 amino acids in these Escherichia phages. In contrast, homology to the C-terminal region of EP75 ORF167 was found in phages that share no significant sequence homology to the rest of the EP75 genome (K1 ind 1 and HK620). Therefore, it is hypothesized that EP75 ORF167 acquired the C-terminal domain of ORF167 from another phage, like K1 ind 1 or HK620, forming an ORF167 protein which seems to be unique in combining these two protein domain sequences. It is suggested that the N-terminal domain of this tailspike protein connects the tailspike to the baseplate of the bacteriophage, while the C-terminal domain provides a specific enzymatic activity to bind and degrade sugar molecules on the bacterial cell. Therefore, the inventors conclude that EP75 contains a tailspike protein (ORF167) which is not found to date in combination with the rest of the genome sequence and structure of EP75. Likely ORF167 contributes to the 0157 specific hostrange of phage EP75. ORF169.1 of phage EP75 is a putative tailspike protein of 708 amino acids in length. Upon a blast search with the complete protein sequence of ORF169.1 , homologous proteins with a sequence coverage of more than 50% exclusively originate from Salmonella phages, while no homologs are found in Escherichia phages. However, high homology to Escherichia phage proteins is found in the first 160 to 200 amino acids of ORF169.1 . This aligns with the general structure of a tailspike protein, in which the N-terminal part binds to the baseplate of the bacteriophage, in this case an Escherichia phage. Therefore, the inventors hypothesized that EP75 acquired the C-terminal region of tailspike ORF169.1 from a Salmonella phage, in its tailspike protein, a combination which seems to be unique.

Sequences analysis revealed that the EP335 genome shares homology with several described Escherichia phages. However, the C-terminal 450 amino acids of ORF12 and the C- terminal 260 amino acids of ORF13 share only little homology with corresponding tail fiber proteins of other Escherichia phages. Other differences in the genomic nucleotide sequence are made in regions which contain hypothetical proteins of which we do not know the function yet. These proteins appear to provide a crucial difference between EP335 and the mentioned phages. The inventors found that the host range of these novel EP75 and EP335 bacteriophages is surprisingly very specifically active against a broad range of E.coli 0157 isolates without affecting other E. coli strains.

In a first aspect, the invention provides for a composition comprising a first bacteriophage and/or a second bacteriophage, wherein:

- the first bacteriophage has a genome with at least 70% sequence identity with SEQ ID NO: 1 , or has at least 70% sequence identity with 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, or has at least 70% sequence identity with the genome of bacteriophage EP335, deposited under number CBS 143859,

wherein the composition has lytic activity against E. coli 0157. The composition and the first and second bacteriophage are herein referred to as a composition according to the invention and a bacteriophage according to the invention.

Bacteriophage EP75 has been deposited under number CBS 143858 under the regulations of the Budapest Treaty at the Westerdijk Fungal Biodiversity Institute (CBS), Uppsalalaan 8, 3508AD Utrecht, The Netherlands.

Bacteriophage EP335 has been deposited under number CBS 143859 under the regulations of the Budapest Treaty at the Westerdijk Fungal Biodiversity Institute (CBS), Uppsalalaan 8, 3508AD Utrecht, The Netherlands.

Preferably, the first bacteriophage according to the invention has a genome comprising at least one polynucleotide encoding a polypeptide with an amino acid sequence having preferably at least 70, 75, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, at least 99 or 100% sequence identity with SEQ ID NO:1 over the entire length of SEQ ID NO: 1. Preferably, the first bacteriophage according to the invention has a genome that has at least 70, 75, 80, 81 , 82, 83,

84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, at least 99 or 100% sequence identity with the genome of bacteriophage EP75 over the entire length of the genome of bacteriophage EP75, deposited under number CBS 143858 and represented herein by SEQ ID NO: 1.

Preferably, the second bacteriophage according to the invention has a genome comprising at least one polynucleotide encoding a polypeptide with an amino acid sequence having preferably at least 70, 75, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO:2 over the entire length of SEQ ID NO: 2. Preferably, the first bacteriophage according to the invention has a genome that has at least 70, 75, 80, 81 , 82, 83, 84,

85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the genome of bacteriophage EP335 over the entire length of the genome of bacteriophage EP335, deposited under number CBS 143859 and represented herein by SEQ ID NO: 2.

