Therapeutic phages

Abstract

Provided is a bacteriophage of the family Myoviridae, comprising a genome of circularly arranged double-stranded DNA of 200 kbp or longer in length; characterized in that the bacteriophage has any one or more of the following characteristics: a) does not have a perforin gene; and / or b) binds to Salmonella lipopolysaccharide; and / or c) has only one tail fiber protein; and d) has a sequence having at least 95% sequence identity with SEQ ID NO. 7 and / or a sequence having at least 80% sequence identity with SEQ ID NO. 8. Suitably, the bacteriophage can be used to treat Salmonella, particularly in pigs and poultry.

Description

Technical Field

[0001] The present invention relates to therapeutic bacteriophages useful for preventing or treating Salmonella infections, kits of such bacteriophages, and pharmaceutical compositions of such bacteriophages. The present invention also relates to methods for drying bacteriophage solutions and methods for preparing heat-stable bacteriophages. Background Art

[0002] Non-typhoidal Salmonella spp. are a leading cause of food poisoning worldwide and in the UK, 33,000 cases of human salmonellosis are reported each year. The European Food Safety Authority (EFSA) estimates that 11.7% of reported cases of human salmonellosis are caused by the consumption of pork products. Epidemiological surveillance by the UK Animal and Plant Health Agency (APHA) has determined that the five most prevalent serotypes of Salmonella associated with pigs in the UK are: S. Typhimurium, S.4:5:12:i>, S.4:5:12, and S. Derby, S. Derby. S. Derby, S.4,5,12:i>, and S. Typhimurium are also among the five most prevalent serotypes in the United States. Furthermore, in the EU, S. Typhimurium, S.4,5,12:i>, S.4:5:12 and S. Derby are the most prevalent Salmonella serotypes reported to EFSA.

[0003] Studies have shown that the prevalence and spread of Salmonella increases particularly during the weaning period and when market-weight pigs leave the farm and are transported to slaughterhouses for slaughter, which increases the likelihood of carcass contamination of the pork product that is ultimately consumed. This problem is particularly concerning because multidrug-resistant (MDR) Salmonella strains have been isolated from pigs. The range of antibiotics available to treat Salmonella in pigs is decreasing, and there are major concerns about the spread of MDR or the presence of antibiotic residues in the human food chain. Summary of the invention

[0004] Clearly, alternatives to antibiotics are urgently needed to control Salmonella infections.

[0005] The present inventors have isolated and characterized bacteriophages (natural viruses that target and kill bacteria of a specific bacterial species) that target multidrug-resistant strains of Salmonella associated with pigs and chickens.

[0006] Therefore, the present invention provides a bacteriophage of the family Myoviridae, comprising a genome of circularly arranged double-stranded DNA of 200 kbp or longer; characterized in that the bacteriophage has any one or more of the following characteristics:

[0007] a) does not have the perforin gene; and / or

[0008] b) binds to Salmonella lipopolysaccharide; and / or

[0009] c) have only one tail fiber protein;

[0010] as well as

[0011] d) having a sequence having at least 95% sequence identity with SEQ ID NO.7 and / or a sequence having at least 80% sequence identity with SEQ ID NO.8.

[0012] Suitably, at least two of features a) to d) are provided, suitably, at least three of features a) to d) are provided, suitably, at least four of features a) to d) are provided.

[0013] a) and b); a) and c); a) and d); b) and c); b) and d); or c) and d), or

[0014] a), b) and c); or a), b) and d); or b), c) and d); or a), c) and d).

[0015] Preferably, the phage is capable of lysing 3 or more strains of Salmonella associated with poultry, pigs and / or cattle. For example, any one or more of the following: a) 25 strains of S. Typhimurium; b) 15 strains of S.4,12:i>; c) 10 strains of S.4.5,12:i>; d) 10 strains of S. bovis; e) 10 strains of S. Delbe; f) 3 strains of S.13,23:i>; g) 5 strains of S. Enteritidis; h) 4 strains of S. Infantis; i) 3 strains of S. Ohio; and j) 3 strains of S. Seftenberg.

[0016] Suitably, the phage is capable of lysing 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more strains of Salmonella associated with poultry, pigs and / or cattle.

[0017] The phage may have 80%-100% sequence identity with any of the phages deposited with Nos. NCTC 18080701; NCTC 18080702; NCTC 18080703; NCTC 18080704; NCTC 18080705; or NCTC 18080706.

[0018] The deposits were made on August 7, 2018, at the European Collection of Certified Cell Cultures and are numbered 18080701; 18080702; 18080703; 18080704; 18080705; or 18080706.

[0019] Suitably, the phage can be selected from the phages deposited with the European Collection of Recognized Cell Cultures on August 7, 2018 with accession numbers 18080701; 18080702; 18080703; 18080704; 18080705; or 18080706.

[0020] The phage may be present in a set comprising two, three, four, five, six or more of the above phage. For example, NCTC 18080703 and 18080704. Suitably, the set may include any one of 18080701; 18080702; 18080703; 18080704; 18080705; or 18080706 or a combination thereof, wherein

[0021]

[0022] Table 1

[0023] Suitably, the phage may be selected from SPFM 1, 3, 10, 14, 15, 17 and / or 19 or a combination thereof. Suitably, the phage may be selected from any one or more of SPFM 1 to 22. Suitably, the phage may be selected from any one or more of SPFM 1 to 22, excluding SPFM 9 and SPFM 11. Suitably, the phage may be selected from any one or more of SPFM 2, 4, 10, 14, 17 and / or 19. Suitably, at least 2, at least 3, at least 4 phage may be selected from SPFM 2, 4, 10, 14, 17 and / or 19. Suitably, a combination of SPFM 4 and 17 or SPFM 10 and 14 may be provided. Suitably, a combination comprising any two of SPFM 4, 10, 14 and 17 may be selected. Suitably, at least one phage stable at pH 3 may be provided in the kit. Suitably, the phage used in the kit may be selected from at least one of 2, 4 or 19 or a combination thereof. Suitably, phages resistant to thermal degradation may be used. Suitably, phages selected from SPFM 10 and 17 may be selected. Suitably, SPFM 10- may be selected.

[0024] Suitably, a combination of three phages selected from the group consisting of:

[0025] SPFM4, 10 and 19

[0026] SPFM10, 17 and 19

[0027] SPFM2, 17 and 19.

[0028] Suitably, a combination of four phages selected from the group consisting of:

[0029] SPFM10, 14, 17 and 19

[0030] SPFM2, 10, 14 and 19

[0031] SPFM2, 4, 10 and 19.

[0032] Table 1 provides the nucleotide accession numbers of these phages and details of the phage deposits that have been made under the Budapest Treaty discussed herein.

[0033] The phage may be part of a pharmaceutical composition comprising one or more of the above-mentioned phage or kit.

[0034] Alternatively, the bacteriophage may be an animal feed or disinfectant comprising any of the bacteriophage, pharmaceutical composition or kit described above.

[0035] In another aspect, the above-mentioned bacteriophage, kit, pharmaceutical composition or animal feed can be used in a method of treatment, such as a preventive method of treatment.The disease to be treated or prevented can be a Salmonella infection in poultry, pigs or cattle.

[0036] Thus, there is provided a bacteriophage for use in treating Salmonella according to the present invention. Suitable for treating Salmonella in pigs and / or poultry (eg chickens).

[0037] Also provided is a method for drying a bacteriophage solution to form a powder, the method comprising:

[0038] a) mixing the bacteriophage with any one or more of the following;

[0039] (i) sugars; and / or

[0040] (ii) sugar alcohols; and / or

[0041] (iii) amino acids; and / or

[0042] (iv) polymers;

[0043] as well as

[0044] b) drying the mixture.

[0045] Suitably, features of the method include wherein:

[0046] drying by spray drying; and / or b) wherein the method further comprises preparing tablets or pellets from the dried powder;

[0047] The sugar is a glucose-based sugar, preferably trehalose.

[0048] ·Sugar alcohol is mannitol.

[0049] The amino acid is an aliphatic amino acid, preferably leucine.

[0050] The phage solution used in the drying method may comprise any of the above-mentioned phage, the claimed kit or the pharmaceutical composition.

[0051] In another aspect, a bacteriophage composition is provided, comprising bacteriophage, sugar, sugar alcohol, amino acid and polymer. The bacteriophage, sugar, sugar alcohol, amino acid or polymer may be any one of those described above.

[0052] Also provided is a thermostable bacteriophage comprising:

[0053] a) polymorphism 1 in a sequence having at least 80% sequence identity with SEQ ID NO. 1; and / or

[0054] b) polymorphism 2 in a sequence having at least 80% sequence identity with SEQ ID NO. 2; and / or

[0055] c) polymorphism 3 in a sequence having at least 80% sequence identity with SEQ ID NO. 2; and / or

[0056] d) Polymorphism 4 in a sequence having at least 80% sequence identity with SEQ ID NO.2.

[0057] Suitably, the phage may have polymorphism 1, 2, 3 or 4 as described herein and have a sequence identity of at least 85%, at least 90%, at least 95%, at least 97% and at least 99% to SEQ ID No.2.

[0058] The thermostable phage may be any one of the above-mentioned phages.

[0059] In another aspect, a method for preparing a thermostable bacteriophage is provided, comprising mutating the nucleotide sequence of the bacteriophage genome at the following position:

[0060] a) polymorphism 1 is at a position from base a, g or c to t in a sequence having at least 80% sequence identity with SEQ ID NO. 1;

[0061] b) polymorphism 2 is at a position from base a, t or c to g in a sequence having at least 80% sequence identity with SEQ ID NO. 2;

[0062] c) the position of polymorphism 3 from base a, g or t to c in a sequence having at least 80% sequence identity with SEQ ID NO. 2; and

[0063] d) polymorphism 4 at a position from base a, g or c to t in a sequence having at least 80% sequence identity with SEQ ID NO. 2,

[0064] The method optionally comprises a first step of introducing SEQ ID NO. 1 and / or SEQ ID NO. 2 into the phage genome.

[0065] Suitably, the phage may have polymorphism 1, 2, 3 or 4 as described herein and have at least 85%, at least 90%, at least 95%, at least 97% and at least 99% sequence identity to SEQ ID No. 1 or 2, respectively.

[0066] The invention is further described below, by way of example only, and with reference to the accompanying Examples. DETAILED DESCRIPTION

[0067] Bacteriophage

[0068] Bacteriophages, also commonly referred to as phages, are viruses that infect and replicate in their target bacteria. Phages can be prophages, temperate / lysogenic phages, phage-like particles (such as plasmids), or lytic phages.

[0069] Prophage / temperate / lysogenic phages are phage particles composed of double-stranded or single-stranded DNA or RNA. The phage genome can be inserted and integrated into the circular bacterial DNA chromosome, or exist as an extrachromosomal plasmid. This is a latent form of phage in which the viral genes are present in bacteria without causing the destruction of the bacterial cell, which can sometimes provide a competitive advantage to the overall fitness of the bacterial host.

[0070] Lytic or virulent phages contain viral DNA / RNA that exists and replicates separately from the host bacterial DNA. Lytic phages are released when the infected cell and its membrane are destroyed.

[0071] The bacteriophage is a lytic phage and is isolated and / or purified from its natural environment.

[0072] Phage morphology

[0073] The 22 phages described here have equal-length heads and retractable tails and are classified as members of the Myoviridae family within the Caudoviridae family.

[0074] The tail of the bacteriophage varies from 130 to 260 nm, for example, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 nm.

[0075] The capsid diameter is about 70 nm to 160 nm, for example, 80, 90, 100, 110, 120, 130, 140 or 150 nm.

[0076] Genetic analysis of bacteriophages

[0077] Bacteriophages have a double-stranded DNA genome in a linear, circular arrangement. The genome length is about 200-260 kbp. For example, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250 or 255 kbp. That is, bacteriophages are classified as giant phages.

[0078] The phage may not have a perforin gene; and / or the phage may have only one tail fiber protein; and / or the phage receptor may be Salmonella lipopolysaccharide (LPS).

[0079] The genome of the phage may additionally have any one or more of the following characteristics, including any combination of these characteristics:

[0080] Encodes only one tRNA; and / or

[0081] Encodes more than one RNAP (RNA polymerase) β subunit with different DNA sequences.

[0082] That is, the RNAP β subunits have less than 50% sequence identity; and / or

[0083] Genes encoding folate synthesis pathways, such as dihydrofolate reductase and / or thymidylate synthase and / or

[0084] or thymidylate kinase; and / or

[0085] DNA adenine methylase gene; and / or

[0086] · Free radical S-adenosylmethionine gene.

[0087] The phage shares sequence identity of several core genes and hypothetical proteins with other members of the Myrovidae family, Salmonella phages SPN3US and SEGD1 (although no core gene shares 100% sequence identity with these known phages).

[0088] The differences between several core sequences are listed below:

[0089] SEQ ID No. 3: Endolysin. 95% similar to SPN3US and SEGD1. Function: An enzyme used by bacteriophages at the end of the replication cycle to degrade the peptidoglycan of the bacterial host from the inside, leading to cell lysis and release of progeny virions.

[0090] >MFRTILFLLVAMTFNSNASYQTTSNKHQKEFVLIQDQFNEIKPLIVTASRKQGVHAGMLATTIYRESRFNKNVGKNKSSSASSVVQMTTGTKRSMIRLYGKQLNIPKNADLNKPKYAV QLAAVYMKHIEEHLTKQLKRKPSTAEIALGYRFGEGAAVAMIKKKSPVGKRWMASYRKDAAFYGAKMTPPKAETRQLAFAKEDHDQRVAELQKIWDTLYTKISPAAGTLLANNTIMKGALL

[0091] SEQ ID No. 4: Acetyltransferase (GNAT) family protein. 95% similar to SPN3US and 63% similar to SEGD1. Function: GNAT may have a range of enzyme functions, including resistance to antibiotics.

[0092] >MKTVAKLFRSSIRSAEFQNNVGTDTVRARRLKEFLKDETYSVYVVRNKETWEGYLVATYRHGDPTLTCHYFEEFVPGAFDKVGLALVLDHTGFEYMSCPAPTGDVEQKLVEKGFVLSDIVWACHSKLFDKVITASEIKIISGDAYAELSKDDKDVILNLVSGAICE ELEYDVRGHYTHIGPLAETVIARIQHANVETAIWRDGRDIVGIAQMRLVAPGVVELSGLTVAPTHRKRGIGRRLMGALFDFAQRHSDQVYIETAADNNPANHLYGNILRCKQVLRTLTLKRDPELISWKRVKGNRADRVLKEEPSLPVETNPRPTINDMFQSGMRGC

[0093] SEQ ID No.5: N-acetyltransferase GCN5. 67% similar to SPN3us and 99% similar to SEGD1

[0094] >MKIVFVTKPNSPYGKKYYADLLGLHNKLYDDQKIKLADLNGREGFNQISKLEEVWGEGTLCACAVDDRGVAVGFITFGYTKKGQRFLWVYNFFVDESVRSQGVGHELIEAVENYGKSKGCAWMQLNVLGNNDRAIAFYERFGFRTEYQDMVKEL

[0095] SEQ ID No. 6: Phikz-like internal noggin. 35% similar to SPN3 US and 99% similar to SEGD1 Function: Similar to the noggin of giant bacteriophage PhiKZ.

[0096] >MANFVKSKLARESVEATGDVIDGLSNVEPAEENIDVQLAEVASIDGQLEQLDGDQETLAADTERTEAAIDAADEKIENGEEMPEEAIAHTEVAQESIRKRWAIDR TKLARESYRRGRGMTKAAQEGWKETLKDLFKRFVELCKAVIAKAKELKLKYINVGKSAQKRAKAYQEMLRKLGKQKKENISGGFISKLSIEGDFDVANSIAIAKELT GGKAKDAINKLSSQASESAVAITKTAEGTATAAKGAVDVALFGTAAKKLRTLPQFEDGEGGQKVLALPGNAYVQIGSKTLAGGQDFTAVAFLSTGDSTDTKEIATP AITALASAATALDAIGKGFEKVLQDFRSYDADVEKLEQAASKAAAALDKSNDEGEWEALRNARTAADQAVRNYQTLNRAVAHVGNTVISGLNGYIGAGIGAYEKSKA

[0097] SEQ ID No. 7: Putative ATP-dependent DNA helicase from bacteriophage: 41% similar to SEGD1 and 99% similar to SPN3US

[0098] >MVEIQMTYTSMGVRVEVPIRQAERAILAWAEENMHAPKMGKQHGRITTERGDAYYAHIPSLRTFIFHKVFAEKIKHILQRAAIEYAFQYKLTEHHPERGTPYSCTFDNYGFSMVESDPESRFYYQNEVVDAASDPNRPQTI FAIQTGRGKTKSCMKSMVKRGTRTALIHRPSYVPKWLFDVCDDETGLRIILRDEVLVCTGVQAIYDALEMGKSGELDRRGIKVIILPTVSLQRFLKEYINTAATNPVDLDTFYDVLGVGLVAMDEVHEHFHLVYMAGIMLNPP ASIEMSATLKPGSSKAFIAERYLERFPMEYRISIPIIPVVDVKALYYRLDDKKFAWWASKMTPYNHKLFEGKLIKENLHLEYANMIWDVVEKSFLKRYQPGQKCLLLFATVAMCEFFTEYVKDKLSRDPVFHPLMVAKYNAG DSYDDFIQADFSISTPGKAGTAVDKPGLVHMYISTPVEDQQLNEQMAGRPRKILHNEWGEIDPTVWLFHAYNVPKHCNYLNARQKSLKDVVLSFRIATSPYVVRKSHAHSAASSRATAALHRNDFSKFSRKHSKGVSRRRRRR

[0099] SEQ ID No. 8: Putative peptidase of HsIV family protein Not present in SPN3US or SEGD1.

