Novel compound

Abstract

The present invention provides for enrichment of animal foodstuffs using endolysin polypeptides or fragments thereof, which possess antimicrobial activity (e g., against Clostridium septicum). 'The invention also provides for nucleic acid molecules encoding the endolysin, and methods of their production. Also provided are compositions comprising the endolysins, or comprising any of the means for producing the endolysin (e.g., a host cell, lysate, or dried product, etc.) that has antimicrobial activity, for the use of said compositions in a method of reducing the population of Clostridium septicum bacteria in a foodstuff, or preventing or treating a disease or disorder in an animal.

Description

Attorney Docket: 125930-848037 NOVEL COMPOUND SEQUENCE LISTING

[0001] The Sequence Listing submitted with this application, entitled “125930-848037_SL.xml”, was created on August 25, 2025, having a file size of 95,369 bytes, and is hereby incorporated by reference thereto in its entirety. FIELD OF THE INVENTION

[0002] The present invention relates to improvements to animal foodstuffs via endolysin enrichment and uses thereof. BACKGROUND

[0003] Concerns over antibiotic resistance have resulted in public and regulatory pressure to ensure the judicious use of antibiotics in both human and veterinary medicine. Despite these initiatives on antibiotic use, the prevention of animal diseases caused by microorganisms (e.g., Clostridium septicum) remains a considerable problem for industries such as animal production. Cellulitis in broiler chickens and other poultry, such as “gangrenous dermatitis” (GD), is a disease that affects commercial broiler chickens and turkeys, and is responsible for severe economic losses in the poultry industry worldwide due to high mortality, medication costs, and increased condemnations at the processing plants. Clostridial dermatitis (CD) is a gangrenous dermatitis caused by Clostridial bacteria (e.g., C. septicum; C. perfringens; C. sordelli), which is found in both chickens and turkeys. Clostridium perfringens and Clostridium septicum play a role in causing cellulitis and mortality in poultry breeder hens. The prevalence and severity of this condition has increased globally over the past two decades, and is one of the most significant diseases impacting the commercial poultry industry in the United States and globally.

[0004] Cleaning and disinfection are insufficient to eliminate Clostridial spores from a farm environment, and antibiotics are often unsuccessful as therapeutics after onset of disease. Antibiotics (e.g., bacitracin, virginiamycin, penicillin, ionophores (e.g., including monensin or salinomycin)) may be unsuccessful in mitigating Clostridial dermatitis in poultry. A recent increase in antimicrobial resistance has reduced the reliability of existing antibiotics, and the increase in both the prevalence and range of multi-drug resistant strains is of particular concern. In addition to the increasing occurrence of resistance against conventional antibiotics, most antibiotics have a broad-spectrum activity against a range of bacterial groups and can detrimentally affect the beneficial and protective bacterial flora in the chicken gut. This occurs 1 105506204.3Attorney Docket: 125930-848037 even when low doses of antibiotics are added to poultry environments, altering gut biome in the host, and exposing the poultry to risk of infection from bacteria gaining resistance to the antimicrobial used. The emergence of microbial resistance to first-line antibiotics and the increasing frequency of multidrug-resistant pathogens is a disturbing trend particularly as there is evidence that resistant strains are transmitted to humans through food. Due to increasing concern regarding antimicrobial resistance and the consumer push for antibiotic- free production, the use of antibiotics is declining and farmers may face the future challenge of raising healthy animals without growth-promoting antibiotics.

[0005] Consequently, there is thus an urgent unmet need for antibacterial agents which specifically target Clostridium septicum and which are capable of being used in the poultry industry as a prophylactic, and / or treatment for Clostridial dermatitis, and which do not contribute to the increasing problem of antibiotic resistance. SUMMARY OF THE INVENTION

[0006] In an aspect, provided herein is an endolysin polypeptide having antimicrobial activity, comprising an amino acid sequence of a Clostridium septicum cell wall peptidoglycan catalytic domain, or a fragment thereof; or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof, optionally SEQ ID NO:50.

[0007] The endolysin polypeptide may further comprise an amino acid sequence of a Clostridium septicum cell wall binding domain, or a fragment thereof; or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof, optionally SEQ ID NO:80.

[0008] The endolysin polypeptide may further comprise a linker between the amino acid sequence of a Clostridium septicum cell wall peptidoglycan catalytic domain, or a fragment thereof; or the amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 2 105506204.3Attorney Docket: 125930-848037 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof, optionally SEQ ID NO:50, and the amino acid sequence of a Clostridium septicum cell wall binding domain, or a fragment thereof; or the amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof, optionally SEQ ID NO:80.

[0009] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0010] In an aspect, provided herein is an endolysin polypeptide having antimicrobial activity, comprising an amino acid sequence of a Clostridium septicum cell wall binding domain, or a fragment thereof; or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof, optionally SEQ ID NO:80.

[0011] The endolysin polypeptide may further comprise an amino acid sequence of a Clostridium septicum cell wall peptidoglycan catalytic domain, or a fragment thereof; or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof, optionally SEQ ID NO:50.

[0012] The endolysin polypeptide may further comprise a linker between the amino acid sequence of a Clostridium septicum cell wall binding domain, or a fragment thereof; or the amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof, optionally SEQ ID NO:80, and the amino acid sequence of a Clostridium 3 105506204.3Attorney Docket: 125930-848037 septicum cell wall peptidoglycan catalytic domain, or a fragment thereof; or the amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof, optionally SEQ ID NO:50.

[0013] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0014] The endolysin polypeptide may be purified or isolated. The antimicrobial activity of an endolysin may be bacteriolytic activity and / or bacterial growth inhibitory activity. The antimicrobial activity may be against Clostridium septicum. The antimicrobial activity may be determined by a cell viability assay; cell turbidity reduction assay; or minimum inhibitory concentration (MIC) assay or minimum bactericidal concentration (MBC) assay, optionally wherein the assay is performed against Clostridium septicum. The antimicrobial activity may be determined by endolysin activity, optionally wherein the endolysin activity is assessed by the rate of degradation of peptidoglycan (PGN).

[0015] In an aspect, provided herein is a recombinant nucleic acid molecule comprising a first nucleic acid sequence encoding the endolysin polypeptide as described herein, optionally wherein the recombinant nucleic acid molecule further comprises a second nucleic acid sequence encoding a promoter and wherein the first nucleic acid sequence is operably linked to the second nucleic acid sequence; further optionally wherein the first nucleic acid sequence of the recombinant nucleic acid molecule encodes a cDNA. The recombinant nucleic acid molecule may comprise the first and second nucleic acid sequences. Also provided herein is an expression vector comprising a nucleic acid molecule as described herein. The recombinant nucleic acid molecule or the expression vector may comprise a promoter that may be capable of promoting expression of the endolysin polypeptide in a cell of a unicellular microorganism. The promoter may be capable of promoting expression of the endolysin polypeptide in Bacillus subtilis.

[0016] In an aspect, provided herein is a host cell comprising a nucleic acid molecule or expression vector as described herein. The host cell may be a Bacillus subtilis cell. 4 105506204.3Attorney Docket: 125930-848037

[0017] In an aspect, provided herein is a cell lysate comprising the one or more endolysin polypeptides as described herein. The one or more endolysin polypeptides may be expressed from a nucleic acid molecule or expression vector as described herein, or is expressed by a population of one or more host cells as described herein.

[0018] In an aspect, provided herein is a dried whole culture comprising one or more endolysin polypeptides as described herein, optionally wherein the dried whole culture is spray dried or freeze dried. The dried whole culture may comprise one or more endolysin polypeptides that are expressed from the nucleic acid molecule or expression vector as described herein, or are expressed by a population of one or more host cells as described herein.

[0019] In an aspect, provided herein is a dried medium comprising one or more endolysin polypeptides as described herein, optionally wherein the dried medium is spray dried or freeze dried. The dried culture medium may comprise one or more endolysin polypeptides that are expressed from the nucleic acid molecule or expression vector as described herein, or is expressed by a population of one or more host cells as described herein.

[0020] In an aspect, provided herein is a dried material comprising one or more endolysin polypeptides as described herein, a population of one or more host cells as described herein, and / or a cell lysate as described herein, optionally wherein the dried material is spray dried or freeze dried.

[0021] In an aspect, provided herein is a combined material comprising two or more agents selected from the one or more endolysin polypeptides as described herein, a population of one or more host cells as described herein, a cell lysate as described herein, a dried whole culture as described herein, a dried culture medium a described herein, and / or a dried material as described herein, optionally wherein the combined material is dried, further optionally wherein the combined material is spray dried or freeze dried.

[0022] In an aspect, provided herein is a method for producing an endolysin polypeptide having antimicrobial activity, the method comprising culturing a host cell comprising a nucleic acid or expression vector as described herein under conditions in which the endolysin polypeptide is expressed. The method may further comprise lysing the host cell to produce a cell lysate comprising the endolysin polypeptide. The method may further comprise purifying or isolating the endolysin polypeptide. The method may not comprise further purification or isolation of the endolysin polypeptide. 5 105506204.3Attorney Docket: 125930-848037

[0023] In an aspect, provided herein is an animal foodstuff comprising: (a) one or more foodstuffs; and (b) one or more endolysin polypeptides as described herein. The animal foodstuff may comprise one or more foodstuffs and a population of one or more host cells as described herein, a cell lysate as described herein, a dried whole culture as described herein, a dried culture medium a described herein, a dried material as described herein, and / or a combined material as described herein.

[0024] In an aspect, provided herein is a supplemented water comprising: (a) water; and (b) one or more endolysin polypeptides as described herein. The supplemented water may comprise water and a population of one or more host cells as described herein, a cell lysate as described herein, a dried whole culture as described herein, a dried culture medium a described herein, a dried material as described herein, and / or a combined material as described herein.

[0025] In an aspect, provided herein is a supplemented hydrogel comprising: (a) hydrogel; and (b) one or more endolysin polypeptides as described herein. The supplemented hydrogel may comprise hydrogel and a population of one or more host cells as described herein, a cell lysate as described herein, a dried whole culture as described herein, a dried culture medium a described herein, a dried material as described herein, and / or a combined material as described herein.

[0026] In an aspect, provided herein is a method for reducing a population of Clostridium septicum bacteria in a foodstuff, the method comprising admixing with the foodstuff an endolysin polypeptide as described herein, a population of one or more host cells as described herein, a cell lysate as described herein, a dried whole culture as described herein, a dried culture medium a described herein, a dried material as described herein, and / or a combined material as described herein.

[0027] In an aspect, provided herein is a method for the prevention or treatment of a disease or disorder in an animal, the method comprising administering to the animal an endolysin polypeptide as described herein, a population of one or more host cells as described herein, a cell lysate as described herein, a dried whole culture as described herein, a dried culture medium a described herein, a dried material as described herein, and / or a combined material as described herein, an animal foodstuff as described herein, a supplemented water as described herein, and / or a supplemented hydrogel as described herein, optionally wherein the disease or disorder may be caused by Clostridium septicum. Further optionally wherein the disease or disorder caused by Clostridium septicum may be cellulitis or gangrenous dermatitis. 6 105506204.3Attorney Docket: 125930-848037

[0028] The animal subject for a method described herein may be selected from a turkey, optionally a domestic turkey; a chicken, optionally a broiler chicken; a duck, optionally a domestic duck, further optionally a domestic duck derived from a mallard duck or a domestic duck derived from a Muscovy duck; a goose, optionally a domestic goose, further optionally a domestic goose derived from a Greylag goose or a domestic goose derived from a Chinese goose; a pigeon, optionally a domestic pigeon; a quail, optionally a domestic quail; or a pig, optionally a domestic pig.