The first and second bacteriophages of the invention may be mutant, chimeric and/or recombinant bacteriophages. The person skilled in the art may construct a bacteriophage starting from either EP75 or EP335 by placing mutations in the genome and/or deleting and/or inserting coding sequences or parts thereof into the genome using methods known in the art. Most preferably, a composition according to the invention comprises EP75 and EP335 as deposited at the CBS Fungal Biodiversity Centre under numbers CBS 143858 and CBS 143859.

The composition comprising the first and second bacteriophage according to the invention has lytic activity against E. coli 0157. Lytic activity can be assessed by any suitable method known by the person skilled in the art. In an embodiment, lytic activity can be assessed spectrophotometrically by measuring a decrease in turbidity of substrate cell suspensions. Turbidity is assessed by measuring optical density at a wavelength of 600 nm, typically a culture is turbid when it exhibits an optical density of at least 0.3 OD at a wavelength of 600 nm. Preferably, lytic activity can be assessed spectrophotometrically measuring a decrease in turbidity of an E. coli suspension, wherein turbidity is quantified by measuring OD600 spectrophotometrically (Libra S22, Biochrom). More preferably, 1 ul of a composition according to the invention is incubated together with an E. coli suspension having an initial OD600 of 1 ± 0.05, as assessed spectrophotometrically (Libra S22, Biochrom), in PBS buffer pH 7.4, 120 mM sodium chloride for 30 min at 37°C. The decrease in turbidity is calculated by subtracting the OD600 after 30 min of incubation from the OD600 before 30 min of incubation. Within the context of the invention, a bacteriophage according to the invention is said to have lytic activity when using this assay a drop in turbidity of at least 10, 20, 30, 40, 50 or 60% is detected. Preferably, a drop of at least 70% is detected.

In an embodiment, the first bacteriophage of the composition according to the invention comprises: - a tailspike protein that has at least 70, 75, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, at least 99 or 100% sequence identity with SEQ ID NO: 3 over the entire length of SEQ ID NO: 3, or SEQ ID NO: 4 over the entire length of SEQ ID NO: 4, and/or

- a tailspike protein that has at least 70, 75, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, at least 99 or 100% sequence identity with SEQ ID NO: 5 over the entire length of SEQ ID NO: 5.

In another embodiment, which can be combined with the previous embodiment, the second bacteriophage of the composition according to the invention comprises:

- a tail fiber protein that has at least 70, 75, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, at least 99 or 100% sequence identity with SEQ ID NO: 6 over the entire length of SEQ ID NO: 6, SEQ ID NO: 7 over the entire length of SEQ ID NO: 7, and/or

- a tail fiber protein that has at least 70, 75, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, at least 99 or 100% sequence identity with SEQ ID NO: 8 over the entire length of SEQ ID NO: 8 or SEQ ID NO: 9 over the entire length of SEQ ID NO: 9.

In a second aspect, the invention provides for a composition that comprises progeny of the first bacteriophage as described herein, and/or the second bacteriophage as described herein. Preferably, said progeny has the same phenotypic features and the same lytic activity against E. coli 0157 as the respective first and second bacteriophage. The invention thus provides for the progeny of the bacteriophages described herein, said progeny is described as having minor variation(s) in the genomic sequence and polypeptides encoded thereby while retaining the same general genotypic and/or phenotypic characteristics as the bacteriophages described herein. In particular these progeny are the result of successive passaging of the bacteriophages described herein where the variants accumulate silent mutations, conservative mutations, minor deletions, and/or minor replications of genetic material. The progeny retains the phenotypic characteristics of bacteriophages according to the invention, in a preferred embodiment, the progeny retain lytic activity against E. coli 0157. Preferably, the progeny has at least 90, 91 , 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity with the genome of the parent bacteriophage over the entire length of the genome of the parent bacteriophage.