[0100] MTTIAFDGKVLAADGQLTRGSNICNLNTQKLFICPSDEEWWITGERIVAFGVSGDITGQLALLETVRLRPGYKGLTQSSQLPAKMDFTFLMLLASGKAIVGGKREDDVTLWWSEATPPLATGSGYEYALGAMKMGANAVRAVEIASECDIYTGGEISTYELR

[0101] Accession number

[0102] The phage contained in the kit of the present invention may include mutants and variants of the deposited phage.

[0103] In this application the bacteriophages of the invention are referred to according to the following table:

[0104]

[0105] The deposits were made at NCTC / ECACC under the Budapest Treaty, following the rules of the International Depositary Agencies (IDAs) operating under the Budapest Treaty, and were made on August 7, 2018.

[0106] Such deposited phage mutants and variants retain the ability to kill Salmonella. Compared to the genome of at least any deposited phage of the present invention, the mutants and variants have at least 80%, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleotide sequence identity throughout the genome.

[0107] Such mutants and variants may be generated by adding, deleting or substituting nucleotides from the nucleic acid sequence of the deposited phage (eg, addition, deletion or substitution of 1, 2, 3, 4 or 5 nucleotides, optionally consecutive nucleotides).

[0108] Optionally, the mutant or variant does not include an integrase gene and / or a toxin gene.In addition to retaining the ability to kill Salmonella, the mutants and variants may have any of the further properties described for the deposited phage from which they were characterized.

[0109] Set

[0110] The kit of phage can include any combination of phage provided herein. The phage can be prepared for separate, sequential or simultaneous administration.

[0111] All combinations of phage that can be selected from the above groups are contemplated for inclusion in the set.

[0112] The kit may have additional components, such as components that may not be bacteriophage, such as carriers, excipients, therapeutic agents (e.g., non-bacteriophage agents such as antibiotics).

[0113] Optionally, the set may comprise 2 or more different phage, 3 or more different phage, 4 or more different phage, 5 or 6 or more different phage.

[0114] For example, a set may contain two phages, for example, SPFM 10 and 14; SPFM 2 and 4; SPFM 10 and 17 or SPFM 2 and 10.

[0115] For example, the set may contain three phages, such as SPFM4, 10, 19; SPFM10, 17 and 19; or SPFM2, 17 and 19.

[0116] For example, a set may include four phages, such as SPFM10, 14, 17, 19; SPFM2, 10, 14, and 19; or SPFM2, 4, 10, and 19.

[0117] For example, the set may contain more than four phages. For example, SPFM 10, 14, 17 and 19 and another or other; SPFM 2, 10, 14 and 19 and another or other; or SPFM 2, 4, 10 and 19 and another or other.

[0118] For example, a set may include SPFM 2, 4, 10, 14, 17, and 19.

[0119] Serotype

[0120] Salmonella strains are classified by serotyping. Serotyping involves the analysis of the size and number of copies of the ribosomal RNA genes, which establishes a genetic profile for each strain (called a serotype). The standard protocol for serotyping uses PCR combined with agarose gel electrophoresis, or capillary analysis after incorporation of fluorescent primers.

[0121] The phages described herein target the following five Salmonella serotypes: a) 25 S. Typhimurium; b) 15 S.4,12:i>; c) 10 S.4.5,12:i>; d) 10 S. Bovis; and e) 10 S. Derby. These are the five most prevalent serotypes in pigs in the UK. All of these serotypes exhibit multidrug resistance. Therefore, new technologies, such as the phages described herein, are urgently needed.

[0122] Propagation and isolation of bacteriophages

[0123] Propagation and isolation of phage are described in the Examples section.

[0124] Thermostable phage

[0125] The stability of the phage may be increased at high temperatures. The phage described herein may be stable at 50°C, 55°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C.

[0126] Thermostability was tested by exposing the phage to temperatures of 4°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C for one hour and incubating in a polymerase chain reaction (PCR) machine. To determine the phage titer, the phage lysate was serially diluted 10-fold and a small drop plaque assay was used on LB 1% agar plates with SL1344 bacterial populations. The final phage titer was expressed in PFU / ml.

[0127] Stable means minimal loss of titer when exposed to high temperature. For example, the loss is 0.0 to 3.5 log 10 PFU / ml or less at 60°C, 70°C, 80°C or 90°C using the above thermal stability test. For example, the loss is 0.0-0.05, 0.1, 0.3, 0.6, 0.9, 1.0, 1.3, 1.6, 1.9, 2.0, 2.3, 2.6, 2.9, 3 or 3.5 log 10 PFU / m.

[0128] Although the phages themselves may not be stable, the storage solution can improve their thermostability. Thus, thermostability can also refer to minimal titer loss when exposed to high temperatures after immersion in the storage solution. Phages having sequence identity to SPFM10, 14 and / or 17 are particularly thermostable. For example, the phages have 80, 85, 90, 95 or 100% sequence identity to SPFM10, 14 or 17.

[0129] Genetic analysis has revealed specific single nucleotide differences in SPFM10 that may be responsible for the stability of this phage. These are described below.

[0130] In the following sequence of SPFM10 (predicted to be a pectate lyase), there is a t instead of an a (which is present in the less thermostable SPFM phage identified in this application) at position 70890 of the genome. This polymorphism is located in the first line of the following SEQ ID NO. 1, shown in bold and underlined. This is referred to as polymorphism 1 (from a to t) in the claims.

[0131]

[0132] The amino acid sequence of SEQ ID NO. 1 is as follows. The SNP from adenine to threonine changes serine to threonine as shown underlined below (the second to last line of SEQ ID No. 9). This mutation from serine to threonine may make the phage more thermostable.

[0133] SEQ ID NO.9:

[0134]

[0135] Further polymorphisms have been found in another putative pectate lyase. The sequence is as follows (SEQ ID NO. 2). In SPFM10, single nucleotide polymorphisms exist at the following positions:

[0136] Position 74291: In SPFM10, there is a g at this position. The guanine at this position is referred to as polymorphism 2 in the claims.

[0137] Position 74294: In SPFM10, there is a c at this position. The guanine at this position is referred to as polymorphism 3 in the claims.

[0138] Position 74295: In SPFM10, there is a g at this position. The guanine at this position is referred to as polymorphism 4 in the claims.

[0139] These are shown in bold and underlined in SEQ ID NO. 2 of SPFM10 below (see second line indicated by arrow).

[0140] SEQ ID No: 2 (3' to 5')

[0141]

[0142]

[0143] The amino acid sequence of SEQ ID NO.2 is as follows:

[0144] SEQ ID No.10:

[0145]

[0146] Polymorphism 2: A SNP at position 74291 from adenine (as found in SPFM 4 and 17) to guanine (as found in SPFM10) results in a change from leucine to proline (SSQWYNSPPY (SEQ ID No: 11) above). Mutation of this proline can make SPFM10 more thermostable.

[0147] Polymorphism 3: A SNP from adenine (as found in SPFM2) to cytosine (as found in SPFM10) at position 74294 results in a change from isoleucine to serine (SSQWYNSPPY (SEQ ID No: 12) above). Mutation of this proline may render SPFM10 more thermostable.

[0148] Polymorphism 4: A SNP from adenine (as found in SPFM2) to cytosine (as found in SPFM10) at position 74295 results in a change from isoleucine to serine at the same position as above polymorphism 3. Likewise, the serine at this position in SEQ ID NO. 2 may render SPFM10 more thermostable.

[0149] Salmonella infection

[0150] Salmonella infection is a common bacterial illness that affects the intestines.

[0151] This kit is for veterinary use, that is, the objects of treatment are animals.

[0152] The animals may be pigs, chickens, turkeys and / or cattle.

[0153] Salmonella infections can be caused by any one or more of the following serotypes: S. Typhimurium, S.4,12:i>, S.4.5,12:i>, S. Bovis, and S. Derby. These strains all exhibit multidrug resistance. Phages can kill 80%, 85%, 90%, 95%, or 100% of isolates from each of these strains.

[0154] The Salmonella infection may be caused by any one or more of the following serotype strains: S. 13, 23:i>, S. Enteritidis, S. Infants, S. Ohio, and S. Seftenberg. The phage may kill 33%, 100%, 50%, 60%, or 33% of isolates from each of these strains.

[0155] Pharmaceutical composition

[0156] The composition may include a pharmaceutically acceptable excipient and / or carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drugs, drug stabilizers, gelling agents, adhesives, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, similar materials and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition. Mack Printing Company, 1990, pp. 1289-1329).

[0157] Treatment

[0158] The phage can be used in methods of treating Salmonella, for example, in animals such as pigs, cattle, and poultry, such as chickens.

[0159] The composition can be prepared for various routes of administration. For example, the phage or pharmaceutical composition can be added to animal feed, such as pig or chicken feed. For example, the animal feed can include compressed feed, such as pellets. Other oral compositions can be used. Alternatively, the phage can be added to a spray to disinfect a fence or carcass.

[0160] Preventive treatment methods

[0161] The phage or pharmaceutical composition of phage can be administered to an animal before it is infected with Salmonella. For example, young pigs during the weaning period are very susceptible to infection. Therefore, in this possible situation, a preventive treatment can be administered.

[0162] Treatment is effective

[0163] The phage is generally used in an effective dosage to achieve the intended purpose. For use in treating or preventing a disease condition, the molecules of the invention or their pharmaceutical compositions are administered or applied in a therapeutically effective amount. A therapeutically effective amount is an amount effective to improve or prevent the symptoms of the treated animal or to prolong its survival. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0164] Detection

[0165] Laboratory-based methods are currently used to detect Salmonella, such as ELISA and PCR-based methods, but these methods have been reported to lack specificity. The bacteriophages described in this application can be used as an alternative, more specific method for diagnosing Salmonella strains.

[0166] The sample taken from a pig or other animal for such a diagnostic test may be feces. A tag may then be added to the phage. For example a fluorescent tag. Once the phage has infected / bound the target Salmonella, the target Salmonella to which the tag is bound may be detected. For example, by washing the remaining phage from the sample and detecting the bound tag or tags within the Salmonella. The amount of tag present in the remaining sample may then be assessed. This may indicate the number of bacterial cells present. For example, the amount of fluorescence remaining after washing the remaining unbound phage from the sample.

[0167] Sequence identity

[0168] The sequence identity can be consistent with a phage deposit, for example, 80%, 85%, 90%, 95% or 100% nucleotide or amino acid sequence identity to the deposited phage.

[0169] Alternatively, sequence identity may be with reference to any one of SEQ ID NOs 1 to 10. For example, 80%, 85%, 90%, 95% or 100% nucleotide or amino acid sequence identity to any one of SEQ ID NOs 1 to 10. Sequence identity is calculated over the entire length of the sequence relative to the SEQ ID NO number.

[0170] Polymorphism

[0171] A single nucleotide polymorphism, also referred to herein as a SNP, represents a difference in a single nucleotide. For example, as a result of a SNP, the nucleotide cytosine (C) may be replaced by the nucleotide thymine (T) in a certain length of DNA.

[0172] Method for drying bacteriophage

[0173] The phage can be dried to provide a more convenient way of administration to animals and patients. Drying also increases the storage life of the phage. For example, if the dried phage is added to feed, no additional moisture is added. This also extends the shelf life of the product.

[0174] When ready to dry, the phage can be concentrated to about 10 10 pfu / ml or above.

[0175] The phage are mixed with a preservation solution to protect them during drying. The preservation solution may contain sugars and optionally amino acids and polymers as described below.

[0176] The phage may be mixed with the solution at a concentration of 1:10 v / v (phage:mixture).

[0177] Sugar / Sugar Alcohols

[0178] One or more sugars may be used in the preservation composition. For example, glucose or a glucose-based sugar such as mannitol. Glucose-based refers to a disaccharide or polysaccharide containing glucose.

[0179] The sugar may be a disaccharide.

[0180] The storage solution may contain sugar alcohol alone or in addition to one or more sugars. Suitable sugar alcohols include mannitol. Other 6-carbon sugar alcohols are also suitable.

[0181] For example, the storage solution may include one or more of trehalose, mannitol, and leucine.

[0182] Amino Acids

[0183] In addition to sugars and / or sugar alcohols, one or more amino acids may also be added to the storage solution.

[0184] The amino acid can be a non-polar aliphatic amino acid. For example, the amino acid can be leucine, isoleucine, glycine, alanine, proline, valine, methionine or phenylalanine.

[0185] polymer

[0186] Polymers can also be added to the preservation solution to help coat the phages and ensure they are not killed in the animal's intestines. The polymers can also help target the phages to the intestines where they are needed to work.

[0187] The polymer may comprise a copolymer derived from acrylates and methacrylates.The polymer may comprise acidic or basic groups which enable a pH-dependent release of the active ingredient.

[0188] Sample storage solution

[0189] The storage solution may contain trehalose, mannitol and leucine.

[0190] For example, the storage solution can contain 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% or more trehalose, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% mannitol and 0.5%, 1%, 2%, 3%, 4% or 5% leucine. For example, 8% trehalose, 8% mannitol, 1% leucine.

[0191] A polymer called Eudragit S100 (available from Evonik Health Care, Germany) was found to also help protect phages. For example, 2% Eudragit was added to a storage solution containing 8% trehalose, 8% mannitol and 1% leucine.

[0192] Dry mixture

[0193] The resulting solution of bacteriophage and sugar and optionally amino acids is then dried to form a powder which can be compacted to form tablets or pills.

[0194] To dry the solution, spray drying can be used.

[0195] The temperature used for drying the powder may be 70°C, 75°C, 80°C, 85°C, 90°C, 95°C or 100°C. The powder of bacteriophage is produced by drying the bacteriophage.

[0196] Phages produced by this method

[0197] Therefore, the bacteriophage / group / pharmaceutical composition or animal feed may additionally comprise any of the above-mentioned sugars, sugar alcohols, amino acids and / or polymer combinations.

[0198] For example, the phage solution or powder (after drying) can contain any of the above-mentioned sugars, sugar alcohols, amino acids and / or polymer combinations. For example, a phage solution or powder containing trehalose, mannitol and leucine.

[0199] Throughout the specification, unless the context requires otherwise, the term "comprises" or "comprising" or variations such as "comprising" or "comprising", will be understood to imply that the method or kit includes the stated integer or group of integers but not excluding any other integer or group of integers.