[0029] In an aspect, provided herein is a kit comprising an agent having antimicrobial activity, wherein the agent is selected from: an endolysin polypeptide as described herein, a host cell as described herein, a cell lysate as described herein, a dried whole culture as described herein, a dried culture medium as described herein, a dried material as described herein, and / or a combined material as described herein, and instructions for admixing the agent with a foodstuff. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Embodiments of the present inventive concept are illustrated by way of example in which like reference numerals indicate similar elements and in which:

[0031] FIG.1 depicts a schematic describing a generalized DNA fragment , which was used in the transformation of Bacillus subtilis strain strain bAL265 for expression of endolysin polypeptides of the invention. The AmyE gene in strain bAL265 was disrupted by the integration of a gene conferring resistance to the antibiotic spectinomycin. The DNA fragment carrying the endolysin was flanked by sequences of homology to the AmyE gene (RHA: right homologous arm; LHA: left homologous arm). The DNA fragment features the erythromycin resistance gene (Ery), a promoter located in the intergenic region between the erythromycin resistance and endolysin genes, an additional terminator, and the lactose repressor gene (lacI). FIGS.2A-2C show tryptose sulfite cycloserine (TSC) plate bioassay results of endolysins against Clostridium septicum strains bAL274 (Clostridium septicum Ramsay) and bAL275 Clostridium septicum. TSC is an indicator agar that turns black in the presence of Clostridium septicum, whilst bacterial free areas are indicated by white / pale zones. FIG.2A shows results of plate A replicates I and II, for endolysin strains bAL340 (e.g., endolysin Sep-EL6 of SEQ ID NO:6); bAL371 (e.g., endolysin Sep-EL2 of SEQ ID NO:2); bAL373 (e.g., endolysin Sep-EL3 of SEQ ID NO:3); bAL375 (e.g., endolysin Sep-EL9 of SEQ ID NO:10); bAL377 (e.g., endolysin Sep-EL4v2 of SEQ ID NO:7), and bAL379 (e.g. endolysin Sep-EL25 of SEQ ID NO25). FIG.2B shows results of plate B replicates I and II, for endolysin strains bAL381 7 105506204.3Attorney Docket: 125930-848037 (e.g., endolysin Sep-EL7 of SEQ ID NO:8); bAL383 (e.g., endolysin Sep-EL8 of SEQ ID NO:9); bAL385 (e.g., endolysin Sep-EL18 of SEQ ID NO:18); and bAL265 (negative control strain), as well as lysozyme and PBS positive and negative controls. FIG.2C shows results for ampicillin positive control. DETAILED DESCRIPTION OF THE INVENTION Bacteriophage endolysins

[0032] Bacteriophage-encoded endolysins may be employed as antimicrobial agents that are specific to their host species of bacteria. Bacteriophages are viruses that infect bacteria. The expansion of understanding on bacteriophage biology, including the increase in antibiotic resistant strains of bacteria, has led to increased interest in these specific bacteriolytic enzymes. Endolysins are used by bacteriophage to degrade the bacterial cell wall peptidoglycans from within the bacteria in order to facilitate the release of bacteriophage progeny. Endolysins can also have antimicrobial effects when applied externally to gram-positive bacteria. Endolysins that are specific for gram-positive bacteria usually have a modular organisation. Many endolysins have a two-domain structure with an N-terminal catalytic domain and a C-terminal cell wall binding domain connected by a linker. Endolysins with different domain architectures have also been discovered, and are contemplated for use herein.

[0033] The N-terminal catalytic domain of the endolysin is responsible for the enzymatic cleavage of the peptidoglycan layer of the cell wall. The peptidoglycan layer comprises of linear carbohydrate backbones with alternating (β1-4)-linked N-acetylglucosamine and N- acetylmuramic acid monomers. The carbohydrate backbones are cross-linked by species- specific peptide side chains. Depending on the type of bond that they cleave, endolysins can be grouped into at least five classes of distinct enzymatic activities: N-acetyl-β-D-muramidases and N-acetyl-β-D-glucosaminidases break down the polysaccharide backbone of peptidoglycan layer but N-acetyl-β-D-muramidases cleave glycosidic bonds between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc), while N-acetylβ-D-glucosaminidases hydrolyze bonds between GlcNAc and MurNAc, instead; lytic transglycosylases also cleave β-1,4 bonds between MurNAc and GlcNAc but do not require a water molecule for their catalytic activity; N-acetylmuramoyl-L-alanine amidases hydrolyze the amide bond between MurNAc and the peptide moiety; and lastly, endopeptidases hydrolyze peptide bonds between two amino acids in the peptide stem or in the cross-bridge that links the stems. 8 105506204.3Attorney Docket: 125930-848037

[0034] The C-terminal cell wall binding domain is largely responsible for the specificity of the endolysin against its target species or genera. The domain binds noncovalently to the cell envelope, which can be part of the peptidoglycan or other cell wall associated molecules. The binding to the cell wall may be tight and irreversible, which also minimizes the endolysins from attacking the surrounding cells of the lysed bacterium. In addition, the catalytic domains also have an influence on the specificity of an endolysin. The peptidoglycan structures differ between Gram-types and also between different bacterial species. Therefore, the absence or presence of a specific target bond may contribute to the specificity of endolysins.

[0035] The antibacterial specificity of endolysins can be wide (e.g., the specificity of endolysin from the moderate bacteriophage ϕ0303), or narrow (as the specificity of endolysin PlyG from phage γ, which is specific for Bacillus anthracis). In practice, both specific and nonspecific endolysins are of use: the former can be used as drugs with a wide spectrum of action and the latter are handy for diagnostics. The bacterial affinity of modular endolysins from the bacteriophages infecting gram-positive hosts is determined by a separate CBD - cell wall binding domain, which recognizes components of the cell wall of a particular strain or species. The EAD (enzymatically active domain) is the catalytic domain which degrades peptidoglycan (PG). In case of single-domain globular endolysins from the phages infecting gram-negative bacteria, there is almost no information about the nature of their bacterial selectivity and the mechanisms of their interaction with the cell wall. It is only clear that in these endolysins, both functions (recognition and hydrolysis) must be carried out by the same domain – meaning that spatially, these functions cannot be differentiated too much.

[0036] The inventors have identified and characterised bacteriophage-derived endolysins with high specificity and bacteriolytic activity against a broad spectrum of C. septicum strains. Endolysin Polypeptides, Fragments and Variants Thereof

[0037] In an aspect, provided herein are endolysin polypeptides useful as antimicrobial agents. As described herein, an endolysin polypeptide and all variant endolysins having antimicrobial activity may be referred to interchangeably herein as “endolysins”, “endolysins of the invention”, “endolysin polypeptides”, or “endolysin polypeptides of the invention”. An endolysin polypeptide may be a polypeptide comprising the amino sequence set forth in any one of the SEQ ID NOs of the disclosure (such as disclosed in TABLE 6), or a variant thereof. The endolysins provided herein have surprising and advantageous antimicrobial functional characteristics and activity. The endolysins as described herein have antibacterial activity 9 105506204.3Attorney Docket: 125930-848037 against Clostridium septicum species, and its different strains. Clostridium septicum may be referred to herein as “C. septicum” or “Cs”.

[0038] In an aspect, provided herein are variants of the endolysins disclosed herein (e.g., a variant of the amino acid sequence set forth in any one of the SEQ ID NOs disclosed herein, or a polypeptide described herein as an endolysin). A “variant” may be a fragment or truncated form thereof, or a mutant or derivative of a specific endolysin having an amino acid sequence as set forth any one of the SEQ ID NOs disclosed herein. For example, a variant may differ from a specific endolysin having an amino acid sequence as set forth in SEQ ID NOs disclosed herein (such as disclosed in TABLE 6) by one or more amino acid substitutions. The amino acid sequence of an endolysin variant may relate to the amino acid sequence of a particular SEQ ID NO by reference to a percentage identity, as defined in more detail herein. A variant endolysin may have antimicrobial activity, particularly against Clostridium septicum. The variant endolysin may have antibacterial activity against Clostridium septicum, or a strain thereof. A variant amino acid sequence of an endolysin described herein, wherein the variant does not have antimicrobial or antibacterial activity (e.g., such as by an assay as described herein) is not an endolysin polypeptide.

[0039] A “fragment” of an endolysin polypeptide, or “fragment” of a domain of an endolysin polypeptide, as described herein, may be any polypeptide which has an amino acid sequence that is shorter than the full-length endolysin polypeptide or full-length of a domain of an endolysin polypeptide (e.g., N-terminally truncated, C-terminally truncated, or both). The fragment may be a fragment of an amino acid sequence set forth in TABLE 6. Endolysins are well- characterised enzymes and the skilled person may readily delineate the sequence characteristics which define a full-length endolysin polypeptide or full-length domain of the endolysin polypeptide, such as by sequence alignment and analysis. Accordingly, a skilled person may readily delineate such a “fragment”. Structural Features

[0040] An endolysin as described herein may comprise a Clostridium septicum cell wall peptidoglycan catalytic domain, also referred to herein as a peptidoglycan catalytic domain or as a catalytic domain. An endolysin may comprise one or more Clostridium septicum cell wall peptidoglycan catalytic domain (e.g., two, three, four, or more). In some embodiments, the catalytic domain comprises the amino acid sequence set forth in any one of SEQ ID NOS:26-50, or a variant thereof having at least 80% or greater sequence identity to any one of SEQ ID NOS:26-50, or a fragment thereof. 10 105506204.3Attorney Docket: 125930-848037

[0041] An endolysin as described herein may comprise a Clostridium septicum cell wall binding domain, also referred to herein as cell wall binding domain or binding domain. An endolysin comprising a Clostridium septicum cell wall binding domain, but not a Clostridium septicum cell wall peptidoglycan catalytic domain, may comprise a further component (e.g., amino acid sequence) having antimicrobial or antibacterial activity (e.g., as determined by an assay as described herein). An endolysin may comprise one or more Clostridium septicum cell wall binding domain (e.g., two, three, four, or more). In some embodiments, the binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOS:51-75, 80, 81, 82, or 83, or a variant thereof having at least 80% or greater sequence identity to any one of SEQ ID NOS:51-75, 80, 81, 82, or 83, or a fragment thereof.

[0042] An endolysin as described herein may comprise a Clostridium septicum cell wall peptidoglycan catalytic domain and a Clostridium septicum cell wall binding domain. An endolysin may comprise one or more Clostridium septicum cell wall peptidoglycan catalytic domains (e.g., two, three, four, or more) and one or more Clostridium septicum cell wall peptidoglycan catalytic domains (e.g., two, three, four, or more). In some embodiments, the endolysin comprising a catalytic domain and a binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOS:1-25, or 79, or a variant thereof having at least 80% or greater sequence identity to any one of SEQ ID NOS:1-25, or 79, or a fragment thereof.

[0043] An endolysin as described herein may further comprise one or more linkers connecting the catalytic domain and the binding domain, and / or one or more linkers connecting multiple binding domains, when present, and / or one or more linkers connecting multiple catalytic domains, when present.

[0044] Reference made herein to an “isolated” endolysin polypeptide or fragment thereof. may distinguish between the endolysin polypeptides or fragments thereof of the invention and endolysin polypeptides or fragments thereof which occur in nature. In the context of the present invention the term “isolated” may be used when referring to such endolysin polypeptides or fragments of the invention when expressed in host cells from nucleic acids and / or from expression vectors, or when present in lysates derived from such host cell and compositions derived from such host cells and lysates. The term “isolated” may mean that the endolysin polypeptide, variant, or fragment thereof is artificially produced and purified by the hand of man, such as is routine for exogenous protein production using recombinant expression systems. 11 105506204.3Attorney Docket: 125930-848037 Endolysins Comprising Catalytic Domains and Binding Domains

[0045] An endolysin as described herein may comprise a catalytic domain (e.g., a bacterial cell wall peptidoglycan catalytic domain) and a binding domain (e.g., a bacterial cell wall binding domain), wherein the endolysin comprises the amino acid sequence set forth in any one of SEQ ID NOS:1-25, or 79. For SEQ ID NOS:1-25, or 79, the N-terminal domains represent Clostridium septicum peptidoglycan catalytic domains and the C-terminal domains represent Clostridium septicum cell wall binding domains.

[0046] In some embodiments, an endolysin polypeptide comprises a Clostridium septicum cell wall peptidoglycan catalytic domain and a Clostridium septicum cell wall binding domain. In some embodiments, an endolysin polypeptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2425, or 79. In some embodiments, an endolysin polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2425, or 79, or a fragment thereof each.

[0047] For a fragment of said amino acid sequence, the endolysin polypeptide may comprise or consist of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a fragment of the amino acid sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2425, or 79, when each sequence is aligned to each other according to their full length.