In a third aspect of the invention, the composition according to the invention preferably is an aqueous liquid or a lyophilized aqueous liquid. Preferably, the composition according to the invention comprises 1 x 10 ⁷ PFU/ml to 1 x 10 ¹³ PFU/ml of bacteriophage. In an embodiment, the composition according to the invention comprises 1 x 10 ¹ ° PFU/ml, 1 x 10 ¹¹ PFU/ml, 1 x 10 ¹² PFU/ml or 1 x 10 ¹³ PFU/ml of bacteriophage. Preferably, the composition according to the invention comprises at least 1 x 10 ¹ ° PFU/ml of bacteriophage, more preferably at least 1 x10 ¹¹ PFU/ml. Preferably, when comprising two bacteriophages, the bacteriophages are present in a ratio of 50/50 (+ 20%) in the composition according to the invention. In an embodiment, 1 x 10 ¹¹ PFU/ml of bacteriophage EP75 and 1 x 10 ¹¹ PFU/ml are present in a composition according to the invention. Plaque Forming Unit (PFU) is a term known in the art and is a standard unit for depicting the concentration of bacteriophages. The concentration can be assessed by any assay known in the art, typically by a plaque assay.

In an embodiment, a composition according to the invention further comprises an additional active ingredient, preferably in a concentration known to be effective. Preferably said active ingredient is present in a concentration known in the art to result in a significantly reduced number, such as one, two or three logs, of foodborne pathogens that would otherwise be present. Preferably, said one or more additional active ingredients are selected from the group consisting of a further bacteriophage, a bacteriostatic agent, a bactericidal agent, an antibiotic, a surfactant and/or an enzyme. An antibiotic can be any antibiotic known in the art including antibiotics and chemotherapeutic agents, and including but not limited to vancomycin, nisin, danofloxacin and neomycin. An enzyme useful in a composition of the invention includes but is not limited to enzymes that aid in breaking up biofims (e.g. biofilms found in food processing equipment) such as but not limited to polysaccharide depolymerise enzymes and protease. A surfactant useful in a composition of the invention helps to wet the surface so that bacteriophages are properly distributed over the various surfaces, and to solubilise and remove dirt so that the E. coli are accessible to the bacteriophage. Suitable surfactants include but are not limited to polysorbate (tween) 80, 20 and 81 and Dobanols (Shell Chemical Co. RTM). An antimicrobial disinfectant composition of the invention may further comprise surface disinfectants known in the art such as but not limited to benzoic acid and PBT, preferably disinfectants with which a bacteriophage of the invention is compatible. A further bacteriophage in a composition according to the invention can be any phage known in literature, other than the bacteriophage of the invention. Preferably, such a further bacteriophage includes, but is not limited to, a tailed phage of the order of Caurdovirales, consisting of Myoviridae, Siphoviridae and Podoviridae. Preferably, the compostion according to the invention is buffered. An additional active ingredient of the composition according to the invention may be an inorganic salt, preferably selected from the group consisting of lactic acid, preferably L-lactic acid, acetic acid, propionic acid and mixtures thereof. The salt of the organic acid according to the invention is preferably selected from the group consisting of the sodium salt, potassium salt, ammonium salt and mixtures thereof, preferably K-(L)lactate, Na-(L)lactate, K-acetate, Na-acetate, K-diacetate, Na-diacetate, K-propionate, Na-propionate and mixtures thereof.

Preferably, the organic acid in the composition according to the invention is acetic acid in a buffered aqueous solution, preferably comprising 2% to 30% acetate, more preferably comprising 5% to 20% acetate, more preferably comprising 10% to 20% acetate, more preferably comprising 15% to 20% acetate, more preferably comprising 15, 16, 17, 18, 19 or 20% acetate, most preferably comprising 17% acetate. The preferred pH of the acetic acid in a buffered aqueous solution is 2 to 7, more preferably 5 to 6.5 and most preferably to 5.7 to 6.3. Preferred buffering is performed using sodium acetate, acetic acid, sodium hydroxide, sodium carbonate and/or sodium bicarbonate.