[0200] Each document, reference, patent application or patent cited in this article is expressly incorporated herein by reference in its entirety, which means that the reader should understand and consider it as part of this article. The documents, references, patent applications or patents cited in this article are not repeated and are only for the purpose of conciseness in this article. Reference to the material or information contained in this article should not be understood as part of the general knowledge or known in any country. Sequence Listing <110> University of Leicester <120> Therapeutic phages <130> P193346.WO.01 <150> 1815483.1 <151> 2018-09-24 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 903 <212> DNA <213> PhiKZ-like giant phage <400> 1 tacagctaat agaatttgac ctttgttgtc ggtccggtct gaaatctaag gtaactttag 60 atagcaatgc tgttggggatc tgtttcacaa ttgggtcatc gtggccccct tggataacat 120 tgtgatggtc cattgtgttg ctgcatgctc ctgttctggt ggcttcgcat attttgcata 180 gttatagcgt agcaccgcca tttcccgtga cttttccaat accccgacgg ctaacatgtc 240 ccgctgaaag gttactgtcc tgggccgggt gtccttgaat aagcaccgct gaccgtagca 300 ccgatgaaac cgtgtgactg tttgcttctt aaataagaat tggtacggct cgacttacca 360 attaaccga attacggac cttggttcga ggttgtaaca aggtatttaa gcaaaagtttt 420 cctgcgtaag acaagtaagg actgtgccac tgggcaaatc cctgctgcac cttgcttgtt 480 atggtcgtc cagaccggac cccttgcta ctgatgccaa agggcggtgc ctcgcagaga 540 tggtgattg tcgcagcctg taaattattt ctgccaatgc tcatacacca tgcaaacgga 600 gctgagcctc tgatgtcaat acccgtgtcg tggatggatt cgtcgccacc aataaaagtt 660 ctgccgctca ccgtgttgtg caagcgttcg aaaggcga tgcgcccgca gcattctgca 720 aaaccgctga atggctgcgc gaggccgcca tcaggttggc taagttcgcg ttgtaacaaa 780 cggttgccaa tactgttgcg gtgggggacc atgatagcgt cgttgagtgg tctgagtggt 840 agcatgtggt gaagctggtt atcgcgcagc catcaggtgc aatagcttga ccaagaatcg 900 att 903 <210> 2 <211> 900 <212> DNA <213> PhiKZ-like giant phage <400> 2 tacggataat gtaatgtgaa acgtgttgcc ttcggtgtcc gcgagctatc gtagctttag 60 atagcgttct gcggcccgtt gcggtggtaa ctacaattac gtggcccttg cggagaccgt 120 tgggaaggcc cactgcgatg tagcatgctt ctgttgcgac accttttgtt gtggtgaatg 180 gcaataacct atcgacgcca atttctgcca ctcgcacaga agttgtgggg atggcgggtt 240 cccaagaaaa atctgtggcc cggaccgggg gtctttgaca atatgccgct gaccgtgcga 300 cctatgaaac cttgtcattg cggtcgtctc aataagtgat tgtgccttga cttgccggag 360 caccacttat acaagtcaag gcggcaacca cggaccgtat ttaagtaaat gtttttggca 420 taagacaaat acaggctatt gagctattat ttgcgatcgg gcgtcaagta aatattggtc 480 ccctactaca tgccggtcct accgttacct atacatggcc cgacccgatg acgtgcattg 540 gttgccgcgt gccactgctt tttgccaatg cttatgcacc acgcgaacgg cataaacaag 600 ctgatgttta agacctgcag aatgcaacct tcgccattgt gactcaacat agagctgcca 660 ctcaccaaat tgtggaagcg cgcggacatg ttgatgtcac ccgtcaaagg cgcgaaactg 720 ctgtaaggcc agccaggtta gctaagccaa tgacgggttg ttagacgtaa gaaacgccga 780 tacttattgt gcaggtcagt caccatgttg tcaggtggaa tgggactcag cggcagcacc 840 tcattgatgc catggtgatg ttatgcgagg taacgccatg aacttgagca agacggaatt 900 <210> 3 <211> 240 <212> PRT <213> Myoviridae <400> 3 Met Phe Arg Thr Ile Leu Phe Leu Leu Val Ala Met Thr Thr Phe Asn 1 5 10 15 Ser Asn Ala Ser Tyr Gln Thr Thr Ser Asn Lys His Gln Lys Glu Phe 20 25 30 Val Leu Ile Gln Asp Gln Phe Asn Glu Ile Lys Pro Leu Ile Val Thr 35 40 45 Ala Ser Arg Lys Gln Gly Val His Ala Gly Met Leu Ala Thr Thr Ile 50 55 60 Tyr Arg Glu Ser Arg Phe Asn Lys Asn Val Gly Lys Asn Lys Ser Ser 65 70 75 80 Ser Ala Ser Ser Val Val Gln Met Thr Thr Gly Thr Lys Arg Ser Met 85 90 95 Ile Arg Leu Tyr Gly Lys Gln Leu Asn Ile Pro Lys Asn Ala Asp Leu 100 105 110 Asn Lys Pro Lys Tyr Ala Val Gln Leu Ala Ala Val Tyr Met Lys His 115 120 125 Ile Glu Glu His Leu Thr Lys Gln Leu Lys Arg Lys Pro Ser Thr Ala 130 135 140 Glu Ile Ala Leu Gly Tyr Arg Phe Gly Glu Gly Ala Ala Val Ala Met 145 150 155 160 Ile Lys Lys Lys Ser Pro Val Gly Lys Arg Trp Met Ala Ser Tyr Arg 165 170 175 Lys Asp Ala Ala Phe Tyr Gly Ala Lys Met Thr Pro Pro Lys Ala Glu 180 185 190 Thr Arg Gln Leu Ala Phe Ala Lys Glu Asp His Asp Gln Arg Val Ala 195 200 205 Glu Leu Gln Lys Ile Trp Asp Thr Leu Tyr Thr Lys Ile Ser Pro Ala 210 215 220 Ala Gly Thr Leu Leu Ala Asn Asn Thr Ile Met Lys Gly Ala Leu Leu 225 230 235 240 <210> 4 <211> 333 <212> PRT <213> Myoviridae <400> 4 Met Lys Thr Val Ala Lys Leu Phe Arg Ser Ser Ile Arg Ser Ala Glu 1 5 10 15 Phe Gln Asn Asn Val Gly Thr Asp Thr Val Arg Ala Arg Arg Leu Lys 20 25 30 Glu Phe Leu Lys Asp Glu Thr Tyr Ser Val Tyr Val Val Arg Asn Lys 35 40 45 Glu Thr Trp Glu Gly Tyr Leu Val Ala Thr Tyr Arg His Gly Asp Pro 50 55 60 Thr Leu Thr Cys His Tyr Phe Glu Glu Phe Val Pro Gly Ala Phe Asp 65 70 75 80 Lys Val Gly Leu Ala Leu Val Leu Asp His Thr Gly Phe Glu Tyr Met 85 90 95 Ser Cys Pro Ala Pro Thr Gly Asp Val Glu Gln Lys Leu Val Glu Lys 100 105 110 Gly Phe Val Leu Ser Asp Ile Val Trp Ala Cys His Ser Lys Leu Phe 115 120 125 Asp Lys Val Ile Thr Ala Ser Glu Ile Lys Ile Ile Ser Gly Asp Ala 130 135 140 Tyr Ala Glu Leu Ser Lys Asp Asp Lys Asp Val Ile Leu Asn Leu Val 145 150 155 160 Ser Gly Ala Ile Cys Glu Glu Leu Glu Tyr Asp Val Arg Gly His Tyr 165 170 175 Thr His Ile Gly Pro Leu Ala Glu Thr Val Ile Ala Arg Ile Gln His 180 185 190 Ala Asn Val Glu Thr Ala Ile Trp Arg Asp Gly Arg Asp Ile Val Gly 195 200 205 Ile Ala Gln Met Arg Leu Val Ala Pro Gly Val Val Glu Leu Ser Gly 210 215 220 Leu Thr Val Ala Pro Thr His Arg Lys Arg Gly Ile Gly Arg Arg Leu 225 230 235 240 Met Gly Ala Leu Phe Asp Phe Ala Gln Arg His Ser Asp Gln Val Tyr 245 250 255 Ile Glu Thr Ala Ala Asp Asn Asn Pro Ala Asn His Leu Tyr Gly Asn 260 265 270 Ile Leu Arg Cys Lys Gln Val Leu Arg Thr Leu Thr Leu Lys Arg Asp 275 280 285 Pro Glu Leu Ile Ser Trp Lys Arg Val Lys Gly Asn Arg Ala Asp Arg 290 295 300 Val Leu Lys Glu Glu Pro Ser Leu Pro Val Glu Thr Asn Pro Arg Pro 305 310 315 320 Thr Ile Asn Asp Met Phe Gln Ser Gly Met Arg Gly Cys 325 330 <210> 5 <211> 154 <212> PRT <213> Myoviridae <400> 5 Met Lys Ile Val Phe Val Thr Lys Pro Asn Ser Pro Tyr Gly Lys Lys 1 5 10 15 Tyr Tyr Ala Asp Leu Leu Gly Leu His Asn Lys Leu Tyr Asp Asp Gln 20 25 30 Lys Ile Lys Leu Ala Asp Leu Asn Gly Arg Glu Gly Phe Asn Gln Ile 35 40 45 Ser Lys Leu Glu Glu Val Trp Gly Glu Gly Thr Leu Cys Ala Cys Ala 50 55 60 Val Asp Asp Arg Gly Val Ala Val Gly Phe Ile Thr Phe Gly Tyr Thr 65 70 75 80 Lys Lys Gly Gln Arg Phe Leu Trp Val Tyr Asn Phe Phe Val Asp Glu 85 90 95 Ser Val Arg Ser Gln Gly Val Gly His Glu Leu Ile Glu Ala Val Glu 100 105 110 Asn Tyr Gly Lys Ser Lys Gly Cys Ala Trp Met Gln Leu Asn Val Leu 115 120 125 Gly Asn Asn Asp Arg Ala Ile Ala Phe Tyr Glu Arg Phe Gly Phe Arg 130 135 140 Thr Glu Tyr Gln Asp Met Val Lys Glu Leu 145 150 <210> 6 <211> 425 <212> PRT <213> Myoviridae <400> 6 Met Ala Asn Phe Val Lys Ser Lys Leu Ala Arg Glu Ser Val Glu Ala 1 5 10 15 Thr Gly Asp Val Ile Asp Gly Leu Ser Asn Val Glu Pro Ala Glu Glu 20 25 30 Asn Ile Asp Val Gln Leu Ala Glu Val Ala Ser Ile Asp Gly Gln Leu 35 40 45 Glu Gln Leu Asp Gly Asp Gln Glu Thr Leu Ala Ala Asp Thr Glu Arg 50 55 60 Thr Glu Ala Ala Ile Asp Ala Ala Asp Glu Lys Ile Glu Asn Gly Glu 65 70 75 80 Glu Met Pro Glu Glu Ala Ile Ala His Thr Glu Val Ala Gln Glu Ser 85 90 95 Ile Arg Lys Arg Trp Ala Ile Asp Arg Thr Lys Leu Ala Arg Glu Ser 100 105 110 Tyr Arg Arg Gly Arg Gly Met Thr Lys Ala Ala Gln Glu Gly Trp Lys 115 120 125 Glu Thr Leu Lys Asp Leu Phe Lys Arg Phe Val Glu Leu Cys Lys Ala 130 135 140 Val Ile Ala Lys Ala Lys Glu Leu Lys Leu Lys Tyr Ile Asn Val Gly 145 150 155 160 Lys Ser Ala Gln Lys Arg Ala Lys Ala Tyr Gln Glu Met Leu Arg Lys 165 170 175 Leu Gly Lys Gln Lys Lys Glu Asn Ile Ser Gly Gly Phe Ile Ser Lys 180 185 190 Leu Ser Ile Glu Gly Asp Phe Asp Val Ala Asn Ser Ile Ala Ile Ala 195 200 205 Lys Glu Leu Thr Gly Gly Lys Ala Lys Asp Ala Ile Asn Lys Leu Ser 210 215 220 Ser Gln Ala Ser Glu Ser Ala Val Ala Ile Thr Lys Thr Ala Glu Gly 225 230 235 240 Thr Ala Thr Ala Ala Lys Gly Ala Val Asp Val Ala Leu Phe Gly Thr 245 250 255 Ala Ala Lys Lys Leu Arg Thr Leu Pro Gln Phe Glu Asp Gly Glu Gly 260 265 270 Gly Gln Lys Val Leu Ala Leu Pro Gly Asn Ala Tyr Val Gln Ile Gly 275 280 285 Ser Lys Thr Leu Ala Gly Gly Gln Asp Phe Thr Ala Val Ala Phe Leu 290 295 300 Ser Thr Gly Asp Ser Thr Asp Thr Lys Glu Ile Ala Thr Pro Ala Ile 305 310 315 320 Thr Ala Leu Ala Ser Ala Ala Thr Ala Leu Asp Ala Ile Gly Lys Gly 325 330 335 Phe Glu Lys Val Leu Gln Asp Phe Arg Ser Tyr Asp Ala Asp Val Glu 340 345 350 Lys Leu Glu Gln Ala Ala Ser Lys Ala Ala Ala Ala Leu Asp Lys Ser 355 360 365 Asn Asp Glu Gly Glu Trp Glu Ala Leu Arg Asn Ala Arg Thr Ala Ala 370 375 380 Asp Gln Ala Val Arg Asn Tyr Gln Thr Leu Asn Arg Ala Val Ala His 385 390 395 400 Val Gly Asn Thr Val Ile Ser Gly Leu Asn Gly Tyr Ile Gly Ala Gly 405 410 415 Ile Gly Ala Tyr Glu Lys Ser Lys Ala 420 425 <210> 7 <211> 568 <212> PRT <213> Myoviridae <400> 7 Met Val Glu Ile Gln Met Thr Tyr Thr Ser Met Gly Val Arg Val Glu 1 5 10 15 Val Pro Ile Arg Gln Ala Glu Arg Ala Ile Leu Ala Trp Ala Glu Glu 20 25 30 Asn Met His Ala Pro Lys Met Gly Lys Gln His Gly Arg Ile Thr Thr 35 40 45 Glu Arg Gly Asp Ala Tyr Tyr Ala His Ile Pro Ser Leu Arg Thr Phe 50 55 60 Ile Phe His Lys Val Phe Ala Glu Lys Ile Lys His Ile Leu Gln Arg 65 70 75 80 Ala Ala Ile Glu Tyr Ala Phe Gln Tyr Lys Leu Thr Glu His His Pro 85 90 95 Glu Arg Gly Thr Pro Tyr Ser Cys Thr Phe Asp Asn Tyr Gly Phe Ser 100 105 110 Met Val Glu Ser Asp Pro Glu Ser Arg Phe Tyr Tyr Gln Asn Glu Val 115 120 125 Val Asp Ala Ala Ser Asp Pro Asn Arg Pro Gln Thr Ile Phe Ala Ile 130 135 140 Gln Thr Gly Arg Gly Lys Thr Lys Ser Cys Met Lys Ser Met Val Lys 145 150 155 160 Arg Gly Thr Arg Thr Ala Leu Ile His Arg Pro Ser Tyr Val Pro Lys 165 170 175 Trp Leu Phe Asp Val Cys Asp Asp Glu Thr Gly Leu Arg Ile Leu Arg 180 185 190 Asp Glu Val Leu Val Cys Thr Gly Val Gln Ala Ile Tyr Asp Ala Leu 195 200 205 Glu Met Gly Lys Ser Gly Glu Leu Asp Arg Arg Gly Ile Lys Val Ile 210 215 220 Ile Leu Pro Thr Val Ser Leu Gln Arg Phe Leu Lys Glu Tyr Ile Asn 225 230 235 240 Thr Ala Ala Thr Asn Pro Val Asp Leu Asp Thr Phe Tyr Asp Val Leu 245 250 255 Gly Val Gly Leu Val Ala Met Asp Glu Val His Glu His Phe His Leu 260 265 270 Val Tyr Met Ala Gly Ile Met Leu Asn Pro Pro Ala Ser Ile Glu Met 275 280 285 Ser For Thr Leu Lys Pro Gly Ser For Lys For Phe Ile Ala Glu Arg 290,295,300 Tyr Leu Glue Arg Phe Pro Met Glue Tyr Arg Ile Ser Ile Pro Ile Ile 305 310 315 320 Pro Val Val Asp Val Lys Ala Leu Tyr Tyr Arg Leu Asp Asp Lys Lys 325 330 335 Phe Ala Trp Trp Ala Ser Lys Met Thr Pro Tyr Asn His Lys Leu Phe 340 345 350 Glu Gly Lys Ile Lys Glu Asn Leu His Leu Glu Tyr Ala Asn Met 355 360 365 The Trp Asp Val Val Glu Lys Served By Leu Lys Arg Tyr Gln Pro Gly 370 375 380 Gln Lys Cys Leu Leu Leu Phe Ala Thr Val Ala Met Cys Glu Phe Phe 385 390 395 400 Thr Glu Tyr Val Lys Asp Lys Leu Ser Arg Asp Pro Val Phe His Pro 405 410 415 Leu Met Val Ala Lys Tyr Asn Ala Gly Asp Ser Tyr Asp Asp Phe Ile 420 425 430 Gln Ala Asp Phe Ser Ile Ser Thr Pro Gly Lys Ala Gly Thr Ala Val 435 440 445 Asp Lys Pro Gly Leu Val His Met Tyr Ile Ser Thr Pro Val Glu Asp 450 455 460 Gln Gln Leu Asn Glu Gln Met Ala Gly Arg Pro Arg Lys Ile Leu His 465 470 475 480 Asn Glu Trp Gly Glu Ile Asp Pro Thr Val Trp Leu Phe His Ala Tyr 485 490 495 Asn Val Pro Lys His Cys Asn Tyr Leu Asn Ala Arg Gln Lys Ser Leu 500 505 510 Lys Asp Val Val Leu Ser Phe Arg Ile Ala Thr Ser Pro Tyr Val Val 515 520 525 Arg Lys Ser His Ala His Ser Ala Ala Ser Ser Arg Ala Thr Ala Ala 530 535 540 Leu His Arg Asn Asp Phe Ser Lys Phe Ser Arg Lys His Ser Lys Gly 545 550 555 560 Val Ser Arg Arg Arg Arg Arg Arg 565 <210> 8 <211> 162 <212> PRT <213> Myoviridae <400> 8 Met Thr Thr Ile Ala Phe Asp Gly Lys Val Leu Ala Ala Asp Gly Gln 1 5 10 15 Leu Thr Arg Gly Ser Asn Ile Cys Asn Leu Asn Thr Gln Lys Leu Phe 20 25 30 Ile Cys Pro Ser Asp Glu Glu Trp Trp Ile Thr Gly Glu Arg Ile Val 35 40 45 Ala Phe Gly Val Ser Gly Asp Ile Thr Gly Gln Leu Ala Leu Leu Glu 50 55 60 Thr Val Arg Leu Arg Pro Gly Tyr Lys Gly Leu Thr Gln Ser Ser Gln 65 70 75 80 Leu Pro Ala Lys Met Asp Phe Thr Phe Leu Met Leu Leu Ala Ser Gly 85 90 95 Lys Ala Ile Val Gly Gly Lys Arg Glu Asp Asp Val Thr Leu Trp Trp 100 105 110 Ser Glu Ala Thr Pro Pro Leu Ala Thr Gly Ser Gly Tyr Glu Tyr Ala 115 120 125 Leu Gly Ala Met Lys Met Gly Ala Asn Ala Val Arg Ala Val Glu Ile 130 135 140 Ala Ser Glu Cys Asp Ile Tyr Thr Gly Gly Glu Ile Ser Thr Tyr Glu 145 150 155 160 Leu Arg <210> 9 <211> 300 <212> PRT <213> PhiKZ-like jumbo phage <400> 9 Met Ser Ile Ile Leu Asn Trp Lys Gln Gln Pro Gly Gln Thr Leu Asp 1 5 10 15 Ser Ile Glu Ile Tyr Arg Tyr Asp Asn Pro Arg Gln Ser Val Asn Pro 20 25 30 Val Ala Pro Gly Glu Pro Ile Val Thr Leu Pro Gly Asn Thr Thr Thr 35 40 45 Tyr Glu Asp Lys Thr Thr Glu Ala Tyr Lys Thr Tyr Gln Tyr Arg Ile 50 55 60 Val Ala Val Lys Gly Thr Glu Lys Val Met Gly Leu Pro Ile Val Gln 65 70 75 80 Gly Asp Phe Pro Met Thr Gly Pro Gly Pro Gln Glu Leu Ile Arg Gly 85 90 95 Asp Trp His Arg Gly Tyr Phe Gly Thr Leu Thr Asn Glu Glu Phe Ile 100 105 110 Leu Asn His Ala Glu Leu Asn Gly Leu Ile Gly Phe Asn Ala Trp Asn 115 120 125 Gln Ala Pro Thr Leu Phe His Lys Phe Val Phe Lys Gly Arg Ile Leu 130 135 140 Phe Ile Pro Asp Thr Val Thr Arg Leu Gly Thr Thr Trp Asn Glu Gln 145 150 155 160 Tyr Gln Gln Gly Leu Ala Trp Gly Thr Asp Asp Tyr Gly Phe Pro Pro 165 170 175 Arg Ser Val Ser Thr Thr Asn Gln Arg Arg Thr Phe Asn Lys Asp Gly 180 185 190 Tyr Glu Tyr Val Val Arg Leu Pro Arg Leu Gly Asp Tyr Ser Tyr Gly 195 200 205 His Ser Thr Tyr Leu Ser Ser Gly Gly Tyr Phe Gln Asp Gly Glu Trp 210 215 220 His Asn Thr Phe Ala Ser Leu Phe Arg Tyr Ala Gly Val Val Arg Arg 225 230 235 240 Phe Gly Asp Leu Pro Thr Arg Ser Gly Gly Ser Pro Thr Asp Ser Ser 245 250 255 Ala Thr Leu Phe Ala Asn Gly Tyr Asp Asn Ala Thr Pro Trp Tyr Tyr 260 265 270 Arg Ser Asn Ser Pro Asp Ser Pro Ser Tyr Thr Thr Ser Thr Asn Ser 275 280 285 Ala Ser Val Val His Val Ile Glu Leu Val Leu Ser 290 295 300 <210> 10 <211> 299 <212> PRT <213> PhiKZ-like jumbo phage <400> 10 Met Pro Ile Thr Leu His Phe Ala Gln Arg Lys Pro Gln Ala Leu Asp 1 5 10 15 Ser Ile Glu Ile Tyr Arg Lys Thr Pro Gly Asn Ala Thr Ile Asp Val 20 25 30 Asn Ala Pro Gly Thr Pro Leu Ala Thr Leu Pro Gly Asp Ala Thr Ser 35 40 45 Tyr Glu Asp Asn Ala Val Glu Asn Asn Thr Thr Tyr Arg Tyr Trp Ile 50 55 60 Ala Ala Val Lys Asp Gly Glu Arg Val Phe Asn Thr Pro Thr Ala Gln 65 70 75 80 Gly Phe Phe Leu Asp Thr Gly Pro Gly Pro Gln Lys Leu Leu Tyr Gly 85 90 95 Asp Trp His Ala Gly Tyr Phe Gly Thr Val Thr Pro Ala Glu Leu Phe 100 105 110 Thr Asn Thr Glu Leu Asn Gly Leu Val Val Asn Met Phe Ser Ser Ala 115 120 125 Val Gly Ala Trp His Lys Phe Ile Tyr Lys Asn Arg Ile Leu Phe Met 130 135 140 Ser Asp Asn Ser Ile Ile Asn Ala Ser Pro Gln Phe Ile Tyr Asn Gln 145 150 155 160 Gly Met Met Tyr Gly Gln Asp Gly Asn Gly Tyr Val Pro Gly Trp Ala 165 170 175 Thr Ala Arg Asn Gln Arg Arg Thr Val Thr Lys Asn Gly Tyr Glu Tyr 180 185 190 Val Val Arg Leu Pro Tyr Leu Phe Asp Tyr Lys Phe Trp Thr Ser Tyr 195 200 205 Val Gly Ser Gly Asn Thr Glu Leu Tyr Leu Asp Gly Glu Trp Phe Asn 210 215 220 Thr Phe Ala Arg Leu Tyr Asn Tyr Ser Gly Gln Phe Pro Arg Phe Asp 225 230 235 240 Asp Ile Pro Val Gly Pro Ile Asp Ser Val Thr Ala Gln Gln Ser Ala 245 250 255 Phe Phe Ala Ala Met Asn Asn Thr Ser Ser Gln Trp Tyr Asn Ser Pro 260 265 270 Pro Tyr Pro Glu Ser Pro Ser Trp Ser Asn Tyr Gly Thr Thr Thr Ile 275 280 285 Arg Ser Ile Ala Val Leu Glu Leu Val Leu Pro 290 295 <210> 11 <211> 10 <212> PRT <213> PhiKZ-like jumbo phage <400> 11 Ser Ser Gln Trp Tyr Asn Ser Pro Pro Tyr 1 5 10 <210> 12 <211> 10 <212> PRT <213> PhiKZ-like giant phage <400> 12 Ser Ser Gln Trp Tyr Asn Ser Pro Pro Tyr 1 5 10 BRIEF DESCRIPTION OF THE DRAWINGS