[0048] In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:1, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:2, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:3, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:4, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:5, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:6, or a variant thereof. In some embodiments, the endolysin polypeptide having 12 105506204.3Attorney Docket: 125930-848037 antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:7, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:8, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:9, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:10, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:11, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:12, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:13, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:14, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:15, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:16, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:17, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:18, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:19, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:20, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:21, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:22, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:23, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:24, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:79, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:25, or 79, or a variant thereof. 13 105506204.3Attorney Docket: 125930-848037

[0049] Also provided is a combination of endolysins of the invention, comprising two or more different Clostridium septicum endolysin polypeptides. The different endolysin polypeptides present in a combination may be present as separate polypeptides, e.g., the combination represents a mixture of two or more different polypeptides. Alternatively, the different endolysin polypeptides present in a combination may be present as physically linked polypeptides, e.g., the combination represents at least one chimeric protein or fusion protein.

[0050] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:1, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0051] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:2, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0052] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:3, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof. 14 105506204.3Attorney Docket: 125930-848037

[0053] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:4, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0054] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:5, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0055] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:6, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0056] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:7, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at 15 105506204.3Attorney Docket: 125930-848037 least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0057] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:8, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0058] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:9, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0059] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:10, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0060] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at 16 105506204.3Attorney Docket: 125930-848037 least 99%, or 100% identical to the sequence forth in SEQ ID NO:11, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0061] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:12, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0062] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:13, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0063] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:14, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof. 17 105506204.3Attorney Docket: 125930-848037

[0064] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:15, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0065] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:16, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0066] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:17, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0067] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:18, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at 18 105506204.3Attorney Docket: 125930-848037 least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0068] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:19, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0069] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:20, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

[0070] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:21, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or 79, or a fragment thereof.

[0071] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at 19 105506204.3Attorney Docket: 125930-848037 least 99%, or 100% identical to the sequence forth in SEQ ID NO:22, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, or 79, or a fragment thereof.

[0072] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:23, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, or 79, or a fragment thereof.

[0073] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:24, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, or 79, or a fragment thereof.

[0074] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:25, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24, or a fragment thereof. 20 105506204.3Attorney Docket: 125930-848037

[0075] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:79, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or a fragment thereof. Endolysins Comprising Catalytic Domains

[0076] An endolysin as described herein may comprise a catalytic domain (e.g., a bacterial cell wall peptidoglycan catalytic domain), wherein the endolysin comprises the amino acid sequence set forth in any one of SEQ ID NOS:26-50. A catalytic domain may enzymatically cleave the peptidoglycan layer of a bacterial cell wall. The catalytic domain may have influence on the specificity of the endolysin against its target species, genera or strain. The catalytic domain, or a fragment thereof, may have N-acetyl-β-D-glucosaminidase activity, N-acetyl-β-D-muramidase activity, N-acetylmuramoyl-L-alanine amidase activity and / or endopeptidase activity. SEQ ID NOS:26-50 represent Clostridium septicum peptidoglycan catalytic domains.

[0077] In some embodiments, an endolysin polypeptide comprises a Clostridium septicum cell wall peptidoglycan catalytic domain. In some embodiments, an endolysin polypeptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. In some embodiments, an endolysin polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, or a fragment each.

[0078] For a fragment of said amino acid sequence, the endolysin polypeptide may comprise or consist of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a fragment of the amino acid sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, when each sequence is aligned to each other according to their full length. 21 105506204.3Attorney Docket: 125930-848037

[0079] In one aspect, endolysins may comprise only a catalytic domain when the catalytic domain provides a means for specificity for its target bacterial species, genera or strain.

[0080] In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:26, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:27, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:28, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:29, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:30, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:31, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:32, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:33, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:34, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:35, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:36, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:37, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:38, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:39, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:40, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:41, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:42, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:43, or a variant thereof. In some embodiments, the endolysin polypeptide having 22 105506204.3Attorney Docket: 125930-848037 antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:44, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:45, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:46, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:47, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:48, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:49, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:50, or a variant thereof. Endolysins Comprising Binding Domain

[0081] An endolysin as described herein may comprise a binding domain (e.g., a bacterial cell wall binding domain), optionally wherein the endolysin comprises the amino acid sequence set forth in any one of SEQ ID NOS:51-75, 80, 81, 82, or 83. A binding domain may provide a means for specificity of the endolysin for its target bacterial species, genera or strain. A binding domain may bind noncovalently to the cell envelope, which can be part of the peptidoglycan or other cell wall associated molecules. The binding of a binding domain to the cell wall in the interior of the bacteria cell may prevent the endolysin from lysing other bacteria that the Clostridium septicum bacteriophage has yet to infect. As such, the existence of the C-terminal binding domain(s) may be an evolutionary feature to ensure the survival of bacteriophage progeny. SEQ ID NOS:51-75, 80, 81, 82, or 83 represent Clostridium septicum cell wall binding domains.

[0082] The binding domain may enhance the specificity of the endolysin for its target bacterial species, genera or strain but it may not be essential for antimicrobial activity.

[0083] An endolysin of the invention may also have more than one binding domain in series, such as two C-terminal binding domains in series.

[0084] In some embodiments, an endolysin polypeptide comprises a Clostridium septicum cell wall binding domain. In some embodiments, an endolysin polypeptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 7475, 80, 81, 82, or 83. In some 23 105506204.3Attorney Docket: 125930-848037 embodiments, an endolysin polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 7475, 80, 81, 82, or 83, or a fragment thereof each.

[0085] For a fragment of said amino acid sequence, the endolysin polypeptide may comprise an amino acid sequence which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a fragment of the amino acid sequence of the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 7475, 80, 81, 82, or 83, when each sequence is aligned to each other according to their full length.

[0086] In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:51, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:52, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:53, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:54, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:55, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:56, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:57, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:58, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:59, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:60, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:61, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:62, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:63, or a variant thereof. In some embodiments, the endolysin 24 105506204.3Attorney Docket: 125930-848037 polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:64, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:65, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:66, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:67, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:68, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:69, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:70, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:71, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:72, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:73, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:74, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:75, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:80, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:81, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:82, or a variant thereof. In some embodiments, the endolysin polypeptide having antimicrobial activity comprises the amino acid sequence set forth in SEQ ID NO:83, or a variant thereof. Linkers

[0087] An endolysin as described herein may comprise one or more linkers which connect the catalytic domain with the cell wall binding domain, and / or one or more linkers connecting multiple binding domains, when present, and / or one or more linkers connecting multiple catalytic domains, when present. The linker may comprise a polypeptide comprising an amino acid sequence. In certain embodiments, the linker is a polypeptide. The linker may be cleavable 25 105506204.3Attorney Docket: 125930-848037 or non-cleavable. In certain embodiments, the linker is non-cleavable. A linker may be a linear linker or a branched linker.

[0088] The linker may be a flexible linker. In some embodiments, the flexible linker comprises an amino acid sequence comprising the sequence of (GGGGS)n,(SEQ ID NO:76) where n specifies the number of repeats of the unit. The number of repeats of the flexible linker may be from 1-3.

[0089] The linker may be a rigid linker. In some embodiments, the rigid linker comprises an amino acid sequence comprising the sequence of (EAAAK)n (SEQ ID NO:77), where n specifies the number of repeats of the unit. The number of repeats of the non-flexible linker may be from 1-3.

[0090] The linker may be a portion of an endolysin as described herein (e.g., a non-functional fragment). In some embodiments, the linker comprises an amino acid sequence comprising the sequence of NIEQS (SEQ ID NO:78).

[0091] As a skilled artisan would recognize, any suitable linker may be used to connect the domains of the endolysin as descripted herein. The linker may be a naturally occurring linker, such as a naturally occurring endolysin linker, or may be an artificial linker. Examples of linkers which may be employed are described in Chen et al., Adv Drug Deliv Rev., Oct.15, 2013; 65(10): 1357-1369 and in Chichili et al., Protein Sci., 2013 February; 22(2): 153-167, which are incorporated by reference in their entireties.

[0092] A linker may comprise a hydrocarbon chain. A hydrocarbon chain may comprise of from 2 to about 2000 carbon atoms. The hydrocarbon chain may comprise an alkylene group, e.g., C2 to about 2000 or more alkylene groups. The hydrocarbon chain may have a general formula of —(CH2)n-, wherein n is from 2 to about 2000. The hydrocarbon chain may be optionally interrupted by one or more ester groups (e.g., —C(O)—O—) or one or more amide groups (e.g., —C(O)—N(H)—).

[0093] A linker may be selected from PEG, polyacrylamide, poly(2-hydroxyethyl methacrylate), Poly-2-methyl-2-oxazoline (PMOXA), zwitterionic polymers, e.g., poly(carboxybetaine methacrylate) (PCBMA), poly[N-(3-sulfopropyl)-N-methacryloxyethyl-N, N dimethyl ammonium betaine](PSBMA), glycopolymers, or polypeptides.

[0094] A linker may comprise a polyethylene glycol (PEG) having a general formula of --(CH2- CH2-O)n-, wherein n is from 1 to about 600. A linker may comprise oligoethylene glycol- 26 105506204.3Attorney Docket: 125930-848037 phosphate units having a general formula of —[(CH2-CH2-O)n-PO2-O]m-, where n is from 1 to about 600 and m is from 1 to about 200. Variation in Domain / Linker Architecture

[0095] In the present disclosure, Clostridium septicum cell wall peptidoglycan catalytic domains such as described by SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 or the Clostridium septicum cell wall binding domains such as described by SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83 may be defined according to homology search using the Pfam software. Any of the Clostridium septicum cell wall peptidoglycan catalytic domains, such as described by SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and any one of the Clostridium septicum cell wall binding domains described by SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83 may be defined according to homology search using HHpred software, InterProScan software or the Structural Classification of Proteins (SCOP) database.

[0096] The endolysins of the invention may comprise a Clostridium septicum peptidoglycan catalytic domain. The endolysins of the invention may comprise a Clostridium septicum peptidoglycan catalytic domain and additionally may comprise one or more Clostridium septicum cell wall binding domains. The endolysins of the invention may comprise one or more Clostridium septicum cell wall binding domains.

[0097] Endolysins of the invention encompass various combinations of catalytic and / or cell wall binding domains. Endolysins of the invention may comprise combinations of two or more different Clostridium septicum peptidoglycan catalytic domains. Endolysins of the invention may comprise combinations of two or more different Clostridium septicum peptidoglycan catalytic domains and additionally may comprise two or more different Clostridium septicum cell wall binding domains. The endolysins of the invention may comprise two or more different Clostridium septicum cell wall binding domains.

[0098] Endolysins of the invention may comprise more than one Clostridium septicum peptidoglycan catalytic domain, as described herein. Endolysins of the invention may comprise more than one Clostridium septicum cell wall binding domain, as described and defined herein. Further in addition, they may also contain one or more linker sequences as described and defined herein. 27 105506204.3Attorney Docket: 125930-848037

[0099] The components of an endolysin polypeptide as described herein (e.g., the one or more catalytic domain, or variant or fragment thereof; the one or more binding domain, or variant or fragment thereof; the one or more linker, or variant or fragment thereof) may be structured in any order in the endolysin polypeptide. Thus, for example, a Clostridium septicum cell wall peptidoglycan catalytic domain (or fragment or variant thereof), which in a naturally occurring endolysin polypeptide is positioned at the N-terminus of the polypeptide may instead be positioned at the C-terminus of a polypeptide of this invention. Similarly, a Clostridium septicum cell wall binding domain (or fragment or variant thereof), which in a naturally occurring endolysin polypeptide is positioned at the C-terminus of the polypeptide may instead be positioned at the N-terminus of a polypeptide of this invention. Any one or more linker sequences may connect any two or more catalytic domain sequences or fragments thereof in any order and any two or more binding domain sequences or fragments thereof in any order. Endolysin Polypeptides Comprising Combinations of Catalytic Domains

[0100] An endolysin polypeptide may comprise a first Clostridium septicum cell wall peptidoglycan catalytic domain polypeptide or fragment thereof, a second Clostridium septicum cell wall peptidoglycan catalytic domain polypeptide or fragment thereof and optionally a linker polypeptide or fragment thereof connecting the first and second catalytic domains; wherein the isolated polypeptide comprises: a) a first amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an Clostridium septicum peptidoglycan catalytic domain polypeptide, or a fragment of said polypeptide; or which is an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the catalytic domain of the Clostridium septicum bacteriophage endolysin polypeptide set forth in SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, or an amino acid sequence which is a fragment of said catalytic domain polypeptide and which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the sequence set forth in SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 which corresponds to the amino acid sequence of the fragment; and 28 105506204.3Attorney Docket: 125930-848037 b) a second amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an Clostridium septicum peptidoglycan catalytic domain polypeptide, or a fragment of said polypeptide; or which is an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the catalytic domain of the Clostridium septicum bacteriophage endolysin polypeptide set forth in SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, or an amino acid sequence which is a fragment of said catalytic domain polypeptide and which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the sequence set forth in SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 which corresponds to the amino acid sequence of the fragment; and c) an optional linker amino acid sequence; wherein the linker amino acid sequence connects the amino acid sequence of the peptidoglycan catalytic domain of the first Clostridium septicum bacteriophage endolysin polypeptide or fragment thereof and the second Clostridium septicum bacteriophage endolysin polypeptide or fragment thereof.