Preferably, the salt of the organic acid in the composition according to the invention is in an aqueous solution comprising a mixture of K-(L)lactate and Na-diacetate, preferably comprising 50% to 80% K-(L)lactate and 2% to 10% Na-diacetate, more preferably comprising 60% to 80% K-(L)lactate and 3% to 10% Na-diacetate, more preferably comprising 70% to 75% K-(L)lactate and 4% to 6% Na- diacetate, most preferably comprising 72.8% K-(L)lactate and 5.2% Na-diacetate. The aqueous solution may be buffered as described here above.

In a fourth aspect, the invention provides for a use of a composition according to the invention. Preferably, the composition is used as an antimicrobial agent, more preferably as a food preservative or disinfectant, preferably for controlling a bacterium, preferably by lysing said bacterium, preferably a bacterium of the species E. coli, more preferably E. coli 0157. Preferably, a composition according to the invention, is used to reduce the counts of E. coli 0157 bacteria and/or to prevent their growth in the first place, in food products as well as in food processing plants in which the food products are being processed such as on processing equipment and other sites in food industry facilities, e.g. food storage container.

Preferably, a composition according to the invention is used as an antimicrobial agent, preferably a food preservative or a disinfectant. Preferably said antimicrobial agent is for killing a bacterium, preferably a bacterium of the species E. coli, more E. coli 0157. Preferably, the composition according to the invention exhibits a broad host range E. coli 0157 infection property and has lytic activity as defined herein.

In a fifth aspect, the invention relates to a method for controlling bacterial contamination in a food- or feed environment, on and/or in food- or feed processing equipment, on and/or in food- or feed containers comprising contacting a composition according to the invention with the food- or feed processing equipment and/or the food- or feed containers. Preferably said method is for controlling a bacterium of the species of E. coli, more preferably E. coli 0157. Preferably, said method of controlling includes the reduction of counts of E. coli 0157 bacteria and/or the prevention of their growth in the first place, in food products as well as in food processing plants in which the food products are being processed, such as on processing equipment and other sites in food industry facilities, e.g. food storage containers. A method of the invention encompasses the application of a composition according to the invention on or into food products, and/or into various physical sites within the food processing plants on or in food processing equipment, by a number of means including, but not limited to, admixing, spraying or directly applying said composition. The applications according to the invention significantly reduce the numbers of E. coli bacteria that would otherwise be present.

In a sixth aspect, the invention provides a method of controlling bacterial contamination in a food- or feed product comprising administering to the food product an effective amount of a composition according to the invention to reduce the number of a pathogenic bacterium in said food product. In an embodiment, the food product is a processed, non-processed, cured or uncured food product. Preferably, the food product is selected from the group consisting of meat, fish, shellfish, pastry, dairy products, vegetables, fruit and mixtures thereof. More preferably, 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.

In an embodiment, the method for controlling bacterial growth and/or bacterial contamination on a food- or feed product, a food- or feed environment, on and/or in food- or feed processing equipment, on and/or in food- or feed containers is a method for controlling the bacterial growth and/or bacterial contamination of a pathogenic bacterium. Preferably, the pathogenic bacterium is a species of E. coli. More preferably the species of E. coli is E. coli 0157.

In an embodiment, the method of controlling bacterial contamination in a food- or feed product comprises contacting the food- or feed product with the composition according to the invention wherein the contacting is done by spraying or misting the composition to the food product or by dipping or soaking the food product into the composition.

In an seventh aspect the invention provides for a food product obtainable or obtained by a method according the fifth or sixth aspect of the invention. In an embodiment, the food product obtainable or obtained by a method according to the invention comprises at least 1 x 10 ³ PFU, or at least 1 x 10 ³ PFU equivalents of a first bacteriophage according to the invention and/or a second bacteriophage according to the invention, per average gram of food product. Preferably, the food product according to the invention contains at least 1 x 10 ⁴ PFU, 1 x 10 ⁵ PFU, 1 x 10 ^® PFU, 1 x 10 ⁷ PFU, 1 x 10 ⁸ PFU or PFU equivalents, per average gram of food product. More preferably, the food product contains at least 1 x 10 ⁹ PFU, or at least 1 x 10 ⁹ PFU equivalents, per average gram of food product.