[0201] FIG. 1 shows the sample sites where SPFM Salmonella phages were isolated in the UK and their phenotypic structure.

[0202] A. Multiple samples were sourced from the United Kingdom where phages were isolated. (1) Wild boar nature reserve in Hampshire (SPFM1–SPFM3), (2) Food processing plant in Essex (SPFM4–SPFM11), (3) Pig farm in Warwickshire (SPFM12–SPFM15), (4) Pig farm in Hinckley, Leicestershire (SPFM16–SPFM20), and (5) Pig nature reserve in West Sussex (SPFM21–SPFM22). Map of the United Kingdom from Crown Copyright and Database Right

[2017] licensed. Ordnance Survey (Digital Map License). B. All 22 isolated phages were identified as Myoviridae by transmission electron microscopy and representative micrographs are given. Black bars represent 100 nm.

[0203] Figure 2 shows the host range analysis of 21 SPFM Salmonella phages and their inoculation efficiency. A. The host range analysis was based on the lysis spectrum of SPFM phages, targeting multi-antibiotic resistant strains: 25 Salmonella typhimurium, 15 S.4,12:i>, 10 S.4,5,12:i>, 10 S.bovis and 10 Salmonella delbrueckii, and complete lysis was given by orange bars, red bars, green bars, blue bars and purple bars (from left to right), respectively. All strains were isolated from pigs. The light orange bars (left column) show turbid clear liquid, and the light purple (right bar) shows no infection. B. The inoculation efficiency of SPFM phages against 11 Salmonella isolates and the data given were analyzed by principal component analysis. The biplot represents the two main components with the largest variance (59.3% of the total variance), the EOP of SPFM phages (marked as black circles) from two representative isolates of five swine-associated Salmonella serotypes and the EOP of the phage propagation host, Salmonella Typhimurium SL1344 (11 strains in total). The color of the phages depends on their EOP, where red (left) phages show the same EOP on all isolates and green (right) phages have higher EOP on specific strains and are located closer to individual strains (black arrows and text). For host range analysis (A) and EOP analysis (B), three biological replicates were performed for all strains, and the data presented are the average of all three replicates.

[0204] Figure 3. A representative example of SPFM phages and the genome of phage SPFM1 is shown. The inner black circle indicates the GC content, the outer circle indicates the predicted open reading frame, and the scale unit is base pairs. Functional genes belonging to different categories are colored accordingly: red is structural proteins, green is genes involved in DNA replication and transcription, yellow is packaging genes, orange is lytic genes, purple highlights additional genes, and tRNA is marked in blue (second item from the far right column). Unmarked ORFs represent hypothetical proteins.

[0205] Figure 4 Phages of 21 SPFM Salmonella phages generated by hierarchical clustering analysis based on the presence and absence of auxiliary genes. Jaccard distance, a measure of dissimilarity between phages, was calculated based on the presence and absence data of 46 auxiliary genes and hierarchical clustering. Unit 0 indicates the presence of the same gene and 1 indicates maximum variation.

[0206] Figure 5A. Heatmap of pairwise average nucleotide identity (ANI) values ​​for 158 whole-genome sequenced Salmonella phages in the NCBI database, including the 21 SPFM phages in this study. Values ​​range from 0 (0%) ANI to 1 (100% ANI) and are coded in purple to green and yellow, respectively, with the latter representing highly similar phage clusters and branches representing distinct clusters. The SPFM phages are all clustered together on the heatmap, and the cluster is set in the upper right (solid yellow box, indicated by a white arrow).

[0207] Figure 6 Phylogenomic tree showing the relationships among the 21 SPFM phages and two similar enterobacterial phages within the cluster, SEGD1 and SPN3US. The tree was constructed from single nucleotide polymorphisms (SNPs) obtained from codon alignments of 188 core genes shared by all 23 sequenced phages and is drawn to scale.

[0208] Figure 7. Stability of Salmonella phage at different temperatures (a) and pH (b). Phages were incubated for one hour at different temperatures and pH and titrated on their propagation hosts. The stability of phage SPFM2 is shown in blue B, SPFM4 in purple P, SPFM10 in red R, SPFM14 in green G, SPFM17 in orange O, and SPFM19 in silver S (color references along the top of the figure showing the order). The data given are the average of three biological replicates, each with three technical replicates, and the error bars represent the standard error of the mean (SEM). See also Table 4.

[0209] Figure 8. Efficacy of different combinations of 1, 2, 3 and 4 phage cocktails in reducing MDR S01160-12 Salmonella typhimurium in vitro. Bacterial cultures were infected with different phage cocktails (af) at an MOI of 100 and bacterial counts (CFU / ml) were determined every hour for 6 hours. Data presented are the mean of three biological replicates, each with three technical replicates, and error bars represent SEM.

[0210] Figure 9. Efficacy of phage mixtures against MDR S01160-12 Salmonella typhimurium in a larval infection model. The phage mixtures tested were: 2-phage of SPFM10-SPFM14 (red line / bar (r)); 3-phage of SPFM4-SPFM10-SPFM19 (blue line / bar (b)); and 4-phage SPFM2-SPFM10-SPFM14-SPFM19 (green line / bar (g)). Salmonella was used at 10 5CFU / larvae, or prophylactic treatment P-1 to P-3 with phage cocktail 1 hour before infection (a, c, and e), or simultaneous administration of phage and Salmonella CoI-1 to CoI-3 (b, d, and f). (a) and (b) indicate larval survival over 72 hours. The orange line indicates healthy larvae that were not infected in group C-1, the gray line indicates larvae that were only administered PBS in group C-2, and the black line indicates the survival of larvae that were only infected with Salmonella in group C-6. The box plots (c) and (d) (colors are black, red, blue, green from left to right) show the changes in Salmonella counts in different larval treatment groups, and the black bars indicate the counts of larvae that were only infected with Salmonella. The box plots (e) and (f) (colors are red, blue, green from left to right) show the phage counts of the phage cocktail used for prophylactic and co-infection treatments. Error bars represent SEM and statistically significant differences (*p≤0.05, **p≤0.01 and ***p≤0.001) between larvae infected with Salmonella alone and treated with phages are shown.

[0211] Fig.10 .Plaque formation rate of phages on Salmonella colonies isolated from a larval infection model. Larvae were infected with the MDRS01160-12 Salmonella typhimurium strain and treated with 2-phages (SPFM10 and SPFM14), 3-phages (SPFM4, SPFM10 and SPFM19) or a 4-phage mixture (SPFM2, SPFM10, SPFM14 and SPFM19). If Salmonella was recovered after 72 hours, colonies were picked and for 10 colonies in each group, phage plaque formation rate was assessed for the phages in the mixture. The plaque formation rate was then compared to the plaque formation rate of phages on the wild-type strain. Red error bars represent colonies picked after treating larvae with a 2-phage mixture, blue error bars represent 3-phage mixtures, and green error bars represent 4-phage mixtures. The average plaque formation rate is shown by three biological replicates, and the error bars represent SEM.

[0212] Figure 11 provides Table A. Viral (v) RNAP and non-viral (nv) RNAP subunits present in all SPFM phages when compared to annotated and predicted RNAPs in phage SPN3US.

[0213] Figure 12 provides Table B. Figure 5 Heat A map of all Salmonella phage genomes and their List of order in the figure .

[0214] Example

[0215] Aspects and embodiments of the present invention will now be described, by way of example only, and with reference to the following experiments.

[0216] Data presented Characterization of 21 Salmonella phages isolated in this study, termed SPFM phages. Phage killing activity was tested against prevalent and relevant circulating multidrug-resistant UK swine Salmonella strains isolated from outbreaks on UK farms. Inoculation efficiency and host range of all SPFM phages were determined. They were also sequenced, studied by phylogenomic analysis, compared to all previously sequenced Salmonella phages in the NCBI database and their positive selection genes were identified. This is the first study to comprehensively characterize and sequence a large number of phages targeting UK swine-related Salmonella strains.

[0217] Experimental Procedure

[0218] Bacterial strains and culture conditions

[0219] A total of 68 Salmonella strains were used in this study. Salmonella enterica subsp. enterica serotype SL1344 (accession number FQ312003) was used as a reference strain. The remaining 67 Salmonella enterica subsp. enterica strains were isolated from pigs in the United Kingdom at the Animal and Plant Health Establishment (APHA) in Weybridge, United Kingdom, between 2012 and 2015. The serotypes of these strains were: 22 S. enterica serotype typhimurium; 15 S.4,12:i>; 10 S.4,5,12:i>; 10 S. bovis; and 10 S. delbrueckii. All isolates were resistant to at least one of the following antibiotics: nalidixic acid, tetracycline, neomycin, ampicillin, furazolidone, ceftazidime, sulfamethoxazole, trimethoprim, chloramphenicol, amikacin, amoxicillin / clavulanic acid, gentamicin, streptomycin, trimethoprim, cefotaxime, apramycin, and ciprofloxacin.

[0220] All Salmonella strains were maintained in 50% glycerol broth (Abtek Biologicals Ltd., UK) at -80°C. Strains were routinely cultured on xylose lysine deoxycholate (XLD) agar (Oxoid, UK) at 37°C for 18 hours and then in NZCYM broth (Melford Biolaboratories Ltd, UK) at 100 rpm at 37°C for another 18 hours.

[0221] Isolation, purification and propagation of bacteriophages

[0222] All samples were collected from five geographical locations in the UK between July and December 2015. A total of 15 samples were collected from a wild boar reserve in Hampshire; 10 samples were collected from a food processing plant in Essex; 15 samples were collected from finishing pig farms in Warwickshire; 18 samples were collected from piglet and finishing pig farms in Leicestershire; and seven samples were collected from a wild boar reserve in West Sussex ( Figure 1A ).

[0223] All samples were processed using the same enrichment procedure, 1 ml or 1 gram of sample was mixed with 9 ml of NZCYM broth and 100 μl of exponentially growing Salmonella culture was added. In order to maximize phage separation, the same sample was aliquoted and enriched with 12 different MDR Salmonella host strains. The enrichment was incubated with shaking at 37 ° C for 12 hours (100 rpm), and then the sample was centrifuged at 4000 × g for 15 minutes at room temperature. The supernatant was filtered through a 0.22 μm pore size syringe filter (Millipore, UK), and the filtered sample was stored at 4 ° C until further use. The phage in the filtrate was screened by a small droplet plaque assay (Mazzocco et al., 2009). Briefly, 10 μl of the enriched sample was spotted on Luria-Bertani (LB) 1% (w / v) agar plates (Fisher Scientific, UK) with NZCYM 0.5% (w / v) agar as the top layer and mixed with 100 μl of exponentially growing Salmonella culture. The plates were incubated at 37°C for 18 hours and phage lysis was examined by the presence of clearance or phage plaques.

[0224] For phage purification, a single plaque was picked with a 1 μl loop, mixed with 500 μl of SM buffer containing gelatin (100 mM NaCl, 8 mM MgS04.7H2O, 50 mM Tris-Cl and 0.01% (w / v) gelatin), and centrifuged at 21,000 × g for 10 minutes. The resulting supernatant was used for the next round of phage purification by double agar overlay plaque assay (Kropinski et al., 2009), and the process was repeated 7 times to generate clonal phage stocks.

[0225] 10 infected cells were cultured in NZCYM broth at 37°C with shaking (100 rpm) for 6 hours. 7 PFU / ml of Salmonella phage SL1344 (10 7Purified phage lysate was prepared by mixing exponentially growing liquid cultures of 100 μg / ml (PFU / ml). The phage culture was centrifuged at 4,200 x g for 15 minutes, the supernatant was filtered with a 0.22 μm pore filter, and the phage lysate was stored at 4 ° C. To determine the phage titer, the phage lysate was serially diluted 10 times and plated on 1% LB agar plates using a small droplet plaque assay (Mazzocco et al., 2009). The final phage titer was expressed in PFU / ml.