[0101] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:26, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0102] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:27, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, 29 105506204.3Attorney Docket: 125930-848037 at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0103] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:28, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0104] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:29, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0105] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:30, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0106] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, 30 105506204.3Attorney Docket: 125930-848037 at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:31, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0107] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:32, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0108] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:33, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0109] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:34, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of 31 105506204.3Attorney Docket: 125930-848037 SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0110] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:35, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0111] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:36, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0112] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:37, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0113] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:38, or a fragment thereof, and 32 105506204.3Attorney Docket: 125930-848037 an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0114] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:39, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0115] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:40, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0116] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:41, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each. 33 105506204.3Attorney Docket: 125930-848037

[0117] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:42, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0118] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:43, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0119] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:44, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0120] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:45, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at 34 105506204.3Attorney Docket: 125930-848037 least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49, or 50, or a fragment thereof each.

[0121] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:46, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, or 50, or a fragment thereof each.

[0122] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:47, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 48, 49, or 50, or a fragment thereof each.

[0123] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:48, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, or 50, or a fragment thereof each.

[0124] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at 35 105506204.3Attorney Docket: 125930-848037 least 99%, or 100% identical to the sequence forth in SEQ ID NO:49, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 50, or a fragment thereof each.

[0125] A combination of endolysin polypeptides may comprise an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:50, or a fragment thereof, and an endolysin polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49, or a fragment thereof each.

[0126] In the abovementioned combinations of Clostridium septicum peptidoglycan catalytic domains the different domains may be physically linked by a linker, as described herein. When the linker is an amino acid sequence, the domains may be present in the same polypeptide chain and form a chimeric or fusion protein. Alternatively, the Clostridium septicum peptidoglycan catalytic domains may not be linked and may be present as a mixture of a plurality of different polypeptides. Endolysin Polypeptides Comprising Combinations of Catalytic Domains and Binding Domains

[0127] An endolysin polypeptide may comprise an amino acid sequence comprising or consisting of an Clostridium septicum cell wall peptidoglycan catalytic domain polypeptide or fragment thereof, an amino acid sequence comprising or consisting of a Clostridium septicum cell wall binding domain polypeptide or fragment thereof and an amino acid sequence comprising or consisting of a linker polypeptide or fragment thereof connecting the catalytic and cell wall binding domains; wherein the isolated polypeptide comprises: a) an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the cell wall peptidoglycan 36 105506204.3Attorney Docket: 125930-848037 catalytic domain of the Clostridium septicum bacteriophage endolysin polypeptide set forth in SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, or an amino acid sequence which is a fragment of said catalytic domain polypeptide and which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the sequence set forth in SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 which corresponds to the amino acid sequence of the fragment; and b) an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the Clostridium septicum cell wall binding domain of the Clostridium septicum bacteriophage endolysin polypeptide set forth in SEQ ID NO:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 7475, 80, 81, 82, or 83, or an amino acid sequence which is a fragment of said cell wall binding domain polypeptide and which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the sequence set forth in SEQ ID NO:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 7475, 80, 81, 82, or 83 which corresponds to the amino acid sequence of the fragment; and c) a linker amino acid sequence; wherein the linker amino acid sequence connects the amino acid sequence of the cell wall peptidoglycan catalytic domain of the Clostridium septicum bacteriophage endolysin polypeptide or fragment thereof and the cell wall binding domain of the Clostridium septicum bacteriophage endolysin polypeptide or fragment thereof.

[0128] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:26, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each. 37 105506204.3Attorney Docket: 125930-848037

[0129] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:27, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0130] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:28, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0131] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:29, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0132] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:30, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each. 38 105506204.3Attorney Docket: 125930-848037

[0133] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:31, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0134] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:32, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0135] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:33, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0136] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:34, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each. 39 105506204.3Attorney Docket: 125930-848037

[0137] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:35, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0138] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:36, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0139] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:37, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0140] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:38, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each. 40 105506204.3Attorney Docket: 125930-848037

[0141] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:39, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0142] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:40, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0143] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:41, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0144] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:42, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each. 41 105506204.3Attorney Docket: 125930-848037

[0145] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:43, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0146] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:44, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0147] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:45, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0148] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:46, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each. 42 105506204.3Attorney Docket: 125930-848037

[0149] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:47, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0150] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:48, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0151] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:49, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each.

[0152] The endolysin polypeptide may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence forth in SEQ ID NO:50, or a fragment thereof, and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof each. 43 105506204.3Attorney Docket: 125930-848037

[0153] In the abovementioned combinations of Clostridium septicum peptidoglycan catalytic domains and Clostridium septicum cell wall binding domains the catalytic domains may be physically linked, by a linker as described herein, to the cell wall binding domains. When the linker is an amino acid sequence, the Clostridium septicum peptidoglycan catalytic domains and Clostridium septicum cell wall binding domains may both be present in the same polypeptide chain and form a chimeric or fusion protein. Endolysin Polypeptides Comprising Fragments and Variants

[0154] Any of the fragments described herein may be truncated with respect to the full length amino acid sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2425, or 79 or with respect to domains / linkers defined in the amino acid sequences set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2425, or 79, provided that any such fragment has any antimicrobial activity (e.g., such as determined by an assay described herein). Such a fragment may be truncated by any number of amino acids, at the N-terminal and / or C-terminal ends of the polypeptide.

[0155] An endolysin polypeptide according to the invention may be derived from any one of the C. septicum bacteriophage endolysin polypeptides set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79 by substituting, inserting, deleting, or adding any number of amino acids at any position, such as 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more amino acids, provided that any such fragment has any antimicrobial activity as defined herein. One or more or all amino acid substitutions may be a conservative amino acid substitution.

[0156] An endolysin polypeptide according to the invention may be derived from one of the sequences as identified herein by adding one or more additional N- or C-terminal amino acids or chemical moieties to increase stability, solubility and activity.

[0157] An endolysin polypeptide according to the invention may have a length of at least 100 or more, of from 100 to 150, of from 150 to 200, of from 200 to 250, of from 250 to 300, of from 300 to 350, of from 350 to 400, of from 400 to 450, of from 450 to 500, of from 500 to 550, of from 550 to 600, of from 600 to 650, of from 650 to 700, of from 700 to 750, of from 750 to 800, of from 800 to 850, of from 850 to 900, of from 900 to 950, of from 950 to 1000, of from 44 105506204.3Attorney Docket: 125930-848037 1000 to 1100, of from 1100 to 1200, of from 1200 to 1300 or more, of from 1300 to 1400, of from 1400 to 1500, or 2000 or more amino acids.

[0158] A variant endolysin polypeptide may be any non-naturally occurring or genetically engineered form of the endolysin polypeptide according to the invention, including any fragment or derivative thereof, provided that any such variant has any antimicrobial activity as defined herein. An endolysin polypeptide variant may differ in some engineered way from any of the polypeptides disclosed herein by reference to SEQ ID NO:1-25, or 79. For example, a variant endolysin polypeptide according to the invention may be made by site-directed mutagenesis starting from the nucleotide sequence encoding a polypeptide defined by any one of SEQ ID NO:1-25, or 79 disclosed herein. Site-directed mutagenesis may comprise acid replacement substitution of one or more amino acids.

[0159] A fragment of a variant endolysin polypeptide according to the invention may be defined according to percentage identity with respect to a reference amino acid sequence or a reference polynucleotide sequence. For example a fragment of variant endolysin polypeptide according to the invention may be defined according to percentage identity with respect to the amino acid sequence of an endolysin polypeptide defined by a sequence set forth in any one of SEQ ID NO:1-25, or 79 as the reference amino acid sequence, or by a sub-sequence set forth in any one of SEQ ID NOS:1-25, or 79 as the reference amino acid sequence, such as the sequence of any domain or linker defined therein or a sequence portion of such a domain or linker.

[0160] Amino acid identity may be calculated using any suitable algorithm. For example, the PILEUP and BLAST algorithms can be used to calculate identity or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F., 1993 J Mol Evol 36:290-300; Altschul, S, F et al, 1990 J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, 45 105506204.3Attorney Docket: 125930-848037 due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1992 Proc. Natl. Acad. Sci . USA 89: 10915-10919) alignments (B) of 50, expectation (E) of N=4, and a comparison of bothstrands.

[0161] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul, 1993 Proc. Natl. Acad. Sci . USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence when the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Alternatively, the UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al., 1984 Nucleic Acids Research 12, 387- 395).

[0162] As described herein, the amino acid sequence of a polypeptide of the invention may comprise the amino acid sequence of the whole or a portion of a reference sequence defined by SEQ ID NO in which modifications, such as amino acid additions, deletions or substitutions are made relative to the reference sequence or portion thereof. The modifications may be conservative or non-conservative amino acid substitutions. When there are multiple modifications in a single polypeptide, the modifications in the polypeptide sequence may be a combination of conservative and non-conservative amino acid substitutions. A conservative substitution may replace an amino acid with another amino acid of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acid introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acid it replaces. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in TABLE 1 below. 46 105506204.3Attorney Docket: 125930-848037 TABLE 1: Chemical properties of amino acids for conservative substitutions

[0163] Where amino acids have similar polarity, this can be determined by reference to the hydropathy scale for amino acid side chains in TABLE 1 above.

[0164] One of skill in the art may determine whether any fragment or variant endolysin possesses antimicrobial activity by performing a functional test for antimicrobial activity as described further herein. Functional Characteristics of Endolysins

[0165] An endolysin as described herein may have antimicrobial activity. The antimicrobial activity may be bacteriolytic activity. Bacteriolytic activity can be determined using a turbidity reduction assay, a t50% lysis value (time required for an endolysin to lyse 50% of its initial cell population) and / or a cell viability assay (log10 values plotted against increasing concentrations of endolysin or ratio of control over endolysin-treated cell reactions to calculate the percentage reduction of viable bacteria for each endolysin). Bacteriolytic activity can be defined by minimum bactericidal concentration (MBC). The antimicrobial activity may be bacteriostatic or 47 105506204.3Attorney Docket: 125930-848037 growth inhibitory activity. Bacteriostatic or growth inhibitory activity may be determined by minimum inhibitory concentration (MIC).

[0166] An endolysin of the invention may provide an antimicrobial effect against C. septicum bacteria that is higher compared to the effect observed against relevant control bacteria. A relevant control may be any bacterial strain that is not C. septicum (herein referred to as a “non- Cs” strain). The non-Cs strain with its DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) number in parentheses includes Clostridium colinum (DSMZ 6011), Clostridium leptum (DSMZ 753), Clostridium cellobioparum (DSMZ 1351), and Bifidobacterium adolescentis (DSMZ 20083). These are four non-Cs bacteria strains commonly found in the poultry gut and may be used to determine whether endolysin activity and hence antimicrobial activity is specific to C. septicum.

[0167] Endolysin of the invention C. septicum strains may include NCTC (National Collection of Type Cultures) strains procured from the Public Health England culture collection, with the accession numbers: NCTC 281, NCTC 282, NCTC283, NCTC 284, NCTC 286, NCTC501, NCTC 504, NCTC 547, NCTC 549, NCTC 550 and NCTC 551. These C. septicum strains may be referred herein as “reference strains”.

[0168] An endolysin polypeptide of the invention may exhibit antimicrobial effects to one or more of the C. septicum reference strains disclosed herein. The endolysin of the invention may exhibit antimicrobial effects against NCTC strains: NCTC 281, NCTC 282, NCTC283, NCTC 284, NCTC 286, NCTC501, NCTC 504, NCTC 547, NCTC 549, NCTC 550 and NCTC 551.