In an eight aspect, the composition according to the invention may comprise a bacteriophage with the features as defined previously herein that 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 wherein the composition has lytic activity against E. coli 0157. Preferably, the bacteriophage comprises:

- a tail spike protein that has at least 70% sequence identity with SEQ ID NO: 3 over the entire length of 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.

In an ninth aspect, the composition according to the invention may comprise a bacteriophage with the features as defined previously herein that 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, and wherein the composition has lytic activity against E. coli 0157. Preferably, the 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.

Sequence table

Where applicable, the Genbank accession number is given between brackets {} in column two. Definitions

The terms“homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods.

“Sequence identity” and“sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Sequence identity” and “sequence similarity” are preferably expressed as the sequence identity or sequence similarity in view of the entire length of the subject sequence. E.g. when a sequence of 30 amino acids is identical over said 30 amino acids with a part of a sequence according to the invention SEQ ID NO: X of 300 amino acids, the sequence identity in view of SEQ ID NO: X is 10%. Sequences may be referred to as "substantially identical” or“essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program“needle” (using the global Needleman Wunsch algorithm) or“water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for‘needle’ and for‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred. Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the invention can further be used as a“query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403— 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to oxidoreductase nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at www.ncbi.nlm.nih.gov/.

Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.

The term“homologous” when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and/or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. When used to indicate the relatedness of two nucleic acid sequences the term “homologous” means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed later.

The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but has been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.

Any reference to nucleotide or amino acid sequences accessible in public sequence databases herein refers to the version of the sequence entry as available on the filing date of this document.

As used herein, "progeny" shall mean all bacteriophages, including descendants of EP75 or EP335 created by serial passage of EP75 or EP335 or by other means known in the art, that have a substantive sequence identity with the sequences of EP75 or EP 335. Substantive sequence identity is herein defined as at least 95%, 96%, 97%, 98%, or at least 99% sequence identity. Alternatively, the term progeny includes descendants of EP75 or EP335 created by serial passage of EP75 or EP335 or by other means well known in the art whose RFLP profiles are substantially equivalent to the RFLP profile of EP75 or EP335 in accordance with the standards advanced by Tenover et al. from the United States Centers for Disease Control and Prevention (Tenover, F. C. et al. (1995) Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing. J. Clin. Microbiol. 33:2233-2239). Tenover et al. teaches the acceptable levels of variation that may be seen when the genomes identical propagated organisms are electrophoretically analyzed following restriction enzyme digestion.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb“to consist” may be replaced by“to consist essentially of meaning that a product or a composition may comprise additional component(s) than the ones specifically identified; said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the invention in any way.

Description of the figures

Figure 1. Efficacy after 24 h of PhageGuard E™ on cold fresh beef contaminated with a single Stx(- ) E. coli 0157 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. Figure 2. Efficacy after 24 h of PhageGuard E™ on cold fresh beef contaminated with three different E. coli 0157 mixes, each mix consisting of four different Stx(+) E. coli 0157 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 0157 mix.

Figure 3. E. coli 0157 load on non-treated (control) and treated (3x10 ⁷ and 3x10 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) ( ^*** P0.001 , ^**** P<0.0001 ).

Figure 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 3 x 10 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

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 0157 bacteria and a broad host range spectrum within the E. coli 0157 group.

Example 1

Challenge study I: PhageGuard E™ efficacy on meat samples inoculated with a single Stx(-)) E. coli 0157 isolate.

Material and Methods

Bacterial overnight cultures

One colony of the respective E. coli 0157 Stx(-) strain was inoculated in 5 ml LB broth and incubated overnight at 37 °C shaking.

Preparation of samples

Beef sample pieces of 3x3(x1 ) cm were prepared to achieve a 5 cm ² surface to be contaminated (Aeon) and a surface of 9 cm ² to be treated with phages (Atreated). Samples were placed and stored in sterile petri dishes. Artificial contamination of beef samples

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 1 x10 ⁵ cfu/cm ² (5mI_ liquid/ cm ² ). In the laminar flow hood 5 mI/cm ² of the dilution is transferred to each sample and rubbed in evenly with the pipette tip.