[0226] Transmission electron microscopy

[0227] Prior to transmission electron microscopy (TEM) analysis, phage lysates were concentrated by centrifugation at 21,000 × g for 1 h, the pellets were resuspended in 0.1 M ammonium acetate (Fisher Scientific, UK), centrifuged at 21,000 × g for 1 h, and resuspended in 0.1 M ammonium acetate solution. 11 PFU / ml) were negatively stained with 1% uranyl acetate (w / v) for 10 seconds and applied to 3 mm carbon coated copper grids (Agar Scientific Ltd, UK). Phages were detected using an EOL1220 (Joel UK Ltd, UK) at 80 kV and images were acquired using a SIS Megaview III camera with analysis software (Olympus Software Imaging Solutions, Germany) (Ackermann 2009). TEM analysis was performed by Dr. Ali Ali and Natalie Allcock, Core Biotechnology Services, University of Leicester, UK.

[0228] Phage host range analysis and efficiency of plating

[0229] The host range of individual phages was determined by small drop plaque assay (Mazzocco et al., 2009) on different S. enterica subspecies enterica serovars and incubated for 18 h at 37 °C. Plates were checked for bacterial lysis (by clearing or plaques) or absence of infection, and the average observations were taken from three biological replicates, each with three technical replicates.

[0230] The plaque efficiency (EOP) was performed on two representative MDR strains from the following five Salmonella serotypes: Salmonella typhimurium; S.4,12:i>; S.4.5,12:i>; S. bovis; and Salmonella delbrueckii. EOP was also established on the phage propagation host S1344. For EOP, the droplet plaque assay was used, and the phage lysate was serially diluted 10 times and spotted on the bacterial lawn (Kutter, 2009). The average PFU / ml EOP value was based on three biological replicates, each with three technical replicates. The EOP values ​​of all phages on all strains screened using the prcomp function in the R base package (R Core Team, 2017) were subjected to principal component analysis and plotted as a biplot using the autoplot function in the ggplot2 package (Wickham, 2009).

[0231] Phage DNA extraction, sequencing and annotation

[0232] A modified phenol-chloroform-isoamyl method was used with high titer phage lysate (10 10 PFU / ml) to extract DNA (Nale et al., 2016). After extraction, the final DNA pellet was dissolved in 5mM Tris HCl, quantified using a Qubit fluorometer and Qubit double-stranded HS kit (Thermo Scientific, UK), and sequenced by the Illumina MiSeq platform. NexteraXT libraries were prepared using 1ng input DNA according to the manufacturer's instructions. Reads were trimmed using Sickle v1.33 (Joshi and Fass, 2011) before assembly using SPAdes v3.9.1 (Bankevich et al., 2012). Prokka v1.11 (Seemann 2014) was used for annotation with a custom database built from all phage proteins (December 2017) and hmmscan to identify pVOG (Grazziotin et al., 2017). Table 1 lists the accession numbers assigned to the phage genomes. An error occurred when uploading the phage genomes to the European Nucleotide Archive (ENA), and the phage names were incorrect. Even so, the accession numbers listed in Table 1 are correct and allow differentiation of phages. This issue is currently being addressed at ENA and can be found at this link http: / / s3.climb.ac.uk / Sinfo / SPFM_genome.tar.gz Find the correct file with the correct name.

[0233] Calculation of ANI between Salmonella phage genomes

[0234] A total of 158 genomes from Salmonella-infecting phages were collected from the two newly sequenced phages in this study and phages sequenced in previous studies. The average nucleotide identity defined by (Goris et al., 2007) was measured between all pairwise combinations of phage genomes using the BLASTN alignment option in the pyani package (Pritchard et al., 2016) and plotted in an interactive heatmap using heatmaply (Galili et al., 2017).

[0235] Protein ortholog clustering

[0236] Prokka-annotated phage protein sequences were clustered into Orthologous group Jaccard distances between each phage genome were based on the presence / absence of genes determined by COG clustering of phage protein sequences. Jaccard distances were calculated using the base R dist function, dendrograms were constructed from distances using the base R hclust function, and dendrograms were plotted using the ggdendro package in R (Vries and Ripley, 2016). Ortholog clustering data are shown in Supplementary Table 4 as published in Thanki AM, Brown N, Millard AD, Clokie MRJ. Genomic Characterization of Jumbo Salmonella Phages That Effectively Target United Kingdom Pig-Associated Salmonella Serotypes. Front Microbiol. 2019;10:1491. Published Jul 2, 2019, doi:10.3389 / fmicb.2019.01491.

[0237] Hypothetical core gene-based phylogenomic tree

[0238] Codon recognition alignments of the corresponding nucleotide sequences were constructed using COG containing protein sequences of Seoul virus phage genomes using MUSCLE v3.8.31 (Edgar, NAR 32(5)) and the pal2nal.pl script (Suyama et al., 2006). SNPs from the codon recognition nucleotide alignments were extracted using SNP Sites et al. (Page et al., 2016) and used to construct phylogenetic trees using FastTree v.2.1.10 SSE3 (Price et al., 2010) using a generalized reversible model of nucleotide evolution.

[0239] Measuring positive selection

[0240] Positive selection tests were performed on each codon of each orthologous orthologous cluster using the HyPhy software package (Pond et al., 2005). In particular, protein alignments from each orthologous orthologous cluster of MUSCLE v3.8.31 were used to guide the construction of codon recognition alignments for nucleotide sequences from each cluster. The GARD algorithm (Pond et al., 2006) implemented in the HyPhy software package was used to detect the recombination breakpoints in each codon recognition alignment, and the alignments and trees of the partitions were constructed on either side of each breakpoint. The Bayesian FUBAR algorithm (Murrell et al., 2013) implemented in the HyPhy software package was used to test the positive selection of these partitions at each site, which returns the posterior probability (i.e., dN / dS greater than 1) that each site is under positive selection. Because the sample size is too small to accurately evaluate the dN / dS of a single codon, the posterior probability values ​​are given together with the dN / dS values. This hypothesis test was sensitive enough to detect positive selection, and the PSRF / N effective ratio remained below 0.006, causing the posterior probability of positive selection to converge to a solution.

[0241] result

[0242] Several in vitro and in vivo studies have shown that phages can significantly reduce the number of Salmonella in food dishes and pigs, and may therefore provide a viable option for reducing Salmonella in pigs to improve food safety. However, to date, not all published studies have fully characterized their phages in terms of infectivity and sequencing. Sequencing is useful for phages used in therapy because it can confirm that the phages are obligately lytic, they do not carry toxin genes, they do not belong to specific phage genera (e.g., Felixo1 virus genus) that are known to promote the spread of host antibiotic resistance genes and help to rationally optimize phage therapy mixtures. The inventors have characterized 21 new lytic giant SPFM Salmonella phages isolated from environmental sources, which again target Salmonella commonly associated with pigs. All SPFM phages have a wide host range and high EOP values ​​for clinically relevant UK MDR Salmonella strains isolated from pigs, and can be used for phage therapy, which can help improve food safety.

[0243] Isolation, plaque morphology and propagation of bacteriophages

[0244] Samples for phage isolation were collected from food processing plants, wild and domestic pigs and wild boars in the UK between July and December 2015. Sixty-five samples were screened and 21 phages were isolated, named SPFM1 to SPFM22. Phage SPFM18 was excluded from the analysis due to its incomplete genome sequence. Fifteen SPFM phages were isolated on five different Salmonella enterica serovars Typhimurium strains and the remaining seven were isolated on Salmonella enterica serovars 4,12:i> strains. Three phages were derived from wild boar faeces, eight from food processing plants and ten from faeces from fine pigs and piglets obtained from farms in Warwickshire, Hinckley and West Sussex ( Figure 1A and Table 1).

[0245] In terms of plaque morphology, phages SPFM9, SPFM10 and SPFM11 were isolated on the S. typhimurium strain and produced clear plaques of about 1 mm in diameter, and the remaining 18 phages produced clear plaques of about 0.5 mm in diameter. All phages were propagated on S. typhimurium SL1344 to produce high titers (10 10 The main motivation for this was that the original isolates spontaneously released phages, which complicated downstream characterization, whereas phage release from SL1344 was never observed.

[0246] Table 1. Summary of isolated phages and their corresponding isolation hosts, UK origins and genomic information.

[0247]

[0248]

[0249] a The sample source numbers are related to Figure 1: (1) wild boar nature reserve in Hampshire, (2) food processing factory in Essex, (3) pig farm in Warwick, Warwickshire, (4) pig farm in Hinckley, Leicestershire, and (5) pig nature reserve in West Sussex.

[0250] b All genome sequences can be found at this link: http : / / s3.climb.ac.uk / Sinfo / SPFM_ genome.tar.gz .

[0251] Phage morphology

[0252] TEM analysis revealed that these 21 phages have equal-length heads and retractable tails and are therefore classified as members of the Myoviridae family in the Caudovirales order ( Figure 1B ). The tail length of bacteriophages SPFM1, SPFM2, SPFM6, SPFM7, SPFM10, SPFM12, SPFM14, SPFM15, SPFM16, SPFM17, SPFM20, and SPFM22 is 160 ± 20 nm, and the capsid diameter is 100 ± 15 nm. The tail length of bacteriophages SPFM3, SPFM4, SPFM5, SPFM8, SPFM9, SPFM11, SPFM19, and SPFM21 is 200 ± 20 nm, and the capsid diameter is 105 ± 15 nm. The tail of bacteriophage SPFM13 is the longest, at 140 ± 10 nm, and the capsid diameter is 135 ± 7 nm.

[0253] Host range analysis

[0254] The efficacy of the SPFM phages was tested on 67 MDR enteric Salmonella enterica subsp. enterica strains isolated from pigs. All 67 strains represented the five major UK pig-associated Salmonella serotypes, namely S. Typhimurium; S.4,12:i>; S.4.5,12:i>; S. bovis; and S. derby. All phages had a broad spectrum of activity, with each lysing more than 80% of the strains ( Figure 2A). Phages SPFM1, SPFM3, SPFM10, SPFM14, SPFM15, SPFM17 and SPFM19 could lyse 100% of the screened Salmonella strains. Phages SPFM2, SPFM7, SPFM21 and SPFM22 lysed 67 / 68 strains (99%). In contrast, SPFM9 and SPFM11 only lysed 84% and 81% of the strains, respectively, and even where they lysed bacteria, the lysate was turbid on at least one strain.

[0255] EOP

[0256] To determine the efficiency of phage infection on clinically relevant strains, and thus decide which phages should ultimately comprise the phage cocktail, EOP of SPFM phages was performed on Salmonella serotypes S. Typhimurium; S.4,12:i>; S.4.5,12:i>; S. bovis; and two representative MDR swine strains of S. delbrueckii. EOP of phages on their propagation host, S. Typhimurium SL1344, was included as a control. To identify patterns in the EOP dataset, principal component analysis (PCA) was used, which showed that phages clustered into two unique groups ( Figure 2B ); (i) phages with similar EOP in all strains; and (ii) phages with higher EOP in specific Salmonella strains and serotypes. T-test confirmed that group (i) formed by phages SPFM4, SPFM5, SPFM8, SPFM9, SPFM12, SPFM13, SPFM14, SPFM15, SPFM16, SPFM17 and SPFM20 and their EOPs were not significantly different among all five serotypes. The remaining 10 phages formed group (ii). To expand group (ii), phage SPFM10 had a higher EOP against sick bovine Salmonella isolate A; phages SPFM1, SPFM11 and SPFM12 had higher EOP on all sick bovine Salmonella strains. Phages SPFM19 and SPFM21 both had higher EOPs on the S.4,12:i> strain; SPFM3 had higher EOPs on S. delbrueckii isolate B; SPFM22 had higher EOPs on S.4,5,12:i> isolate A; and phages SPFM6 and SPFM7 both had higher EOPs on S. delbrueckii isolates.

[0257] Genomic characterization of SPFM phages

[0258] The SPFM phage group was sequenced using the Illumina MiSeq platform. All 21 phages have a linear, circularly arranged dsDNA genome of 233 Kb to 242 Kb and encode 258 to 307 coding sequences (CDS) (Table 1). Since all genomes are greater than 200 Kb, they are classified as giant phages and are predicted to be soluble due to the lack of known lysogeny-related genes.

[0259] Although the SPFM genomes vary in size, they all encode a tRNA, with an average genome content of 48.5% (Table 1), an average gene length of 0.860 ± 0.010 Kb, a gene density of 1.074 / kb, and gene coding regions accounting for 93% of their genomes. Since the structure and content of the phages are genetically similar, a representative genome map of SPFM1 is shown in Figure 3. Most of the predicted genes encode proteins with no known function, and putative roles can only be assigned to about 30% of the genes. Genes that can be identified by homology with other phages include those encoding structural proteins, such as the major capsid protein, tail fiber protein, and tail sheath protein. Genes encoding packaging protein terminase were also identified, as were phage lytic enzymes. Several genes encoding products involved in DNA replication and transcription, such as endonucleases, helicases, putative nuclease SbcCD D subunits, putative ribonuclease H, and six RNAPβ (β / β') multisubunits, can be identified. The length of the genes encoding RNAP multi-subunits varies from 0.240 to 4.206 Kb, and all six subunits have an average nucleotide identity (ANI) of 99%, 80%, and 55% with Salmonella SPN3US (accession number: JN641803.1), Erwinia phage vB (accession number: KX397364.1), and Cronobacter phage CR5 (accession number: JX094500) (Lee et al., 2016).

[0260] Additional genes were found in all SPFM phage genomes that have the potential to mitigate their reliance on their bacterial hosts during infection. Genes of particular interest include dihydrofolate reductase, thymidylate synthase, and thymidylate kinase, all of which are predicted to be used for folate synthesis and the radical S-adenosylmethionine (SAM) gene involved in enhancing host metabolism during phage infection (Lee et al., 2011). In addition, a DNA adenine methylase gene was found in SPFM phages, which may protect against host restriction modification systems.

[0261] Hierarchical clustering analysis of isolated SPFM phages

[0262] The genomes were compared using nucleotide BLAST (NCBI, 1988) using pairwise local alignments, and it was observed that all SPFM phages were genetically very similar to each other, with an ANI of approximately 95%. To gain insight into the variation within the genome, a dendrogram was constructed based on the presence or absence of 46 shared auxiliary genes ( Figure 4 ). Hierarchical cluster analysis identified phages SPFM5, SPFM15, SPFM17, SPFM19, SPFM21 as being most similar to each other. Phages SPFM2, SPFM6, SPFM9, SPFM12, and SPFM14 formed the second group and shared the same auxiliary genes and core genes. The remaining 11 phages demonstrated variation between auxiliary genes, and the greatest differences were between phages SPFM1, SPFM13, SPFM16, and SPFM20. All of these also had different genome sizes compared to the other SPFM phages (Table 1).

[0263] Comparison of SPFM phages with previously sequenced Salmonella phages

[0264] To determine the extent to which the SPFM phages were similar to Salmonella phages previously deposited in Genbank (until December 2017), a comprehensive comparative analysis was performed against 158 ​​fully sequenced Salmonella phage genomes ( Figure 5 ). The genome size of all Salmonella phages used for analysis ranged from approximately 33 to 240 Kb, and all phages used for analysis are listed in Table B. Cluster analysis identified 21 different groups, with a cluster defined as phages that shared >50% ANI with other members of the cluster. All 21 SPFM phages were grouped together with phages SPN3US (Accession No.: JN641803.1) (Lee et al., 2011) and SEGD1 (Accession No.: KU726251.1) isolated in different studies. The genomes of phages SPN3US and SEGD1 are also approximately 240 Kb and have an ANI% of approximately 95%-97% with all SPFM phages. Phages SPN3US and SEGD1 are part of the SPN3US virus genus (Adriaenssens et al., 2017), which has now been updated to the Seoul virus genus. This is based on the current standard of more than 95% of ANIs, and SPFM phages are also classified into this genus. Phages in the Seoulvirus genus cluster are classified as phiKZ-like phages. The distinctive feature of phiKZ-like phages is that they all contain two multi-subunit RNA polymerases. The first multi-subunit is packaged in the capsid as a "virion" RNAP that transcribes early genes in the phage transcription program, and the second "non-virion" RNAP is responsible for transcribing late genes in its genome.

[0265] Seoul virus phage cluster

[0266] To determine how SPFM phages are related to Salmonella phages SPN3US and SEGD1, a phylogenetic analysis was constructed based on single nucleotide polymorphisms (SNPs) within 188 shared core genes ( Figure 6 )(Data have been provided in Supplementary Table 3 as published in Thanki AM, Brown N, Millard AD, Clokie MRJ. Genomic Characterization of Jumbo Salmonella Phages That Effectively Target United Kingdom Pig-Associated Salmonella Serotypes. Front Microbiol. 2019;10:1491. Published Jul 2, 2019 doi:10.3389 / fmicb.2019.01491 / ). This shows that phages SPFM5, SPFM9, SPFM10, and SPFM11 share the same SNPs. The clustering of phages based on SNPs in the core genes differed from the hierarchical clustering analysis based on the presence or absence of auxiliary genes. The other phages (except SPFM1) were grouped together with little or no changes in their core genes. SNPs in the SPFM1 core gene caused this phage to be grouped as a separate branch from the other 20 SPFM phages. Phages SPN3US and SEGD1 formed a more compact subclade that was different from that of SPFM phages ( Figure 4 ).