[0169] The antimicrobial activity may be determined by using a standard cell viability assay as disclosed herein to calculate a Δ log10 value. The Δ log10 value exhibited by an endolysin polypeptide or fragment may be determined using a concentration of 5 μg / ml of an endolysin or fragment of the invention against a C. septicum reference strain, NCTC 547. The Δ log10 value may be approximately 0.40 or more, 2.00 or more or 3.70 or more. Approximately is taken to mean+ / −10% or + / −5%.

[0170] The antimicrobial activity may be determined by using a standard cell viability assay as disclosed herein to calculate a % reduction in cell viability. The % reduction cell viability value exhibited by an endolysin polypeptide or fragment of the invention may be determined using a concentration of 5 μg / ml of an endolysin or fragment of the invention against a C. septicum reference strain, NCTC 547. The % reduction in cell viability may be approximately 60% or 48 105506204.3Attorney Docket: 125930-848037 more, 70% or more, 80% or more, 90% or more or 100%, wherein approximately may be + / −10% or + / −5%.

[0171] The antimicrobial activity may be determined by using a standard cell turbidity reduction assay as disclosed herein, to calculate a t50 lysis value. The t50 lysis value exhibited by an endolysin polypeptide or fragment is determined using a concentration of 5 μg / ml of an endolysin of the invention or a fragment against a C. septicum reference strain, NCTC 547. The t50 lysis value provided may be approximately 7 minutes or less; or it may provide a t50% lysis value of 3 minutes or less. Approximately is taken to mean+ / −10% or + / −5%.

[0172] The antimicrobial activity may be determined by using a standard minimum inhibitory concentration (MIC) assay and / or a minimum bactericidal concentration (MBC) assay. A standard minimum inhibitory concentration (MIC) assay or a minimum bactericidal concentration (MBC) assay may be performed against Clostridium septicum strain NCTC 547 at 2.1×104 cells / mL wherein an endolysin of the invention or a fragment exhibits an MIC value or an MBC value of 17 μg / mL or less, preferably 1.7 μg / mL or less; or it exhibits an MIC value or an MBC value of 0.65 μM or less, preferably 0.05 μM or less. Approximately is taken to mean+ / −10% or + / −5%.

[0173] Endolysin activity (e.g. hydrolytic activity), and therefore antimicrobial activity, may be determined by assessing the rate of degradation of purified peptidoglycan (PGN) in vitro. The rate of degradation may be assessed by measuring the spontaneous rate of PGN degradation in any suitable buffer. The rate of degradation of purified PGN in vitro can be assessed by optical density. For use in such an assay, preferably the PGN is purified from a bacterial species, more preferably from a C. septicum strain, such as the C. septicum NCTC 547 strain.

[0174] Any endolysin polypeptide of the invention may be determined to have endolysin hydrolytic activity, and therefore antimicrobial activity, when it promotes a statistically significant increase in the rate of degradation of purified PGN in vitro. The ability of a given endolysin polypeptide to promote a statistically significant increase in the rate of degradation of purified PGN in vitro can be assessed by reference to the background rate of PGN degradation, e.g., by reference to a control preparation which does not contain an endolysin polypeptide.

[0175] The ability of a given endolysin polypeptide to promote a statistically significant increase in the rate of degradation of purified PGN in vitro can be assessed by reference to the rate of PGN degradation by a reference or control polypeptide. A reference or control polypeptide should be a polypeptide which is not capable of promoting a statistically significant 49 105506204.3Attorney Docket: 125930-848037 increase in the rate of degradation of purified PGN in vitro. A reference or control polypeptide can be a polypeptide which is a PGN-catalytically inactive endolysin, or any other suitable polypeptide which is not an endolysin, such as bovine serum albumin.

[0176] Any endolysin polypeptide of the invention may be determined to have endolysin hydrolytic activity, and therefore antimicrobial activity, when it promotes a statistically significant reduction in the absolute amount of purified PGN in vitro (e.g., a statistically significant degradation of purified PGN in vitro) at the endpoint (completion) of a PGN degradation assay.

[0177] The ability of a given endolysin polypeptide to promote a statistically significant reduction in the absolute amount of purified PGN in vitro can also be assessed by reference to the background reduction in the absolute amount of purified PGN, e.g., by reference to a control preparation which does not contain an endolysin polypeptide.

[0178] The ability of a given endolysin polypeptide to promote a statistically significant reduction in the absolute amount of purified PGN in vitro can also be assessed by reference to the reduction in the absolute amount of purified PGN degradation by a reference or control polypeptide. A reference or control polypeptide should be a polypeptide which is not capable of promoting a statistically significant reduction in the absolute amount of purified PGN in vitro. A reference or control polypeptide can be a polypeptide which is a PGN-catalytically inactive endolysin, or any other suitable polypeptide which is not an endolysin, such as bovine serum albumin.

[0179] Endolysin hydrolytic activity can be assessed in this way for purified endolysins, as well as for endolysins comprised in cell extracts.

[0180] In the case of purified endolysins, activity may be assessed by reference to the background rate of degradation, or background reduction in the absolute amount, of purified PGN, e.g., by reference to a control preparation which does not contain a purified endolysin polypeptide. Alternatively, activity may be assessed by reference to a control preparation which contains a purified control polypeptide, which can be a polypeptide which is a PGN-catalytically inactive endolysin, or any other suitable polypeptide which is not an endolysin, such as bovine serum albumin. In the case of endolysins comprised in cell extracts, activity may be assessed by reference to the background rate of degradation, or background reduction in the absolute amount, of purified PGN, e.g., by reference to a control preparation which does not contain a cell extract comprising an endolysin polypeptide. Preferably the control contains a cell extract 50 105506204.3Attorney Docket: 125930-848037 comparable with the test sample but minus the endolysin polypeptide. Alternatively, activity may be assessed by reference to a control preparation which contains a control polypeptide comprised in a comparable cell extract. A control polypeptide can be a polypeptide which is a PGN-catalytically inactive endolysin, or any other suitable polypeptide which is not an endolysin, such as bovine serum albumin.

[0181] Antimicrobial activity such as bacteriolytic activity and bacteriostatic activity, may be determined according to the assays described in the following section.

[0182] Assays for bacteriolytic activity include spot assays in which endolysin polypeptides of the invention and fragments thereof are added to C. septicum cell lawns already established on the appropriate medium (e.g., agar plate). Any relevant negative control, such as PBS, may be used. Any positive control such as any antibiotic (e.g., ampicillin may be used) or any other product that exhibits an antimicrobial and / or bacteriolytic effect on C. septicum may be used. Bacteriolytic activity is indicated by the presence of clearance zones and the extent of bacteriolytic activity can be determined by the size of the clearance zones.

[0183] Bacteriostatic (growth inhibitory) activity may be determined as described herein. Assays for bacteriostatic activity include spot assays in which C. septicum strains are freshly plated on the appropriate medium (e.g., agar plate) and varying concentrations of the endolysin polypeptide of the invention added to the freshly plated bacteria. The extent of the bacteriostatic activity can be determined by lack of growth of C. septicum colonies compared with non-treated controls or controls treated with products that do not affect bacterial growth.

[0184] The relative increase in the bacteriolytic / log10 / t50% lysis value associated with any of the endolysin polypeptides of the invention, or the relative decrease in cell viability associated with any of the endolysin polypeptides of the invention compared with the reference endolysin polypeptide of a relevant control strain may be assessed by any suitable means. Any such relative increase or any such relative decrease associated with any of the endolysin polypeptides of the invention should be a statistically significant increase or decrease in the relevant property being assessed compared to a reference or control. A reference or control may be reference to the background, e.g., by reference to a control preparation which does not contain an endolysin polypeptide. A reference or control may be reference to a reference or control preparation comprising a reference or control polypeptide. A reference or control polypeptide should be a polypeptide which is not capable of increasing or decreasing the relevant property being assessed. A reference or control polypeptide can be a polypeptide which is a PGN-catalytically 51 105506204.3Attorney Docket: 125930-848037 inactive endolysin, or any other suitable polypeptide which is not an endolysin, such as bovine serum albumin.

[0185] The relative increase may be assessed by comparing the endolytic / bacteriolytic / log10 / t50% lysis value / cell viability activity of purified proteins of the present invention against any suitable control protein. For example, the enzyme, lysozyme or bovine serum albumin may be employed as a control protein. Preferably, the relative increase may be assessed by comparing the endolytic / bacteriolytic / log10 / t50% lysis value / cell viability activity in clarified lysates when polypeptides are separately expressed in a host cell, preferably in a Bacillus subtilis or an E. coli host cell. More specifically, the antimicrobial activities of the endolysin polypeptides of the invention can be determined using the assays outlined in the following section: Assays for Antimicrobial Effect (Δ Log 10 and Cell Viability) of Endolysins:

[0186] Δ Log 10, which is the log10 of the ratio of untreated cell control reactions over endolysin-treated cell reactions, may be calculated according to the following method: 1. Inoculate Clostridium septicum strain NCTC 547 into BHI+C media and culture under anaerobic conditions at 37° C. overnight to stationary phase; 2. Dilute the overnight culture into fresh BHI+C media and culture under anaerobic conditions at 37° C to OD 600 nm approximately 0.6; 3. Centrifuge 1.8 mL culture at 16,600× g for 5 minutes at room temperature under anaerobic conditions; 4. remove supernatant and resuspend cell pellet in 0.9 mL of an appropriate cell diluent (peptone water pH 7.0) under anaerobic conditions; 5. dilute cell suspension with cell diluent to OD 600 nm 0.6 under anaerobic conditions; 6. add purified endolysin polypeptide or fragment to a concentration of 5 μg / mL under anaerobic conditions; 7. incubate at 37° C for 1 hour under anaerobic conditions; 8. determine viable cell count by serial dilution, plating cells on RCM solid media and incubating at 37° C until colonies form under anaerobic conditions; and 52 105506204.3Attorney Docket: 125930-848037 9. calculate log 10 reduction value as the log 10 of the ratio of untreated cells control over cells treated with the endolysin polypeptide or fragment.

[0187] Cell viability may be calculated according to the following method: 1. Inoculate Clostridium septicum strain NCTC 547 into BHI+C media and culture under anaerobic conditions at 37° C. overnight to stationary phase; 2. Dilute the overnight culture into fresh BHI+C media and culture under anaerobic conditions at 37° C to OD 600 nm approximately 0.6; 3. Centrifuge 1.8 mL culture at 16,600× g for 5 minutes at room temperature under anaerobic conditions; 4. remove supernatant and resuspend cell pellet in 0.9 mL of an appropriate cell diluent (peptone water pH 7.0) under anaerobic conditions; 5. dilute cell suspension with cell diluent to OD 600 nm 0.6 under anaerobic conditions; 6. add purified endolysin polypeptide or fragment to a concentration of 5 μg / mL under anaerobic conditions; 7. incubate at 37° C for 1 hour under anaerobic conditions; 8. determine viable cell count by serial dilution, plating cells on RCM solid media and incubating at 37° C until colonies form under anaerobic conditions; and 9. calculate log 10 reduction value as the log 10 of the ratio of untreated cells control over cells treated with the endolysin polypeptide or fragment. 10. use log 10 reduction value to calculate the percentage reduction of viable bacteria.