Treatment with PhageGuard E™

To allow the treatment of the beef samples with a final concentration of 3 x10 ⁷ or 3 x10 ⁸ pfu/cm ² , dilutions of PhageGuard E™ were prepared. In the fume hood, 10 mI/cm 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

Figure 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 0157 cultures depending on concentration.

Example 2

Challenge study II: PhageGuard E™ efficacy on meat samples inoculated with three different E. coli 0157 mixes, each mix consisting of four different Stx(+) E. coli 0157 isolates

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 0157 mixes, each mix consisting of four different Stx(+) E. coli 0157 isolates. The E. coli mixes used are described in table 1.

Table 1 Overview of E. coli 0157 strains used in presented challenge studies on roast beef

Results

Figure 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 0157 cultures depending on concentration.

Example 3

Host range analysis

Materials and methods

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 4ml 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 pi of phage dilutions 10-2, 10-4, 10-6, and 10-7 of the respective phage stocks (1*10 ¹⁰ to 1 *10 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

PhageGuard E™ infection was tested on 88 E.coli 0157 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 0157 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 0157.

Table 2: Host range analysis PhageGuard E TM

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

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 4ml 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 m I of phage dilutions 10-2, 10-4, 10-6, and 10-7 of the respective phage stocks (1*10 ¹⁰ to 1 *10 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

EP75 and EP335 infection was tested on 88 E.coli 0157 clinical isolates and on 56 other E. coli strains (table 3).

Table 3: Host range analysis EP75 and EP335

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

Efficacy of the bacteriophage product PhageGuard E™ was performed on vegetable samples in the laboratory. Material and Methods

Bacterial overnight cultures

One colony of each E. coli 0157 strain (see Table 5) was inoculated in LB broth and incubated overnight at 37 °C shaking.

Preparation of samples

Vegetable sample pieces of 6x3 cm were prepared to achieve a 10 cm ² surface to be contaminated (A con ) and a surface of 18 cm ² to be treated with phages (Abated). Samples were placed and stored in sterile petri dishes.

Artificial contamination of vegetable samples

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 5x10 ⁵ cfu/cm ² E. coli 0157 (2.5 pL liquid/ cm ² ). In a laminar flow hood, 2.5 mI/cm ² of the dilution was transferred to each sample and rubbed in evenly with the pipette tip.

Treatment with PhaqeGuard E™

To allow treatment of the vegetable samples with a final concentration of 3 x10 ⁷ or 3 x10 ⁸ pfu/cm 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 mI/cnri 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/nnL 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 5 Overview of E. coli 0157 strains used in presented challenge studies on vegetables

*National Reference Centre for Enteropathogenic Bacteria and Listeria

** Public Health of England

Results

Challenge study 1 : PhageGuard E™ efficacy on shiqa toxin negative (Stx(-D E. coli 0157 inoculated romaine lettuce 24 hours post phage treatment It is evident from Figure 3 that PhageGuard E™ reduces E. coli 0157 on refrigerated romaine lettuce. Romaine lettuce samples contaminated with E. coli strains 1286, 396, or 999/1 were treated with either 3 x 10 ⁷ or 3 x 10 ⁸ PFU/cm 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 0157 reductions of 2.1 to 3.4 log CFU/cm ² , respectively, after incubation of 24 hours at refrigeration temperature.

Challenge study 2: PhageGuard E™ efficacy time trial on shiqa toxin negative (Stx(-D E. coli 0157 inoculated romaine lettuce, spinach, and zucchini

It is evident from Figure 4 that PhageGuard E™ reduces E. coli 0157 on refrigerated romaine lettuce, spinach, and zucchini over time. Romaine lettuce, spinach, and zucchini samples were artificially contaminated with the streptomycin resistant E. coli 0157 strain NC13127, after which half of the samples were treated with tap water (control), while the other half were treated with 3 x 10 ⁸ PFU/cm ² of PhageGuard E™. Subsequently, the E. coli 0157 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 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 Figure 4.