[0267] Phage genes under positive selection

[0268] To determine which genes were under evolutionary selection pressure, genes under positive selection were identified in the Seoulvirus cluster. This was done by determining the ratio of nonsynonymous to synonymous substitutions / (dN / dS) for pairwise comparisons of the core orthologs of 21 SPFM phages, SPN3US, and SEGD1 (Supplementary Table 5, as published in Thanki AM, Brown N, Millard AD, Clokie MRJ. Genomic Characterization of Jumbo Salmonella Phages That Effectively Target United Kingdom Pig-Associated Salmonella Serotypes. Front Microbiol. 2019;10:1491. Published Jul 2, 2019 doi:10.3389 / fmicb.2019.01491 / ). The data shown in Table 2 illustrate the posterior probability values ​​greater than 0.900 for the predicted genes under positive selection and their ortholog cluster numbers. The analysis predicted 33 genes to be under positive selection. Twenty-two of these genes are putative virion structural proteins, one is a putative endodeoxyribonuclease RusA, two encode thymidylate synthases, and eight are hypothetical proteins. For putative virion structural proteins under positive selection, HHpred (Zimmermann et al., 2017) was used to determine whether the structural proteins are involved in phage tail or capsid assembly. The program predicted with more than 75% probability that they are putative baseplate wedge proteins and therefore likely to be involved in phage tail assembly. In addition, orthologous cluster number 201 (related to the orthologous cluster number in Supplementary Table 5) has a 98.26% probability hit for the C-terminal pectate lyase domain, which is also part of phage tail fiber formation. Not all genes under positive selection can be assigned a function, but it can be predicted that the following putative proteins with orthologous cluster numbers 106, 108, 129, 130, and 211 are likely to be putative virion structural proteins because their locations are close to other putative structural proteins on the genome. Similarly, a hypothetical protein with ortholog cluster 92 is located close to a cluster of genes involved in DNA replication and transcription and is therefore likely involved in this function.

[0269] Table 2. Genes under positive selection in the Seoul virus phage cluster, including phages SPN3US, SEGD1, and 21SPFM.

[0270]

[0271]

[0272]

[0273] a Protein sequences were re-annotated using the tool HHPred (Zimmermann et al. 2017), and annotations with more than 75% likelihood were presented.

[0274] b The sample size was too small to accurately estimate the dN / dS of individual codons, but the hypothesis tests (posterior probabilities) were sensitive enough to detect positive selection.

[0275] The inventors have characterized 21 new Salmonella phages. All phages can infect representative MDR strains isolated from pigs. Seven virulent phages were identified that could infect 100% of the tested Salmonella isolates. A possible explanation for the high infectivity of these phages may be that the phages infect Salmonella typhimurium and they can also infect its monophasic variants, which are closely related genetically (Moreno et al., 2013). The phages may use the same bacterial receptors to attach to strains of these serotypes. Based on their host range and plaque formation rate, these seven phages appear to be ideal for therapeutic use.

[0276] Two phages, SPFM9 and SPFM11, could only infect about 80% of the screened strains. In addition, both phages produced turbid clearance on one-fifth of the strains, which may indicate potential lysogeny, as turbid clearance is often a characteristic of temperate phages (Gallet et al., 2011). Although sequence analysis confirmed that the two phages had no known lytic modules and were likely lytic phages, as only about 30% of the genes had known functions, unknown lytic modules may exist. A possible explanation for why turbid clearance was observed could be that the screened bacterial strains may have been partially resistant to the phages, thus infecting only a subpopulation of cells, resulting in turbid clearance (Bull et al., 2014). An alternative explanation could be that the phages induced prophages in these strains, which could have produced turbid clearance (Campoy et al., 2006). Despite the observation of turbid clearance, SPFM9 and SPFM11 were able to infect and replicate on the 11 strains screened for EOP analysis, indicating that they were lytic infections. Phages SPFM9 and SPFM11 would not be good candidates for therapy due to potential incomplete lysis and resistance or induction to phage (Chan et al., 2013; Abedon et al., 2017).

[0277] To further characterize the SPFM phages and narrow down those with ideal properties for therapeutic use, an EOP analysis was performed (Mirzaei and Nilsson, 2015). The data showed that a panel of phages had no differences in EOP across all representative strains from five different serotypes, and since there was no difference in infectivity between strains, these phages appeared to be ideal candidates for therapeutic use. In particular, phages SPFM14, SPFM15, and SPFM17 were good candidates because they could infect all representative strains from the main UK Salmonella serotypes and had high EOP across the selected strains.

[0278] All isolated SPFM phages have genomes larger than 233 Kb and significantly increase the number and diversity of approximately 170 giant phage genomes at NCBI (Yuan and Gao, 2017). SPFM phages also have larger genome sizes compared to other Salmonella phages, such as myovirus vB_SalM_SJ_3 with a genome size of 162,910 bp (Wall et al., 2010; Zhang et al., 2010; Saez et al., 2011; Zhang et al., 2014) and brevicaurus UAB_78 with a genome size of 48,110 bp (Bardina et al., 2016). Giant phages are rarely isolated and cannot be frequently isolated by conventional methods, which may bias phages with smaller genome sizes (Hillyard et al., 2016; Saad et al., 2018; Salmond and Fineran, 2015; Serwer et al., 2009). However, it can be considered that of the total phages described so far, about 2% are giant phages, so it is possible that giant phages are truly rare, as mentioned before, and are actually underrepresented.

[0279] All SPFM phages are genetically similar to each other, even though different Salmonella strains were used for enrichment and samples were collected from various environmental sources (Jurczak-Kurek et al., 2016). SPFM phages do differ in SNPs, which may be present in proteins that interact with the host. This may affect the kinetics of phage attachment to the cell surface, leading to changes in host specificity (Switt et al., 2013). This could potentially explain why differences in host range and EOP were observed between SPFM phages. Similar observations were described with 90% genetically identical Pseudomonas phages, where again SNPs led to phenotypic differences between phages (Ceyssens et al., 2011). It should be noted that phages SPFM5, SPFM9, SPFM10, and SPFM11 share the same SNPs, which may indicate that they represent clones with the SNPs. The SNPs may be induced by the propagation host, which is different from the host from which the phage was initially isolated.

[0280] SPFM phages were compared to all sequenced Salmonella phages and clustered with other known giant Salmonella phages SPN3US (Lee et al., 2011) and SEGD1, which were isolated from chicken feces in South China and in Korea, respectively. Interestingly, SPFM phages were isolated in the UK but clustered and were genetically similar to phages isolated in different continents. Similarly, 87% of genetically similar Pseudomonas phages were isolated from different countries in the United States and Europe (Ceyssens et al., 2011).

[0281] The large genome size of giant phages allows them to carry many genes that are not present in phages with smaller genome sizes 17), such as the six RNAPβ subunits that all SPFM phages have. The multiple RNAP subunits of phage SPN3US have been extensively studied in recent publications (2017) and are very similar to the RNAPβ subunits of phiKZ-like phages. Three additional RNAP subunits were recently predicted in phage SPN3US by constructing amber mutants of phage genes. All three predicted subunits were also identified in SPFM phages: nvRNAPβ', vRNAPβ present at the C-terminus and vRNAPβ' present at the C-terminus (Table A). In addition, the three predicted RNAP subunits are part of the core genes shared between phages. The presence of multiple RNAPβ subunits is consistent with other sequenced giant phages, such as the seven RNAPβ subunits that have been bioinformatically identified in V. corallilyticus phage BONAISHI, Ralstonia solanacearum phages RP12 and Φ8RP31. Overall, the presence of additional genes in giant phages may reduce their dependence on bacterial hosts for essential proteins associated with the phage life cycle and therefore may help expand the phage host range. This may explain why all SPFM phages can infect a variety of clinically relevant Salmonella isolates, which may make SPFM phages ideal candidates for phage therapy.

[0282] In the genus Seoulvirus, genes under positive selection were identified from the core genes shared by phages. Genes under positive selection include host-interacting proteins, such as two putative virion structural proteins that are predicted to be baseplate wedge proteins and are involved in the formation of tail fibers, both of which are residual phage binding to bacterial cells. These host-interacting proteins must be adapted to different bacterial hosts, which may explain why they are under positive selection. These results may also provide a plausible explanation for the differences observed in host range and EOP for SPFM phages. Other phage studies have also identified host-interacting proteins under positive selection, such as the gene GP6 that encodes a baseplate protein and may be involved in host specificity. Further genetic and mutational studies are needed to characterize phage genes under positive selection to understand their importance.

[0283] The inventors' research has described and characterized 21 genetically similar lytic giant phages that can lyse Salmonella strains commonly associated with pigs in the UK. Comprehensive host range analysis and EOP identified a number of phages that are ideal candidates for phage therapy to improve food safety.

[0284] The work described below identified the best phage or phage cocktail for the greatest reduction of Salmonella in vitro and in vivo.

[0285] Methods and Materials

[0286] Bacterial strains and culture conditions

[0287] Two strains of Salmonella enterica subspecies enterica serovar Typhimurium were used in this study: SL1344 (accession number FQ312003), a laboratory reference strain, and S01160-12, a phage DT193-type strain isolated from pigs by the Animal and Plant Health Service (Weybridge, UK) in 2012 (Card et al., 2016). S01160-12 is a multi-antibiotic resistant strain resistant to tetracycline, neomycin, ampicillin, sulfamethoxazole, chloramphenicol, gentamicin, streptomycin, trimethoprim-sulfamethoxazole, and apramycin. Both Salmonella isolates were routinely cultured on xylose lysine deoxycholate (XLD) agar (Oxoid, UK) at 37°C for 18 h, and liquid cultures were inoculated in NZCYM broth (Melford Biolaborators Ltd, UK) and incubated at 37°C at 100 rpm for 18 h.

[0288] Phage propagation and titration

[0289] Phages SPFM2, SPFM4, SPFM10, SPFM14, SPFM17 and SPFM19 were propagated. Brief exponentially growing liquid cultures (10 7 CFU / ml / ) was used as 10 7 Plaque forming units (PFU) / ml phage infection and incubated at 37°C at 100 rpm for 6 hours. The phage culture was centrifuged at 4,200xg for 15 minutes, the supernatant was filtered with a 0.22 μm filter, and the phage lysate was stored at 4°C. To determine the titer of the phage, the phage lysate was serially diluted 10 times and a small drop plaque assay (Mazzocco et al., 2009) was used on LB 1% agar plates with SL1344 bacterial lawns. The final phage titer was expressed as PFU / ml.

[0290] One-step growth assay

[0291] The exponential culture of SL1344 was grown at an optical density (OD) of 0.2. 600 ) and 10 7The cell density of CFU / ml was mixed with phage at a multiplicity of infection of 0.01, i.e., a ratio of bacterial cells to phage of 1:0.01. The phage was allowed to adsorb for 5 minutes at 37°C, and unbound phage was removed by centrifugation at 4200×g for 10 minutes. The precipitate was resuspended in NZCYM broth and incubated at 37°C with shaking at 100 rpm. Aliquots were taken every 10 minutes for 1.5 hours, and the PFU / ml counts were determined using a droplet plaque assay (Mazzocco et al., 2009). Three biological replicates were performed, each with three technical replicates.

[0292] Phage receptor analysis

[0293] Sangryeol Ryu provided the S. Typhimurium SL1344 strain with deletions of the flagella production gene (flgK); the gene encoding the vitamin B12 uptake outer membrane protein (butB); and the gene associated with LPS-associated O antigen production (rfaL). The mutant strain was constructed using the lambda red recombination method (Shin et al., 2012). To determine whether the phage uses any of these three as its host receptor, the phage lysate was diluted 10-fold and plated on the mutant strain using the small drop plaque assay (Mazzocco et al., 2009). Three biological replicates were performed, each with three technical replicates.

[0294] Temperature and pH stability determination

[0295] The thermostability of phage lysates was tested by exposing phages to a temperature range of 4°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for one hour. Phages were incubated in a polymerase chain reaction (PCR) machine. For pH studies, 100 μl of phage lysate was added to 900 μl of SM buffer adjusted to a pH range of 1 to 14. To determine phage titers after exposure to different temperatures and pH, phage lysates were serially diluted 10-fold and plated using a small drop plaque assay on LB 1% agar plates with SL1344 bacterial lawns (Mazzocco et al. 2009). Final phage titers were expressed as PFU / ml and three biological replicates were performed with three technical replicates.

[0296] In vitro bacterial killing assay using phage cocktails

[0297] Killing assays were performed with phages alone and with phage cocktails consisting of two, three, and four phages mixed in equal volumes. For the different killing assays, phage cocktail S01160-12 combination cultures were grown to an optical density (OD 600) was 0.2, at which time phage was added at an MOI of 100. In addition, there were two controls: one was a bacterial control, containing only SL1344, and the other was a phage control, containing only phage. Aliquots of the control and test samples were incubated at 37°C at 100rpm, and aliquots were taken at 0, 1, 2, 3, 4, 5, and 6 hours. For each time point, the bacterial concentration (CFU / ml) was determined on LB 1% agar plates, and the phage concentration (PFU / ml) was determined by droplet plaque assay (Mazzocco et al., 2009). For CFU / ml and PFU / ml, three technical replicates were performed, and the plates were incubated at 37°C for 16 hours. Three biological replicates were performed for each killing assay. GraphPad prism 6 was used to draw the killing assay graph, and the paired t-test was used to determine the significance of the data, where P≤0.5 was considered significant.

[0298] Testing the efficacy of phage cocktails in an in vivo Galleria mellonella infection model

[0299] Preparation of Galleria mellonella

[0300] Larvae were purchased from Live Food UK Ltd. (Rooks Bridge, UK), stored at 4°C and used within 5 days. For all in vivo experiments, larvae weighing approximately 0.25 to 0.30 g were selected and surface sterilized using a cotton swab moistened with 70% ethanol.

[0301] Galleria mellonella infected with Salmonella treated with phage cocktail

[0302] For all larval phage therapy studies, the method described previously was used (Nale, Chutia et al., 2016). The final concentration of 10 5 CFU of Salmonella strain S01160-12 and the final concentration was 10 7 PFU of phage mixture was administered to larvae by first culturing a culture containing 10 9 Strain S01160-12 was prepared by culturing a liquid culture of S01160-12 at 10 CFU / ml, which was then centrifuged at 4,200 x g for 10 minutes. The supernatant was discarded, and the pellet was resuspended in 0.1 M phosphate-buffered saline (PBS) and centrifuged at 4,200 x g for 10 minutes. The supernatant was discarded again, and the pellet was resuspended in PBS and diluted 10-fold to 10 in PBS. 5CFU / ml. 10 μl of bacterial culture and phage mixture were administered to the larvae by the oral route using a Hamilton syringe pump. The phage mixture was administered prophylactically 1 hour before infection or simultaneously with Salmonella (all regimens are detailed in Table 3). Afterwards, the larvae were incubated at 37°C for 3 days. The survival of the larvae was monitored every 24 hours and the larvae were sacrificed to determine the CFU and PFU counts. To this end, the larvae were stored at -20°C for 3 hours, dissected to remove their hemolymph, resuspended in 1 ml PBS and vortexed for 30 seconds (Nale et al., 2016). For PFU counting, the samples were centrifuged at 5,000xg for 5 minutes. The filtrate was then diluted 10 times and titrated by small drop plaque assay (Mazzocco et al. 2009). For CFU counting, the suspension was diluted 10 times and spotted on XLD agar plates. Salmonella colonies recovered after exposure to the phage mixture were picked using a 5 μl loop, streaked onto XLD agar plates, and restreaked three times onto XLD agar. Colonies were inoculated into NZCYM broth and incubated at 37°C at 100 rpm for 2 hours. The cultures were then used to make lawns to determine their susceptibility to phages by spot checks (Mazzocco et al., 2009). A concentration of 10 8 PFU / ml of phage. To determine the plating efficiency of phage on isolated colonies, phage lysates were diluted 10-fold and plated using a small drop plaque assay (Mazzocco et al. 2009).

[0303] For all treatment groups, a total of 60 larvae were used and 20 larvae were assessed for survival and dissected every 24 h. Larvae were considered dead if they did not respond to touch and changed color from light brown to black. Similarly, for the control group, 60 larvae were used and 20 larvae were sacrificed every 24 h to determine the CFU and PFU counts in their hemolymph. The control larvae included in all experiments were: healthy larvae; 7 PFU of phage larvae; 10 administered by oral route 5 larvae with CFU S01160-12; and larvae administered PBS (Table 3). All controls were monitored for survival every 24 hours and their gastrointestinal tracts were dissected for CFU and PFU counts.

[0304] Table 3. Experimental setup to evaluate the effectiveness of phage therapy against larvae infected with Salmonella.

[0305]

[0306]

[0307] 1Sixty larvae were used in each group for the study. Every 24 hours, 20 larvae were checked for survival and culled to calculate PFU and CFU counts.

[0308] 2 The phage mixtures were: 2-phage SPFM10-SPFM14; 3-phage SPFM4-SPFM10-SPFM19; and 4-phage SPFM2-SPFM10-SPFM14-SPFM19.