[0188] The t50% lysis value, which is defined as the time required for an endolysin to lyse 50% of its initial cell suspension may be calculated according to the following method. 1. Inoculate Clostridium septicum strain NCTC 547 in BHI+C media and culture under anaerobic conditions at 37° C. overnight to stationary phase; 2. inoculate the overnight culture into fresh BHI+C media and culture under anaerobic conditions at 37° C. until the culture reaches exponential phase (OD 620 nm approximately 1.0); 53 105506204.3Attorney Docket: 125930-848037 3. centrifuge a volume of the exponential phase culture at 3800× g for 30 minutes at 10° C. and then remove the supernatant; 4. resuspend the cells in PBS, pH 7.0, in a volume which is half of the initial pre-centrifuged culture volume in order to concentrate the cells; 5. transfer 90 μL of the resuspended cells into a well of a 96-microwell plate and add 10 μL of 10× concentrated endolysin polypeptide or fragment in PBS, pH 7.0, to a final concentration of 5 μg / mL; 6. monitor the reduction in turbidity by performing an OD 620 nm kinetic read with suitable intervals on a plate reader at room temperature; 7. monitor turbidity for 30-60 minutes, or until the OD 620 nm reduces such that it begins to plateau; 8. from the turbidity reduction profile, calculate t50% lysis (minutes), e.g., the time for 50% of cells to be lysed, using the following Formula (I): wherein ODb_t=0 is theat the beginning of the test, prior to addition of the endolysin polypeptide or fragment; and wherein ODE_t50% lysis is the optical density of the cell suspension corresponding to 50% reduction in the initial cell density following addition of the endolysin polypeptide or fragment. Wherein X is the optical density of the blank at the beginning of the test. Assays for Minimum Inhibitory Concentration (MIC) / Minimum Bactericidal Concentration (MBC) of Endolysins:

[0189] The MIC / MBC value, which is defined as the minimum concentration of purified endolysin which leads to cell growth arrest for a given Clostridium septicum cell density, may be calculated according to the following method: 1. Inoculate Clostridium septicum strain NCTC 547 in BHI+C media and culture under anaerobic conditions at 37° C. overnight to stationary phase; 2. dilute stationary phase culture in fresh BHI+C media under anaerobic conditions; 54 105506204.3Attorney Docket: 125930-848037 3. prepare stocks of a serial dilution of purified endolysin to 10× more concentrated than the final desired protein loading in PBS, pH 7.0 under anaerobic conditions; 4. mix a serial dilution of purified endolysin with the diluted stationary phase culture across the wells of a microtiter plate wherein for each well 45 μL of diluted stationary phase culture is mixed with 5 μL of the 10× concentrated and purified endolysin to a final cell loading of approximately 104cells / mL under anaerobic conditions; 5. incubate cells overnight for 12-20 hrs at 37° C under anaerobic conditions; 6. determine the extent of growth inhibition by measuring the change in OD on a plate reader and compare to both the media and growth controls; 7. determine the minimum inhibitory concentration (MIC) for the endolysin, wherein MIC is the lowest concentration of endolysin which arrests cell growth and no visible growth is observed; 8. transfer 5 μL of culture from each well into a separate well and make up to 100 μL with BHI+C medium under anaerobic conditions; 9. incubate the transferred cultures overnight for 12-20 hrs at 37° C. under anaerobic conditions; 10. determine the extent of growth, and therefore the extent of residual viable Cs cells following exposure to endolysin, by measuring the change in OD on a plate reader following incubation and compare to both the media and growth controls; 11. determine the minimum bactericidal concentration (MBC) for the endolysin, wherein MBC is the lowest concentration of endolysin which kills all of the initial cell loading and no visible growth is observed. Methods of Producing Endolysins

[0190] The invention further provides for a method for the production of an endolysin polypeptide according to the invention, comprising: culturing a host cell according to the invention under conditions conducive to the production of the endolysin polypeptide, optionally isolating and purifying the endolysin polypeptide from the culture broth, and optionally freeze- drying the endolysin polypeptide.

[0191] A polypeptide as disclosed herein may be produced by any suitable means available to skilled artisan. For example, the polypeptide may be synthesised directly using standard 55 105506204.3Attorney Docket: 125930-848037 techniques known in the art, such as Fmoc solid phase chemistry, Boc solid phase chemistry or by solution phase peptide synthesis. Alternatively, a polypeptide may be produced by transforming a host cell, typically a bacterial cell e.g., Bacillus subtilis or E. coli cell, with a nucleic acid molecule or vector which encodes said polypeptide. Production of polypeptides by expression in bacterial host cells is described herein. A polypeptide of the invention may also be produced by transforming into an algal host cell.

[0192] A host cell has been “genetically modified” or “transformed” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes (e.g., Bacillus subtilis or E. coli ), yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0193] Suitable methods of genetic modification (also referred to as “transformation”) include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, and the like. The choice of method of genetic modification is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place, and would be apparent to the skilled person.

[0194] The invention also includes cells that have been modified to express an endolysin polypeptide of the invention. Such cells may be modified to carry an expression vector encoding an endolysin polypeptide of the invention. Such cells typically include prokaryotic cells such as bacterial cells, for example Bacillus subtilis or E. coli . Such cells may be cultured using routine methods to produce a polypeptide of the invention. Preferably, a cell of the invention is a bacterial cell. 56 105506204.3Attorney Docket: 125930-848037

[0195] An endolysin polypeptide may be derivatised or modified to assist with their production, isolation or purification. For example, where an endolysin polypeptide of the invention is produced by recombinant expression in a bacterial host cell, the sequence of the polypeptide may include an additional methionine (M) residue at the N terminus to improve expression.

[0196] As another example, the endolysin polypeptide of the invention may be derivatised or modified by addition of a ligand which is capable of binding directly and specifically to a separation means. Alternatively, the endolysin polypeptide may be derivatised or modified by addition of one member of a binding pair and the separation means comprises a reagent that is derivatised or modified by addition of the other member of a binding pair. Any suitable binding pair can be used. In a preferred embodiment where an endolysin polypeptide for use in the invention is derivatised or modified by addition of one member of a binding pair, the polypeptide is preferably histidine-tagged or biotin-tagged. Typically the amino acid coding sequence of the histidine or biotin tag is included at the gene level and the polypeptide is expressed recombinantly in Bacillus subtilis or E. coli . The histidine or biotin tag is typically present at either end of the polypeptide, preferably at the C-terminus. It may be joined directly to the polypeptide or joined indirectly by any suitable linker sequence, such as 3, 4 or 5 glycine residues. The histidine tag typically consists of six histidine residues, although it can be longer than this, typically up to 7, 8, 9, 10 or 20 amino acids or shorter, for example 5, 4, 3, 2 or 1 amino acids.

[0197] The amino acid sequence of an endolysin polypeptide may be modified to include non- naturally occurring amino acids, for example to increase stability. When the endolysin polypeptides are produced by synthetic means, such amino acids may be introduced during production. The endolysin polypeptides may also be modified following either synthetic or recombinant production. Endolysin polypeptides may also be produced using D-amino acids. In such cases the amino acids may be linked in reverse sequence in the C to N orientation. This is conventional in the art for producing such polypeptides.

[0198] A number of side chain modifications are known in the art and may be made to the side chains of the endolysin polypeptides, subject to the polypeptides retaining any further required activity or characteristic as may be specified herein. It will also be understood that endolysin polypeptides may be chemically modified, e.g., post-translationally modified. For example, they may be glycosylated, phosphorylated or comprise modified amino acid residues.

[0199] The endolysin polypeptide may be PEGylated. The endolysin polypeptide of the invention may be in a substantially isolated form. It may be mixed with carriers or diluents (as 57 105506204.3Attorney Docket: 125930-848037 discussed below) which will not interfere with the intended use and still be regarded as substantially isolated. It may also be in a substantially purified form, in which case it may generally comprise at least 90%, e.g., at least 95%, 98% or 99%, of the protein in the preparation. Polynucleotides and Vectors Encoding Endolysin Polypeptides of the Invention

[0200] The invention further provides nucleic acid molecules and vectors which encode an endolysin polypeptide of the invention. These sequences may include at the 5′ end a codon for the N-terminal methionine (ATG) and, prior to the stop codon (TAA) at the 3′ end, codons for a 3× Gly linker and a 6× his histidine tag, which may optionally be excluded.

[0201] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide of the invention may be provided in isolated or substantially isolated form. When substantially isolated, there may be substantial, but not total, isolation of the polypeptide from any surrounding medium. The polynucleotides may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. A nucleic acid sequence which “encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences, for example in an expression vector. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. For the purposes of the invention, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence.

[0202] Polynucleotides can be synthesised according to methods well known in the art, as described by way of example in Sambrook et al, 1989, Molecular Cloning—a laboratory manual; Cold Spring Harbor Press. The nucleic acid molecules of the present invention may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the polypeptide of the invention in vivo. These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or 58 105506204.3Attorney Docket: 125930-848037 recombinant viral vectors). Such an expression cassette may be administered directly to a host subject. Alternatively, a vector comprising a polynucleotide of the invention may be administered to a host subject. Preferably the polynucleotide is prepared and / or administered using a genetic vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide of the invention.

[0203] The present invention thus includes expression vectors that comprise such polynucleotide sequences. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for expression of a peptide of the invention. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (e.g., U6 promoter, HI promoter, etc.).

[0204] Thus, the present invention may relate to an expression vector comprising a nucleic acid construct or a polynucleotide according to the invention. An expression vector according to the invention may be a recombinant expression vector. Such vector may constitute a plasmid, a cosmid, a bacteriophage or a virus, or a part thereof, which is transformed by introducing a nucleic acid construct or a polynucleotide according to the invention. Such transformation vectors specific to the host organism to be transformed are well known to those skilled in the art and widely described in the literature. In order to produce a polynucleotide or endolysin polypeptide according to the invention in a host, a process for the transformation of a host organism, and integration of a polynucleotide, nucleic acid construct or expression vector according to the invention may be appropriate. Such transformation may be carried out by any suitable known means which have been widely described in the specialist literature and are well- known to the person skilled in the art.

[0205] As used herein, a “promoter” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3′ direction) coding or non-coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include 59 105506204.3Attorney Docket: 125930-848037 the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Non-limiting examples of suitable promoters include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of skilled person. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator.

[0206] The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include nucleotide sequences encoding protein tags (e.g., 6×His tag, hemagglutinin tag, green fluorescent protein, thioredoxin, etc.) that are fused to the site— directed modifying polypeptide, thus resulting in a chimeric polypeptide. Such tags may also be cleavable, for example using a TEV protease.

[0207] A promoter can be a constitutively active promoter (e.g., a promoter that is constitutively in an active / “ON” state), it may be an inducible promoter (e.g., a promoter whose state, active / “ON” or inactive / “OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein), it may be a spatially restricted promoter (e.g., transcriptional control element, enhancer, etc.) (e.g., sub-tissue or tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (e.g., the promoter is in the “ON” state or “OFF” state during specific stages of development or during specific stages of a biological process, e.g., of the cell cycle.

[0208] Non-limiting examples of inducible promoters include, but are not limited to T7 RNA polymerase promoter, T3 RNA polymerase promoter, Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose induced promoter, heat shock promoter, Tetracycline- regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, oestrogen receptor- regulated promoter, etc. Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; RNA polymerase, e.g., T7 RNA polymerase; an oestrogen receptor; an oestrogen receptor fusion; etc.

[0209] The terms “DNA regulatory sequences,” “control elements,” and “regulatory elements,” used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence (e.g., a promoter) or a coding sequence and / or regulate translation of an encoded polypeptide. 60 105506204.3Attorney Docket: 125930-848037

[0210] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert”, may be attached so as to bring about the replication of the attached segment in a cell. An “expression cassette” comprises a DNA coding sequence operably linked to a promoter. “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence when the promoter affects its transcription or expression. Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other vector may be used so long as it is compatible with the host cell.

[0211] The terms “recombinant expression vector,” or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are usually generated for the purpose of expressing and / or propagating the insert(s), or for the construction of other recombinant nucleotide sequences. The insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences. Affinity Tags and Signal Peptide Sequences

[0212] An endolysin polypeptide of the invention may further comprise the endolysin sequence together with an affinity tag at the N terminus and / or at the C terminus to further assist with isolation from standard expression systems such as those described herein. Any suitable affinity tag may be used.

[0213] Suitable affinity tags may be joined directly to the N or C terminus of a polypeptide or joined indirectly by any suitable linker sequence, such as 3, 4 or 5 glycine residues. One such suitable affinity tag is a histidine tag. The histidine tag typically consists of six histidine residues, although it can be longer than this, for example up to 7, 8, 9, 10 or 20 amino acids or shorter, for example 5, 4, 3, 2 or 1 amino acids. Alternative affinity tags include an AviTag, a FLAG-tag, a HA-tag, a Myc-tag, a Strep-tag, a V5-tag, a Poly-arg, a HAT-tag, a calmodulin- binding peptide tag, a GST tag, a MBP tag, a Fh8 tag, a Strep-II tag, a cellulose binding peptide tag, a chitin-binding peptide tag, a protein A tag, a ubiquitin tag, a DHFR tag, a SBR tag.

[0214] Each endolysin of the invention may further comprise the endolysin sequence together with an expression-enhancing peptide sequence to further assist with expression. When 61 105506204.3Attorney Docket: 125930-848037 assisting with expression, the total amount of endolysin polypeptide produced by a host cell may be increased, or the proportion of soluble endolysin polypeptide produced by the host cell may be increased. Thus an expression-enhancing peptide sequence may be any peptide sequence which can facilitate an increase in the amount of endolysin secreted into the culture medium by the host cell, such as a Bacillus subtilis cell or an E. coli host cell. Any suitable expression- enhancing peptide sequence may be used with any of the endolysins of the invention, including any of the expression-enhancing peptide sequences described herein. The expression-enhancing peptide may be selected from secretion-enhancing peptides including OmpA, e mal, gIII, pelB, phoA, ompC, ompT, dsbA, torT, sufI, torA, STII, EOX, lamb, MglB, SfmC, TolB and MmAp. The expression-enhancing peptide may be selected from solubility-enhancing peptides PDI, GST, Trx, MBP, NusA-tag, SUMO, DsbC, Skp, Fh8, ZZ, GB1, T7PK, DsbA and SET.

[0215] Any of the endolysins of the invention may comprise the endolysin sequence together with any expression-enhancing peptide sequence and additionally any affinity tag. Affinity purification of the endolysins of the invention may be employed as a means to produce purified and concentrated endolysin stocks without contaminant native Bacillus subtilis and / or E. coli lysate proteins. Host Cells and Expression Systems

[0216] The invention also provides a host cell comprising a polynucleotide according to the invention, a nucleic acid construct according to the invention or an expression construct according to the invention. A host cell according to the invention may be any microbial, prokaryotic or eukaryotic, cell which is suitable for expression of the polypeptide of the invention. Preferably, said cell is a Bacillus subtilis cell or an E. coli host cell.

[0217] The E. coli cell may be from the expression strain, Rosetta™ (DE3) pLysS. Thus, the E. coli cell used for expression may be a BL21 E. coli derivative designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli and one that is suitable for production of protein from target genes cloned in pET vectors by induction with IPTG, as well as expressing T7 lysozyme, which further suppresses basal expression of T7 RNA polymerase prior to induction. Non-limiting examples of a host cell of the invention are those of the following strains: DH5alpha, HB101, JM109 BL21 strains, C41 (DE3), C43 (DE3), DH10B, W3110, CyDisCo, K12 strains, Lemo21 (DE3), T7Express strains, Rosetta strains, Shuffle strains, Tuner strains, Origami strains, ArticExpress strains, AD494, BL21 and trxB. 62 105506204.3Attorney Docket: 125930-848037

[0218] The Bacillus subtilis cell may be from the expression strain, 168, or from another strain. Non-limiting examples of the host cell of the invention are those of the following strains VTT E- 68013, 1A976, WS9, WB600, WB800, WB800N, 1A751, 1A751P7, 1A237, 1012, PT5, K07, 3NA, SCK6,

[0219] An endolysin polypeptide of the invention may be capable of being directly expressed in its soluble and functional (catalytically active) form in a host cell, preferably in a Bacillus subtilis or an E. coli host cell. Thus, steps of solubilisation and / or refolding of expressed proteins may be avoided. The capability of expressing soluble and functional endolysin polypeptides of the invention is revealed when a polypeptide of the invention is comprised in any suitable expression vector, when the vector is transformed into a host cell, when transformed host cells are subsequently cultured under suitable conditions to promote the expression of the polypeptide and when the activity of the polypeptide is assessed. Bacterial cultivation systems include continuously stirred tank reactors and batch and fed-batch bioreactors. Cultivation systems also include hybrid production systems.

[0220] The endolysin polypeptides of the invention may also be expressed in an algal expression system. For example, the algal expression system may use an algal host cell for expression such as Chlamydomonas reinhardtii or Synechococcus elongatus. Such algal systems including the associated vectors are known in the art. The polypeptides of the invention may remain within the algal host cell, such as to produce whole-cell algal compositions comprising the said polypeptides. Alternatively the polypeptides may be purified following expression in algal cells. Such algal systems also allow for affinity purification of the endolysin polypeptide of the invention using known tags, such as those disclosed herein.

[0221] Algae may be utilised as host cells and / or expression systems for the expression of any of the polypeptides of the invention disclosed herein, and thus for use in any of the compositions or formulations disclosed herein. Such algae encompass both prokaryotic and eukaryotic algae, which preferably are microscopic algae and more preferably unicellular. Unicellular algae are also known as microalgae. Thus microalgae may be utilised as host cells and / or expression systems for the expression of any of the polypeptides of the invention disclosed herein and thus for use in any of the compositions or formulations disclosed herein. In certain preferred embodiments, the algae is a green algae (Chlorophyta), a brown algae (Phaeophyta), or diatoms (Bacillariophyta).

[0222] Non-limiting examples of green algae, which are especially well-suited for use include members of the Chlamydomonas species, particularly Chlamydomonas reinhardtii; the Chlorella 63 105506204.3Attorney Docket: 125930-848037 species, the Volvox species, and some marine macrophytes. A list of genetically transformable algal species that can be used for the invention is provided in TABLE 2. TABLE 2: Overview of genetically transformable algal species, any of which may be used. S ecies) 64Attorney Docket: 125930-848037

[0223] Further information concerning the use of algae for recombinant polypeptide expression can be found in the following additional resources, which are incorporated herein by reference in their entireties: Dyo, Y. M., & Purton, S., (2018), The algal chloroplast as a synthetic biology platform for production of therapeutic proteins, Microbiology (Reading, England). Doi:10.1099 / mic.0.000599. Spicer, A. & Purton, S., (2017), Genetic engineering of microalgae: Current status and future prospects, Microalgal Production for Biomass and High-Value Products (pp. 139-164). Doi:10.1201 / b19464. Wannathong, T., Waterhouse, J. C., Young, R. E. B., Economou, C. K., & Purton, S., (2016), New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 100 (12), 5467-5477. Doi:10.1007 / s00253-016-7354-6. Purton, S., (2015), Algal chloroplast engineering: new tools, technologies and applications, European Journal of Phycology, 50, 94-95. Gangl, D., Zedler, J. A. Z., Rajakumar, P. D., Martinez, E. M. R., Riseley, A., Wlodarczyk, A., Robinson, C. (2015), Biotechnological exploitation of microalgae. JOURNAL OF EXPERIMENTAL BOTANY, 66 (22), 6975-6990. Doi:10.1093 / jxb / erv426. Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii, B A Rasala, M Muto, P A Lee, M Jager, R M F Cardoso, C A Behnke, P Kirk, Plant biotechnology journal 8(6), 719-733. Micro-algae come of age as a platform for recombinant protein production E Specht, S Miyake- Stoner, S Mayfield, (2010), Biotechnology letters 32 (10), 1373-1383. Regulation of chloroplast gene expression, S P Mayfield, C B Yohn, A Cohen, A Danon, Annual review of plant biology 46 (1), 147-166. Chlamydomonas reinhardtii chloroplasts as protein factories, S P Mayfield, A L Manuell, S Chen, J Wu, M Tran, D Siefker, M Muto, Current opinion in biotechnology 18 (2), 126-133. 65 105506204.3Attorney Docket: 125930-848037

[0224] Accordingly, the invention provides a host cell comprising a population of nucleic acid molecules encoding any endolysin polypeptide of the invention, optionally wherein the host cell is a cell of a unicellular microorganism, optionally a yeast cell, such as Pichia pastoris, an algal cell such as Chlamydomonas reinhardtii, a fungal cell, or a bacterial cell, preferably a bacterial cell such as Bacillus subtilis.

[0225] The host cell may additionally comprise any of the endolysin polypeptides or fragments thereof defined herein and which have been expressed from said vectors. Each one of the molecules or vectors of the population may encode the same endolysin polypeptide or fragment as defined herein, or molecules or vectors of the population may encode two or more different endolysin polypeptides or fragments as defined herein.

[0226] The invention additionally provides a cell lysate comprising a population of any of the endolysin polypeptides or fragments thereof as defined herein, optionally wherein the population comprises the same endolysin polypeptide or fragment, or wherein the population comprises two or more different endolysin polypeptides or fragments. The lysate may be produced following expression of the endolysin polypeptides or fragments thereof in a host cell as defined herein, which may be a cell of a unicellular microorganism, optionally a yeast cell, such as Pichia pastoris, an algal cell such as Chlamydomonas reinhardtii, a fungal cell, or a bacterial cell, preferably a bacterial cell such as Bacillus subtilis. Lysates may include host cell debris following lysis. Alternatively, lysates may comprise host cell extracts wherein host cell debris is removed, for example host cell debris may be removed by centrifugation and the supernatants collected and used whilst the pelleted host cell debris is discarded. The lysate may be produced by breaking the cells enzymatically, or by sonication or use of the French Press or by spray drying.

[0227] The invention additionally provides a dried whole culture comprising a population of any of the endolysin polypeptides or fragments thereof as defined herein, optionally wherein the population comprises the same endolysin polypeptide or fragment, or wherein the population comprises two or more different endolysin polypeptides or fragments. The dried whole culture may be produced following expression of the endolysin polypeptides or fragments thereof in a host cell as defined herein, which may be a cell of a unicellular microorganism, optionally a yeast cell, such as Pichia pastoris, an algal cell such as Chlamydomonas reinhardtii, a fungal cell, or a bacterial cell, preferably a bacterial cell such as Bacillus subtilis. Preferably the dried whole culture may be produced by spray drying the unprocessed whole culture although it may be produced by freeze-drying the unprocessed whole culture. Consequently, the dried whole 66 105506204.3Attorney Docket: 125930-848037 culture may comprise a mixture of cells, lysed and partially-lysed host cell debris, culture medium components and materials which the cells have secreted into the medium during the culturing period or materials which are introduced into the medium due to the lysis of cells during the culture period or through the lysis of cells due to the drying process. Materials which the cells have secreted into the medium during the culturing period may comprise endolysin polypeptides and materials which are introduced into the medium due to the lysis of cells may comprise endolysin polypeptides.

[0228] The invention additionally provides a dried culture medium comprising a population of any of the endolysin polypeptides or fragments thereof as defined herein, optionally wherein the population comprises the same endolysin polypeptide or fragment, or wherein the population comprises two or more different endolysin polypeptides or fragments. The dried culture medium may be produced following expression of the endolysin polypeptides or fragments thereof in a host cell as defined herein, which may be a cell of a unicellular microorganism, optionally a yeast cell, such as Pichia pastoris, an algal cell such as Chlamydomonas reinhardtii, a fungal cell, or a bacterial cell, preferably a bacterial cell such as Bacillus subtilis. Preferably the dried culture medium may be produced by removing the cells from the culture medium according to methods well known to those skilled in the art, such as flocculation, chemical coagulation, combined flocculation, centrifugation, filtration and gravity sedimentation, followed by spray drying or freeze drying of the processed, substantially cell-free culture medium. Consequently, the dried culture medium may comprise a mixture of culture medium components and materials which the cells have secreted into the medium during the culturing period or materials which are introduced into the medium due to the lysis of cells during the culture period. Materials which the cells have secreted into the medium during the culturing period may comprise endolysin polypeptides and materials which are introduced into the medium due to the lysis of cells during the culturing period may comprise endolysin polypeptides.

[0229] The invention additionally provides a dried material. The dried material may comprise a population of any of the endolysin polypeptides or fragments thereof as defined herein, optionally wherein the population comprises the same endolysin polypeptide or fragment, or wherein the population comprises two or more different endolysin polypeptides or fragments. Alternatively, the dried material may comprise a host cell comprising a population of nucleic acid molecules encoding any endolysin polypeptide of the invention, optionally wherein the host cell is a cell of a unicellular microorganism, optionally a yeast cell, such as Pichia pastoris, an algal cell such as Chlamydomonas reinhardtii, a fungal cell, or a bacterial cell, preferably a 67 105506204.3Attorney Docket: 125930-848037 bacterial cell such as Bacillus subtilis, or a host cell which may comprise any of the endolysin polypeptides or fragments thereof defined herein and which have been expressed from said vectors. Each one of the molecules or vectors of the population may encode the same endolysin polypeptide or fragment as defined herein, or molecules or vectors of the population may encode two or more different endolysin polypeptides or fragments as defined herein. Alternatively, the dried material may comprise a cell lysate comprising a population of any of the endolysin polypeptides or fragments thereof as defined herein, optionally wherein the population comprises the same endolysin polypeptide or fragment, or wherein the population comprises two or more different endolysin polypeptides or fragments. The lysate may be produced following expression of the endolysin polypeptides or fragments thereof in a host cell as defined herein, which may be a cell of a unicellular microorganism, optionally a yeast cell, such as Pichia pastoris, an algal cell such as Chlamydomonas reinhardtii, a fungal cell, or a bacterial cell, preferably a bacterial cell such as Bacillus subtilis. The dried material may be produced by spray drying or freeze drying.