[0309] Survival curve

[0310] For all experiments, three biological replicates were performed, and survival data were plotted using the Kapad-Meier method using GraphpadPrism version 6 (GraphPad Software Inc, USA). Differences in survival were assessed using the Log-rank (Mantel-Cox) test.

[0311] Bioinformatics analysis

[0312] The phages have been previously deposited to ENA and their accession numbers are SPFM2 (LR535921), SPFM4 (LR535902), SPFM10 (LR535908), SPFM14 (LR535912), SPFM17 (LR535914) and SPFM19 (LR535916). http: / / s3.climb.ac.uk / Sinfo / SPFM_genome.tar.gz Find annotated phage genomes. For SNP analysis, frequencies above 90% and lower p-values ​​can be considered, but SNPs can also be identified when the frequency decreases.

[0313] result

[0314] Phage lysis

[0315] Phage growth curves were performed for the six SPFM phages. Phages SPFM10 and SPFM17 had lysis capacities of 175 and 162 PFU / cell, respectively, with a short incubation period of 20 minutes. SPFM2, SPFM4, SPFM 14, and SPFM19 had lysis capacities of 165, 162, 162, and 153 PFU / cell, respectively, with a longer incubation period of 30 minutes (Table 4).

[0316] Table 4. Lysis of SPFM phage.

[0317]

[0318] 1 The average results were from three biological replicates, each with three technical replicates.

[0319] Temperature and pH stability of bacteriophages

[0320] To determine the sensitivity of phages SPFM2, SPFM4, SPFM10, SPFM14, SPFM17 and SPFM19 to heat, phages were exposed to temperatures of 4°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C and 90°C for 1 hour (Figure 7). All phages were stable at 50°C, and there was no decrease in phage titer compared to phages stored at 4°C. When the phages were incubated at 60°C, only phages SPFM10 and SPFM17 survived, with an average decrease in titer of 0.0 and 6.5 log10 PFU / ml, respectively. The titer of phage SPFM17 decreased by 7.5 log10 PFU / ml at 70°C, while the phage did not survive at 80°C. In contrast, SPFM10 was more stable, with no significant drop in titer at 60°C, 70°C, and 80°C, but at 90°C, the phage titer dropped by 4.5 log10 PFU / ml, and no live phage was recovered at 100°C. To verify the thermal stability of phage SPFM10, phages were incubated using two different temperature control devices (heat block and water bath) and compared to the original data collected when phages were incubated using a PCR machine. The results were identical for all three machines, further confirming the thermal stability of SPFM10.

[0321] Spray drying of SPFM phage

[0322] Prior to spray drying, phage were concentrated using a 50 ml Amicon ultraconcentrator (Millipore, UK) with a cutoff of 10 kDa. 15 ml of phage lysate was added to the concentrator and spun at 5,000 x g for 20 min. The flow-through was discarded and another 15 ml of phage lysate was added to the concentrator. The concentrated phage collected above the filter was titrated and if the titer was approximately 10 10， It was used for spray drying experiments.

[0323] For phage spray drying, a 200 ml volume of 4% trehalose (Oxoid, UK) excipient solution dissolved in ultrapure water at pH 7.5 was mixed with 0.2 ml of a titer of approximately 5×10 10The concentrated phage of PFU / ml was mixed with / . The excipient phage solution was spray dried using a LabPlant spray dryer (UK) with a two-fluid nozzle for atomization on a laboratory scale, and the orifice diameter of the nozzle was 0.5mm. All runs used a constant feed rate of 3ml / min, and the atomizing airflow was 6l / min. The drying inlet temperature was heated to 80°C, 85°C, 90°C or 100°C, and the corresponding outlet temperature varied from 40°C to 60°C. The dry powdered phage was passed through a cyclone separator, collected in a 100ml glass bottle, and stored at 4°C until use. In order to determine the phage titer, 0.05g of dry powdered phage was suspended in 500μl phage suspension buffer, diluted 10 times and titrated by plaque assay.

[0324] Temperature stability of bacteriophage at different inlet temperatures for spray drying.

[0325]

[0326] Spray drying of SPFM phage

[0327] All six phages were spray dried with the sugar excipient trehalose. During spray drying, the phages were exposed to thermal stress because heated air was used to dry the phage-trehalose liquid into a powder. In order to evaporate the liquid, a high inlet drying temperature (which can be set manually) was used, to which the phage-trehalose solution was exposed for only a few milliseconds. Subsequently, the powder was collected and exposed to a lower outlet temperature, which depended on the set inlet temperature. In this study, the phages were subjected to different inlet drying temperatures of 80°C, 85°C, 90°C and 100°C to optimize the drying process, thereby minimizing the reduction in phage titer.

[0328] Regardless of the inlet temperature used during drying, phage titer losses of at least 1 log10 PFU / g were observed for SPFM2, SPFM4, SPFM17, and SPFM19. SPFM2 and SPFM4 were more stable when the inlet temperature was decreased to 80°C, but for SPFM4, phage titer losses were significant at all inlet temperatures, ranging from 3.00 to 3.16 log10 PFU / g. Phage titer losses were lowest for SPFM17 and SPFM19, with inlet temperatures of 85°C and 90°C, respectively. Phages SPFM10 and SPFM14 were the most stable of the six, with minimal titer losses of 0.02 and 0.35 log10 PFU / g when 80°C was used as the inlet temperature. Additionally, titer losses were noted when the inlet temperature of SPFM14 was increased, but titer losses for SPFM10 phage remained below 0.2 log10 PFU / g. The final yield of recovered dry powdered phage was consistently low at 0.2-0.5 g for all phages, thus giving a phage recovery of 5%-13% for all inlet temperatures tested.

[0329] All six SPFM phages used in this study were genetically 95% similar to each other, however, phenotypically, they had different characteristics as observed from their individual and combined killing activities against Salmonella typhimurium in vitro and in vivo. To further investigate why the phage combination SPFM10-SPFM14; SPFM4, SPFM10, and SPFM19 were the best, all six phages were phenotyped by determining the temperature stability of the phages. Consistent with previously characterized phages, SPFM2, SPFM4, SPFM14, and SPFM19 were stable at temperatures up to 50°C. When phages are exposed to higher temperatures, irreversible damage to phage proteins can occur, which results in the subsequent loss of phage infectivity. Interestingly, despite this, both phages SPFM10 and SPFM17 were thermostable and remained active at temperatures up to 70°C. Despite the use of phage SPFM17, phage titers were significantly reduced after incubation at temperatures above 50°C. In contrast to phage SPFM10, there was no decrease in phage titer at temperatures up to 80°C, and more than 55% of phage activity was retained even after exposure to 90°C for one hour. Phage SPFM10 has enhanced thermostability at extreme temperatures and is more stable than the reported E. coli phage vB_Eco4M-7, which retained only 20.5% of phage activity after exposure to 95°C for 5 minutes. It has been hypothesized that the formation of disulfide cross-links within the phage capsid may be involved in stabilizing the phage at extreme temperatures, which has been demonstrated for the RNA phage PP7. Whether the disulfide cross-links in the capsids of phages SPFM10 and SPFM17 contribute to the increased thermostability is unknown.

[0330] Furthermore, there may be a correlation between the thermostability of SPFM10 and its increased stability during spray drying. Bacteriophage SPFM10 is naturally very resilient and when combined with the excipient trehalose, the phage remained active when exposed to a maximum inlet temperature of 100°C. Surprisingly, phage SPFM14, despite not being particularly thermostable, was very stable during spray drying. In contrast, phage SPFM17 could be predicted to withstand spray drying as it is naturally thermostable, but similar to SPM2, SPFM4 and SPM19, showed up to a 3 log10 PFU / g reduction at the highest tested inlet temperature. This study highlights that phage stability during spray drying depends strongly on the phage used, even though the phages tested were genetically similar and all myoviruses and therefore had the same morphology. It was previously assumed that myoviruses would not spray dry well due to their fragile virion structure, as their long tails might shrink or their capsids might detach under pressure. This could explain why phages SPM2, SPFM4, SPFM17, and SPM19 showed titer loss, which was also observed for the Staphylococcus aureus phage Romulus, where a 2.5 log10 reduction was noted. Exceptions to this hypothesis are the giant phages SPFM10, SPFM14, and the Pseudomonas phage KZ.

[0331] The phages were exposed to a range of pH to determine their sensitivity to pH (Figure 7b). All phages were stable at pH 4 to 12 with no significant loss in phage titer. At pH 3, the phage titers of SPFM10, SPFM14, and SPFM17 decreased by 5.44, 3.12, and 2.51 log10 PFU / ml, respectively. However, phages SPFM2, SPFM4, and SPFM19 were stable at pH 3 with no change in phage titer. All phages were unstable at pH 1, 2, 13, and 14, and no plaques were recovered.

[0332] Identification of phage receptors

[0333] Plaque formation was screened on different receptor mutant strains of SL1344 to determine the bacterial receptor that the phage used to bind to Salmonella (Table 5). The phages were tested on strains with a deletion in the gene encoding the vitamin B12 uptake outer membrane protein (SL1344ΔbutB) and a deletion in the gene producing flagella (SL1344ΔflgK). There was no significant change in plaque formation in either strain compared to wild-type SL1344. However, on strain SL1344ΔrfaL, which has a deletion in a gene involved in the production of LPS-associated O-antigen, all phages were unable to infect and the plaque formation was zero. Based on this data, we expect that all six phages use the O-antigen LPS as host receptor.

[0334] Table 5. Salmonella phage titers of wild-type and receptor mutant strain SL1344.

[0335]

[0336] In vitro infection kinetics of six SPFM phages

[0337] Phages SPFM2, SPFM4, SPFM10, SPFM14, SPFM17 and SPFM19 alone and in combinations of two, three and four mixtures were tested to determine the best combination that caused the highest reduction of multiple antibiotic resistant Salmonella typhimurium strains. Killing was assessed over a six hour time course and compared to uninfected controls. The data shown in FIG8 are killing tests performed at a multiplicity of infection (MOI) of 100.

[0338] Bacterial killing assay of phage suspension alone

[0339] At an MOI of 100, when phages SPFM2, SPFM4, SPFM10, SPFM14, SPFM17, and SPFM19 were added individually, they caused significant reductions at 1 h post infection (p ≤ 0.05). However, maximum bacterial cell lysis was observed at 2 h post infection, with reductions of 1.77, 2.36, 2.15, 2.43, 2.32, and 1.52 log10CFU / ml, respectively, compared to uninfected cultures (Fig. 8a). In particular, the reduction rates of phages phiSPFM2, phiSPFM4, phiSPFM5, and phiSPFM6 were highly significant (p ≤ 0.01). After 6 h, the most effective phages that maintained the reduction in bacterial counts were phiSPFM2, phiSPFM4, phiSPFM5, and phiSPFM6 (p ≤ 0.05), with 0.71, 0.80, 0.83, and 0.85 log10CFU / ml, respectively.

[0340] Bacterial killing assay using a cocktail of two phages

[0341] Fifteen different mixtures of two phages were tested and for all combinations there was a reduction of 1 and 3 log10 CFU / ml of S. Typhimurium at 1 hour post infection compared to uninfected cultures ( Figure 8B and 8C ). Similar to the killing assay with individual phages, the phage combinations SPFM10-SPFM14, SPFM2-SPFM4, SPFM10-SPFM17, and SPFM2-SPFM10 observed the greatest statistically significant reductions after 2 hours (p≤0.001), which were 4.43, 4.46, 4.34, and 4.36 log10 CFU / ml, respectively. In addition, the phage mixtures SPFM2-SPFM14, SPFM2-SPFM6, SPFM10-SPFM19, and SPFM2-SPFM19 caused bacterial reductions of 2.36, 2.60, 2.64, and 3.29 log10 CFU / ml, respectively, after 2 hours (p≤0.001). Phages SPFM10-SPFM14 and SPFM2-SPFM10 maintained significant (p≤0.05) reductions in S. Typhimurium of 1.97 and 2.25 log10 CFU / ml, respectively, at 6 hours post-infection. The other 12 two-phage combinations were less efficient after 6 hours and maintained bacterial reductions between 0.5 and 1 log10 CFU / ml compared to uninfected controls.

[0342] Bacterial killing assay using a cocktail of three phages

[0343] Eleven of the three phage combinations tested cleared bacterial liquid cultures more rapidly at 1 hour post infection compared to individual phages and the two phage mixtures (p≤0.01), by approximately 2.5 log10CFU / ml (Figures 8d and 8e). After 2 hours, killing was also enhanced, with the most effective three phage mixtures being SPFM4-SPFM10-SPFM19, SPFM10-SPFM17-SPFM19, and SPFM2-SPFM17-SPFM19, causing 2.60, 3.50 and 3.53 log10CFU / ml reductions, respectively (p≤0.001). At 6 hours post infection, two of the eleven three phage combinations tested prolonged lysis: SPFM10-SPFM17-SPFM19 and SPFM2-SPFM10-SPFM19 (p≤0.05), by 1.10 and 1.70 log10CFU / ml.

[0344] Bacterial killing assay using a cocktail of four phages

[0345] For all four phage mixtures tested, rapid bacterial lysis was observed causing a 1.5-2.5 log10 CFU / ml reduction in S. Typhimurium at 1 hour post infection (p≤0.01). Of the four combinations, three combinations: SPFM10-SPFM14-SPFM17-SPFM19, SPFM2-SPFM10-SPFM14-SPFM19, and SPFM2-SPFM4-SPFM10-SPFM19 were most effective in reducing bacterial counts at 2 hours post infection, with 3.51, 3.40, and 3.38 log10 CFU / ml, respectively (Figure 8f). After 2 hours, the fourth phage combination, SPFM2-SPFM10-SPFM17-SPFM19, resulted in a 2.61 log10 CFU / ml reduction in bacterial density. Compared to the three phage mixture, bacterial inactivation after 6 h was consistent with the combinations of SPFM2-SPFM10-SPFM14-SPFM19 and SPFM2-SPFM4-SPFM10-SPFM19, at 1.58 and 1.61 log10 CFU / ml, respectively, compared to the uninfected bacterial control.

[0346] Infection dynamics of phage mixtures in vivo

[0347] To determine whether the efficacy results observed in vitro translate into the in vivo efficacy of the phage mixture, SPFM10 and SPFM14 (two phages); SPFM4, SPFM10 and SPFM19 (three phages); SPFM2, SPFM10, SPFM14 and SPFM19 (four phages) were tested in the G. mellonella infection model. Larvae were infected with the MDR Salmonella typhimurium S01160-12 strain. The preventive treatment activity of the phage mixture and the simultaneous application of phages to Salmonella were evaluated (Figure 3). The phage mixture was applied at an MOI of 100, consistent with the in vitro killing assay study.

[0348] Preventive treatment of phage mixtures

[0349] The phage mixtures were administered prophylactically, 1 hour before infection, to determine their efficacy in treating Salmonella infection (Figures 9 and 9c). After 24 hours, the average survival rate of all phage-administered larvae was 90%, while the survival rate of the control larvae C-6, which were infected with Salmonella alone, was 81%. The average bacterial counts of infected larvae treated prophylactically with 2-phage (P-1), 3-phage (P-2), and 4-phage (P-3) mixtures were 2 log10, 3.3 log10, and 3.8 log10 CFU / larvae, respectively (Table 3). The average bacterial count of the control group C-6 group was higher, at 4.6 log10 CFU / larvae. After 48 hours, the Salmonella count of the C-6 group was about 4.8 log10 CFU / larvae, and the survival rate dropped to 59%. A further reduction in Salmonella counts of approximately 3.3 log10 CFU / larvae was observed in larval groups P-1 and P-3, with no Salmonella detected in 12 / 20 larvae in both groups. Comparative group P-2 had a 1.3 log10 CFU / larvae reduction, with no Salmonella detected in 6 / 20 larvae. Survival rates were 95%, 81%, and 78% in P-1, P-2, and P-3, respectively. After 72 hours, bacterial counts in group C-6 increased further to 5.7 log10 CFU / larvae, with survival rates falling to 3%. Significant improvements in survival rates were observed in groups P1, P2, and P3, with 90%, 68%, and 62%, respectively. The additional reduction in bacterial load in the phage group reflects higher larval survival rates. The mean counts of Salmonella were 1 log10 CFU / larva for P-1, 2.4 log10 CFU / larva for P-2, and 2.0 log10 CFU / larva for P-3. Furthermore, no Salmonella was recovered from 14 / 20 larvae in the P-1 group, 10 / 20 in the P-2 group, and 13 / 20 in the P-3 group, highlighting the complete clearance of the infection in these larvae.

[0350] Co-infection with phage cocktails

[0351] Phage mixture and Salmonella were orally administered to larvae simultaneously and monitored every 24 hours for 72 hours (Figures 9B and 9d). After 24 hours, the survival rate of larvae infected with a mixture of 2-phage (CoI-1), 3-phage (CoI-2) and 4-phage (CoI-3) exceeded about 88%, while the survival rate of C-6 was 81% (Table 3). In the larval groups CoI-1 and CoI-2, it was observed that the Salmonella count was reduced by about 2.2 log10CFU / larvae, and in the CoI-3 group, it was reduced by 2.8 log10CFU / larvae. After 48 hours, 60% of the larvae in the C-6 group survived, while 84%, 72% and 78% of the larvae in the CoI-1, CoI-2 and CoI-3 groups, respectively, survived. Among the larvae, the average Salmonella count was 2.4 log10CFU / larvae in the CoI-1 group, 2.8 log10CFU / larvae in CoI-2, and 1.9 log10CFU / larvae in CoI-3, while the average Salmonella count in the C-6 group was 4.8log10CFU / larvae. In addition, in both groups COI-2 and COI-3, no Salmonella was isolated from 12 / 20 larvae, and in the COI-1 group, no Salmonella was isolated from only 3 / 20 larvae. After 72 hours, all phage mixtures resulted in a reduction of Salmonella counts by about 4 log10CFU / larvae compared to the average count of larvae in the C-6 group. The average survival rate of larvae in the CoI-2 and CoI-3 groups was 65%, in the CoI-1 group, 72% of the larvae survived, while in the C-6 group, only 3% of the larvae infected with Salmonella survived. No Salmonella was isolated from 13 / 20, 16 / 20, and 12 / 20 larvae in groups COI-1, COI-2, and COI-3, respectively, but Salmonella was isolated from all larvae in group C-6.

[0352] Control larvae group

[0353] After 72 hours, the survival rate of control larval groups C-1, C-2, C-3, C-4 and C-5 was 100%, and Salmonella was not detected in these groups. Phages were only recovered from C-3, C-4 and C-5 groups and phage counts were performed every 24 hours for 72 hours (Figures 9e and 9F). For prevention and co-infection studies, for the control group C-3, the total phage count increased by 1-log from 24 hours to 48 hours. For the control groups C-4 and C-5, no significant increase in phage titer was observed, and the total phage titer remained constant within 72 hours.

[0354] Susceptibility of Salmonella colonies recovered after exposure to phage cocktails in the larval model

[0355] After 72 hours, 168 Salmonella colonies were recovered from the larvae, 56 colonies after exposure to the 2-phage mixture, 56 colonies recovered with the 3-phage mixture, and 56 colonies recovered with the 4-phage mixture. The sensitivity of these colonies to the individual phages within their respective mixtures was tested. All colonies remained sensitive to the individual phages within the mixtures to which they were exposed, based on whether a clear zone of lysis was observed. Ten 10 Salmonella colonies from each phage treatment were selected, and the plaque rate of the individual phages within the mixtures was determined and compared to the wild-type Salmonella typhimurium S01160-12 strain ( Fig.10 ). Generally, all plaque ratios were around 1.0, indicating that the phages were still effective in this in vivo model.

[0356] For some of the screened colonies, differences in plaque formation rates were observed, for example, for 3 / 10 colonies isolated after exposure to the 2-phage mix, SPFM14 had a 10%-19% reduction in plaque formation rates. Conversely, SPFM10 had a 10%-26% increase in efficiency on 4 / 10 colonies. For colonies isolated after exposure to the 3-phage mix, SPFM4 and SPFM10 had a ~15% reduction in plaque formation rates for SPFM19 on 3 / 10 colonies. However, on 4 / 10 colonies, SPFM4 and SPFM10 had a >10% increase in plaque formation rates. On colonies exposed to the 4-phage mix, SPFM10 and SPFM19 had ~15% reduction in activity on 3 / 10 colonies, but SPFM14 had a consistently higher plaque formation rate on all 10 colonies.

[0357] Genetic differences among the six SPFM phages

[0358] Based on the average nucleotide identity, the isolated Salmonella phages are 95% similar to each other. Single nucleotide polymorphism (SNP) analysis was performed and the thermostable phage SPFM10 was compared with SPFM2, SPFM4, SPFM14, SPFM17 and SPFM19. The SNP data are listed in Table 6, and a total of very few SNPs were identified between the phages. The consistent SNP identified in all five phages is located at position 70,890, and a portion of the gene is annotated as a hypothetical protein. This is the only SNP difference identified between phage SPFM10 and SPFM14. Conversely, when SPFM10 was compared with SPFM2, SPFM4, SPFM17 and SPFM19, a total of 3, 3, 5 and 2 SNPs were identified in the phage genes, respectively. All the identified SNPs are in hypothetical proteins, and the SNP difference between SPFM10 and SPFM17 at position 57985 of the genome is expected to be annotated as the chromosome partition protein Smc. In addition, the phage was compared with two giant phages, SPN3US and SEGD1. Approximately more than 6900 SNPs were identified, and at least one SNP was present in all annotated genes.

[0359] Table 6. Comparison of bacteriophage SPFM10 with SPFM2, SPFM4, SPFM14, SPFM 17, and SPFM19 to identify SNPs between phages

[0360]

[0361]

[0362] Salmonella is a major animal and human food safety issue, and due to the increase in infections caused by MDR Salmonella strains, alternatives to antibiotics are needed to deal with such infections. Bacteriophages may provide an alternative. 21 giant phages have been identified as representative strains of prevalent serotypes associated with pigs in the UK. Six phages were selected from the collection based on their wide host range, as identified herein and deposited as discussed herein. All six phages have a large lytic capacity and a short incubation period, which is one of the characteristics of virulent phages and is very suitable for treatment. A large lytic capacity also increases the probability of contact between the phage and its target pathogen, which is crucial for infection. In addition, a large lytic capacity may mean that the phage kills bacteria faster than it replicates, which may potentially reduce the risk of selecting phage-resistant bacteria. Other giant phages that have been isolated have similar short incubation periods of less than 40 minutes and lytic capacities of more than 140 phages per infected cell, including the Klebseilla phage vB_KleM-RaK2 ( et al., 2013) and Serratia phage MAMA1 (Matilla and Salmond 2014).

[0363] All six phages were genetically 95% similar to each other and bound to the same bacterial receptors, but the pH and temperature stability data highlighted that there were phenotypic differences between the phages. The pH stability data showed that three of the six phages had a significant decrease in phage titer at pH 3. In addition, although all phages were stable at 50°C, consistent with other characterized phages, only phages SPFM10 and SPFM17 remained active after exposure to 70°C. However, phage SPFM17 showed a significant decrease in phage titer after incubation at temperatures above 50°C. In contrast to phage SPFM10, there was no decrease in phage titer until 80°C, and even after exposure to 90°C for 1 hour, and more than 55% of phage activity was maintained. This is highly unusual because usually when phages are exposed to high temperatures it can lead to irreversible damage to phage proteins, followed by loss of phage infectivity (Ahmadi et al. 2017). The natural thermostability of SPFM10 is rare and significantly more stable than that reported for E. coli phage vB_Eco4M-7, which retained only 20.5% of phage activity after exposure to 95°C for 5 minutes (Jurczak-Kurek et al. 2016). Within the phage capsid, the formation of disulfide cross-links can participate in stabilizing the phage during extreme temperatures. Disulfide cross-links in the capsids of phages SPFM10 and SPFM17 may contribute to the increased thermostability.

[0364] Ideal phages or phage cocktails for therapeutic use are those that are effective in eliminating multiple subsets of the target bacterial species and are able to delay the emergence of phage-resistant mutants. SPFM phage combinations were tested in vitro: single, two, three, and four phages, and it was determined which combination could rapidly lyse a representative MDR isolate of Salmonella typhimurium (Agency 2014). When tested individually, all phages resulted in a 1 log10 CFU / ml reduction in bacterial counts after just 1 hour. Salmonella regrowth occurred immediately after 1 hour, and at 6 hours, Salmonella counts in phage-infected cultures were similar to those of uninfected controls, with no significant differences between the two. These results are consistent with previous killing assays of Salmonella with individual phages.

[0365] Phage cocktails of two, three and four phage combinations were tested and bacterial lysis improved by about 3 to 4 log10CFU / ml reduction after 2 hours, with less prominent re-growth of Salmonella typhimurium. A total of 36 different phage combinations were tested in vitro. Phage cocktails SPFM10-SPFM14 and SPFM2-SPFM10 were most effective in lysing Salmonella typhimurium and are ideal candidates for phage therapy. Similarly, in previous studies, three (Bardina et al., 2012), six (Albino et al., 2014) and ten (Zhang et al., 2010) phage cocktails could reduce the number of Salmonella by about 2 log10CFU / ml. This is different from the 4 log10CFU / ml reduction observed for the best two phage cocktails in this study. This in vitro data highlights that the two giant phage cocktails are effective in rapidly lysing Salmonella and that the two combinations were able to lyse 100% of the representative isolates of the % top UK swine serotype-related screening. Interestingly, both mixtures also contained the naturally occurring thermostable phage SPFM10, which may indicate that it is more toxic than the other five phages. A possible explanation as to why bacterial killing was not improved with a three or four phage mixture is that when the phages are mixed in equal volumes, the phages are diluted to reach the final MOI and the toxicity of the phages can be diluted. Additionally, since the phages compete for the same receptor sites on Salmonella cells, one phage may outcompete the others in the mixture. A phage that outcompetes the others may be one that reproduces faster (short incubation period and high lysis volume) and since the other phages in the mixture are less competitive, it may reduce the overall efficacy of the mixture. Evaluating combinations of different phages is important to determine the optimal mixture for reducing target pathogens.

[0366] The efficacy of the phage cocktail to clear MDR Salmonella strains was tested in an in vivo larval infection model to compare with the phage in vitro and in vivo efficacy. Three cocktails were tested: SPFM10-SPFM14; SPFM4-SPFM10-SPFM19; and SPFM2-SPFM10-SPFM14-SPFM19, and were administered prophylactically and co-infected. Similar to the in vitro data, the phage cocktail SPFM10-SPFM14 was the best at clearing infection by both treatments, significantly improving larval survival, and no Salmonella was recovered from more than 65% of the larvae after 72 hours. The results provide further evidence that this cocktail may be ideal for treatment. Similar phage cocktail efficacy in clearing target bacteria has been observed in other larval studies, for example, after 24 hours, the administration of a 6-phage cocktail at an MOI of 100 2 hours before infection with Pseudomonas aeruginosa was able to increase larval survival by 80% (Beeton et al. 2015). Another independent study showed that different six-phage cocktails increased larval survival by 30% when infected with a virulent strain of P. aeruginosa (Forti et al. 2018). Although the three- and four-phage cocktails had lower in vitro efficacy, both cocktails improved larval survival and Salmonella clearance, but the three-phage cocktail was more effective when co-infected. The results highlight that in vitro killing data are not always relevant in vivo and that screening the efficacy of phage cocktails in larval infection models may be an improved method to evaluate the toxicity of phage cocktails (Seed and Dennis 2009; Nale, Chutia et al. 2016; Abbasifar et al. 2014; Manohar, Nachimuthu, and Lopes 2018). To our knowledge, this is the first study to evaluate the activity of giant phages against MDR S. typhimurium in a larval infection model and the first study using giant phages.

[0367] Colonies isolated after exposure to a cocktail of three different phages in the larval infection model remained susceptible to the individual phages of the cocktail, indicating that we did not isolate any phage-resistant Salmonella cells. Furthermore, the phages still had high plating rates on recovered colonies and could infect to the same extent as the wild-type strain.

[0368] Since there are phenotypic differences between the six SPFM phages, SNP analysis was performed to identify genetic differences between the phages. When the thermostable phage SPFM10 was compared with the other five SPFM phages, there were very few SNPs between them. In all five phages, the hypothetical protein has a SNP at position 70890 of the genome, and this SNP is present in a protein that is under positive selection and naturally prone to mutation. Further analysis of HHpred predicted that the gene has hit the C-terminal pectate lyase domain (98% probability) and may be involved in the formation of the phage tail fiber. The pectate lyase domain function is expected to cut glycosidic bonds and may be involved in degrading polysaccharides on the surface of Salmonella, and therefore may play an important role in the phage host range. In addition, this SNP may play a role in thermostability, which is the only difference between SPFM10 and SPFM14, the latter phage is only stable at 50°C, while SPFM10 is thermostable until 90°C. Additional SNPs between SPFM10 and phages SPFM2, SPFM4, SPFM17, and SPFM19 were identified in another predicted pectate lyase protein that was also shown to be under positive selection. This protein is likely a host interaction gene that must be susceptible to mutation. Two additional SNPs were identified in a chromosome partitioning protein and a hypothetical protein only in SPFM17 when compared to SPFM10. SPFM17 can also tolerate high temperatures up to 70°C and the addition of these two SNPs could potentially play a role in stabilization as they are unique to the phage. Again further mutagenesis analysis is needed to determine the importance of the presence of the two SNPs for thermostability.

[0369] The work described below characterizes the infectivity of phages against the top serotypes associated with British chickens.

[0370] Experimental Procedure

[0371] Bacterial strains and culture conditions

[0372] In this study, a total of 18 Salmonella strains were examined. All Salmonella enterica subspecies enterica strains were isolated from chickens in the United Kingdom between 2015 and 2017 by the Animal and Plant Health Agency (APHA) in Weybridge, United Kingdom (Table 7). The serotypes of these strains were: 3 S.13,23:i>; 5 S.Enteridis; 4 S.Infantil; 3 S.Ohio and 3 S.Seftenberg.

[0373] Table 7. Salmonella Gallinarum strains.

[0374]

[0375]

[0376] Isolation, purification and propagation of bacteriophages

[0377] By infecting 10 7 PFU / ml of Salmonella SL1344 (10 7 Purified phage lysates of increasing volumes were prepared by shaking (100 rpm) an exponentially growing liquid culture of 1000 μg (0.1% 50 μg / ml) at 37°C in NZCYM broth for 6 hours. The phage culture was centrifuged at 4,200 x g for 15 minutes, the supernatant was filtered with a 0.22 μm pore size filter, and the phage lysate was stored at 4°C. To determine the phage titer, the phage lysate was serially diluted 10 times and a small drop plaque assay (Mazzocco et al., 2009) was used on LB 1% agar plates. The final phage titer was expressed as PFU / ml.

[0378] Phage host range analysis and plaque formation rate

[0379] The host range of individual phages was determined by small drop plaque assay (Mazzocco et al., 2009) on different S. enterica subspecies enterica serovars and incubated for 18 h at 37° C. Plates were examined for bacterial lysis or absence of infection by clearing or plaques and the average observations were noted from three biological replicates, each with three technical replicates.

[0380] Host range analysis

[0381] The efficacy of SPFM 2, 4, 10, 14, 17 and 19 phages was tested on 18 Salmonella enterica subspecies enterica strains isolated from chickens. All 18 strains represented Salmonella serotypes associated with UK top pigs, namely S.13, 23:i>; S. Enteritidis; S. Infants; S. Ohio and S. Seftenberg. All phages could infect all S. Enteritidis strains. Phages SPFM2, SPFM14 and SPFM19 produced turbid clarification on all S. Infants and could not infect representative strains of the S. Seftenberg serotype. Phages SPFM4 and SPFM17 could lyse all S. Infants and only lyse 1 / 3 of the S. Seftenberg strains.

[0382] Table 8. Host range of SPFM phages in Salmonella chicken isolates, and the data presented are the average of three replicates. The strain numbers relate to those listed in Table 7.

[0383]

[0384] While the invention has been particularly shown and described with reference to particular embodiments, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the scope of the invention.

Claims

1. A phage panel comprising two or more phages, wherein the phages are members of the Myoviridae family and are selected from the group consisting of LR535921, deposited as NCTC 18080701; LR535902, deposited as NCTC 18080702; LR535908, deposited as NCTC 18080703; LR535912, deposited as NCTC 18080704; LR535914, deposited as NCTC 18080705; or LR535916, deposited as NCTC 18080706.

2. The kit of claim 1, comprising the bacteriophages deposited under NCTC Nos. 18080703 and 18080704.

3. A pharmaceutical composition comprising any one of the kits according to claims 1-2.

4. Animal feed comprising the kit according to any one of claims 1-2, or the pharmaceutical composition according to claim 3.

5. A composition for use in a method of treatment, comprising the kit according to any one of claims 1-2, the pharmaceutical composition according to claim 3 or the animal feed according to claim 4.

6. A composition for use in a preventive treatment method comprising a kit according to any one of claims 1 to 2, a pharmaceutical composition according to claim 3, an animal feed according to claim 4 or a composition according to claim 5.

7. The composition of claim 5 or 6, wherein the method is for treating Salmonella infection in poultry and the prophylactic method is for preventing Salmonella infection in poultry.

8. The composition of claim 5 or 6, wherein the method is for treating a Salmonella infection in a pig or cattle and the prophylactic method is for preventing a Salmonella infection in a pig or cattle.

9. A method for drying a bacteriophage solution to form a powder, wherein the bacteriophage solution comprises the kit of any one of claims 1 to 2 or the pharmaceutical composition of claim 3, the method comprising: a) mixing the bacteriophage with any one or more of the following; (i) a sugar, wherein the sugar is trehalose; and / or (ii) a sugar alcohol, wherein the sugar alcohol is mannitol; and / or (iii) an amino acid, wherein the amino acid is leucine; and / or (iv) a polymer, wherein the polymer is Eudragit; as well as b) drying the mixture.

10. The method of claim 9, wherein: a) drying by spray drying; and / or b) wherein the method further comprises preparing tablets or pellets from the dried powder.

11. A bacteriophage composition comprising: i. any one of the kits according to claim 1-2 or the pharmaceutical composition according to claim 3; ii.Sugar; iii.Sugar alcohol; iv. amino acids; and v.Polymer.

12. A thermostable bacteriophage, wherein the thermostable phage is LR535908 with a deposit number of NCTC 18080703.

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