[0230] The invention additionally provides a combined material. The combined material is a mixture and may comprise at least two ingredients selected from the following; the one or more endolysin polypeptides of the invention, the population of one or more host cells of the invention, the cell lysate of the invention, the dried whole culture of the invention, the dried culture medium of the invention and the dried material of the invention. The combined material may be dried, preferably by spray drying or freeze drying.

[0231] Reagents and systems for expressing proteins in yeast are described in: Protein expression-yeast, Nielsen K. H., Methods Enzymol. 2014; 536:133-47. Reagents and systems for expressing proteins in fungi are described in: Gene expression in fungi, Kalkanci, A et al. IMA Fungus. 2011 June; 2(1): 29-32. Reagents and systems for expressing proteins in yeast in particular are described in: Micro-algae come of age as a platform for recombinant protein production, Specht, E. et al. Biotechnol Lett. 2010 October; 32(10): 1373-1383. Reagents and systems for expressing proteins in plants are described in: Plants as Expression Systems for Recombinant Proteins, Kawaka et al., Asian Journal of Biology 3(3): 1-8, 2017. All of these resources of which are incorporated by reference in their entireties by reference thereto.

[0232] A host cell may be a cell of a host organism that is a GRAS organism (“Generally Regarded as Safe”), or of a host organism that is listed on the Qualified Presumption of safety (QPS) list published by the European Food Safety Authority, including any organism routinely included in poultry diets. 68 105506204.3Attorney Docket: 125930-848037 Compositions Comprising Endolysins

[0233] In another aspect, the present invention provides compositions comprising any of the endolysin polypeptides of the invention. A composition may comprise any endolysin polypeptide / s of the invention, any host cell / s of the invention, lysate / s of the invention, any dried whole culture / s of the invention, any dried culture medium or media of the invention, any dried material of the invention and / or any combined material of the invention.

[0234] The composition may comprise a population of any of the endolysin polypeptides or fragments defined herein. The population may comprise the same endolysin polypeptide or fragment defined herein. The population may comprise two or more different endolysin polypeptides or fragments as defined herein, in which case the exhibited antimicrobial activity may be a synergistic activity.

[0235] The composition may comprise a population of host cells, wherein host cells of the population comprise any of the endolysin polypeptides or fragments as defined herein.

[0236] The composition may comprise a whole-cell composition comprising a population of whole cells, wherein cells of the population comprise any of the endolysin polypeptides or fragments as defined herein. Cells of the population may be cells of a unicellular microorganism, optionally a yeast cell, such as Pichia pastoris, an algal cell such as Chlamydomonas reinhardtii, a fungal cell, or a bacterial cell, preferably a bacterial cell such as Bacillus subtilis and which cells comprise any of the endolysin polypeptides or fragments as defined herein, e.g., by expression of said polypeptides or fragments. The composition may comprise a cell lysate comprising a population of any of the endolysin polypeptides or fragments thereof as defined herein. The lysate may be produced following expression of the endolysin polypeptides or fragments thereof in a host cell as defined herein, which may be a cell of a unicellular microorganism, optionally a yeast cell, such as Pichia pastoris, an algal cell such as Chlamydomonas reinhardtii, a fungal cell, or a bacterial cell, preferably a bacterial cell, such as Bacillus subtilis. The lysate may be produced by breaking the cells enzymatically, or by sonication, or use of the French Press or by spray drying the cells.

[0237] The composition may comprise a dried culture medium material comprising a population of any of the endolysin polypeptides or fragments thereof as defined herein. The dried culture medium material may be produced following expression of the endolysin polypeptides or fragments thereof in a host cell as defined herein, which may be a cell of a unicellular microorganism, optionally a yeast cell, such as Pichia pastoris, an algal cell such as 69 105506204.3Attorney Docket: 125930-848037 Chlamydomonas reinhardtii, a fungal cell, or a bacterial cell, preferably a bacterial cell, such as Bacillus subtilis. The dried culture medium material may be produced by harvesting or separating the cells from the culture medium and drying the culture medium preferably by spray drying or freeze drying, to produce the dried culture medium material.

[0238] Any of the above-described compositions may comprise the polypeptides or fragments, host cells, whole cells or cell lysates together with a suitable carrier or excipient, such as water or a physiological acceptable buffer.

[0239] Any of the above-described compositions may be encapsulated in a material which surrounds and protects the endolysin from the effects or heat and / or digestive enzymes and / or pH changes. In one embodiment, the above-described compositions of the invention are formulated as an enteric-coated formulation. Enteric-coated formulations can be formulated as capsules, tablets, caplets, pills, troches, lozenges, powders, granules, beads, microbeads, or microparticles. Preferably the enteric-coated formulations are formulated as spherical beads 0.25-3 mm in diameter when manufactured. The coating of the above-described compositions of the invention is not particularly limited, and any such material known in the art of foodstuff and pharmaceutical manufacture may be used. Enteric polymers known in the art can be used, for example, enteric polymers such as poly(methacrylic acid-co-methyl methacrylate) or Eudragit (e.g., EUDRAGIT L30 D-55). any encapsulating material known in the art may be used, for example, PVA, trehalose, chitosan, starch, cellulose derivatives, maltodextrin, pectin, carrageenan, Gum Arabic, gelatin or whey protein. Encapsulating materials may be used singly or combinations of such materials may be used. The combinations of materials may involve them being applied to compositions of the invention in a mixture or as concentric layers successively applied to compositions of the invention.

[0240] The abovementioned compositions, unencapsulated or encapsulated, may be incorporated into an animal feed, preferably a poultry feed. The compositions may be mixed with ground, mashed or crushed cereal, moistened with water or steam and the mixture forced through an extruder plate to form poultry feed pellets.

[0241] In another aspect, the present invention provides formulations comprising any of the endolysin polypeptides of the invention. Preferably the formulations are antimicrobial formulations, antibacterial formulations and / or pharmaceutical formulations. A formulation may comprise a population of host cells, wherein the host cells of the population comprise any of the endolysin polypeptides of the invention. A formulation may comprise a cell lysate, wherein the lysate comprises any of the endolysin polypeptides of the invention. A formulation 70 105506204.3Attorney Docket: 125930-848037 may comprise a dried culture medium material, wherein the dried culture medium material comprises any of the endolysin polypeptides of the invention. Any of the formulations may comprise the same endolysin polypeptide or fragment as defined herein, or may comprise two or more different endolysin polypeptides or fragments as defined herein. The exhibited antimicrobial activity may be a synergistic activity.

[0242] Any such formulation may comprise any of the endolysin polypeptides of the invention and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient. The carrier, diluent, vehicle or excipient must be ‘acceptable’ in the sense of being compatible with the other ingredients of the composition and not deleterious to a subject to which the composition is administered. Typically, carriers and the final composition, are sterile and pyrogen free.

[0243] Formulation of a suitable composition can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan. For example, the agent can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, reducing agents and the like, may be present in the excipient or vehicle. Suitable reducing agents include cysteine, thioglycerol, thioreducin, glutathione and the like. Excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, thioglycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J., 1991).

[0244] Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in 71 105506204.3Attorney Docket: 125930-848037 dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.

[0245] Other parentally administrable compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. The compositions may be suitable for administration by any suitable route including, for example, intradermal, subcutaneous, percutaneous, intramuscular, intra-arterial, intraperitoneal, intraarticular, intraosseous or other appropriate administration routes. Preferred compositions are suitable for administration by intravenous infusion.

[0246] Non-limiting examples of formulations include topical lotions, creams, soaps, wipes, and the like. They may be formulated into liposomes, to reduce toxicity or increase bioavailability. Other methods for delivery include oral methods that entail encapsulation of the endolysins in microspheres or proteinoids, aerosol delivery (e.g., to the lungs), or transdermal delivery (e.g., by iontophoresis or transdermal electroporation). Other routine methods of administration are known to those skilled in the art.

[0247] Pharmaceutical formulations suitable for oral administration may be provided in convenient unit forms including capsules, tablets, gels, pastes, ointments etc.

[0248] Formulations and compositions may comprise a thickener. Suitable thickeners include synthetic hectorite, Irish moss, iota carrageenan, gum tragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose, hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, and colloidal silica. 72 105506204.3Attorney Docket: 125930-848037

[0249] Formulations and compositions may comprise a solubilising agent. Solubilising agents may include agents such as humectant polyols such propylene glycol, dipropylene glycol and hexylene glycol, cellosolves such as methyl cellosolve and ethyl cellosolve, vegetable oils and waxes containing at least about 12 carbons in a straight chain such as olive oil, castor oil and petrolatum and esters such as amyl acetate, ethyl acetate and benzyl benzoate.

[0250] Pharmaceutically acceptable excipients which are suitable for use in tablet formulations include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. Tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

[0251] For hard gelatin capsule formulations, the active ingredient can be mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. For soft gelatin capsule formulations the active ingredient can be mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

[0252] Excipients suitable for the manufacture of aqueous suspensions include suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl methylcellulose, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.

[0253] Aqueous suspensions may also contain one or more preservatives, for example benzoates, such as ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0254] Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example Arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid 73 105506204.3Attorney Docket: 125930-848037 paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavouring agents may be added. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0255] Any of the compositions or formulations described herein may also be formulated for parenteral administration, such as by injection, for example bolus injection or continuous infusion, and may be provided in unit dose form in ampules, pre-filled syringes, small volume infusion or in multi-dose containers, e.g., with an added preservative.

[0256] Preparations for parenteral administration of formulations and compositions described herein include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Non- limiting examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Non-limiting examples of aqueous carriers include water, saline, and buffered media, alcoholic / aqueous solutions, and emulsions or suspensions. Non-limiting examples of parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives such as, other antimicrobial, antioxidants, cheating agents, inert gases and the like also can be included.

[0257] For topical administration to the epidermis, any of the formulations and compositions described herein may be formulated as an ointment, cream, or lotion. Ointments and creams, may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and / or gelling agents. Lotions may be formulated with an aqueous or oily base and may also contain one or more emulsifying agents, stabilizing agents, suspending agents, thickening agents, or colouring agents. Formulations suitable for topical administration in the mouth include lozenges, e.g., in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouth washes comprising the active ingredient in a suitable liquid carrier.

[0258] For topical administration to the eye, any of the formulations and compositions described herein can be made up in solution or suspension in a suitable sterile aqueous or non-aqueous vehicle. Additives such as buffers (e.g., sodium metabisulphite or disodium edeate) and thickening agents such as hypromellose can also be included.

[0259] For intra-nasal administration, any of the formulations and compositions described herein can be provided in a liquid spray or dispersible powder or in the form of drops. Drops 74 105506204.3Attorney Docket: 125930-848037 may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilising agents, or suspending agents.

[0260] For administration by inhalation, any of the formulations and compositions described herein can be delivered by insufflator, e.g., a nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurised packs may comprise a suitable propellant. In the case of a pressurised aerosol the dosage unit may be determined by providing a value to deliver a metered amount.

[0261] Any of the formulations and compositions described herein can take the form of a dry powder composition, for example a powder mix of the active component and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules, cartridges or blister packs of gelatins, from which the powder can be administered with the aid of an inhalator or insufflator.

[0262] Any of the formulations and compositions described herein can be incorporated into a liquid disinfecting solution. Such solutions may further comprise antimicrobials or antifungals such as alcohol, povidone-iodine solution and antibiotics as well as preservatives. These solutions can be used, e.g., as disinfectants of the skin or surrounding area, e.g., prior to insertion or implantation of a device such as a catheter, as catheter lock and / or flush solutions, and as antiseptic rinses for any medical device including, but not limited to catheter components such as needles, Luer-Lok connectors, needleless connectors and hubs as well as other implantable devices. These solutions can also be used to coat or disinfect surgical instruments.

[0263] The amount of endolysin polypeptide required for use in treatment may vary not only with the particular polypeptide but also with the route and form of administration, the nature and severity of the condition being treated, and the type, age and condition of the organism. Thus, appropriate concentrations of the active agent(s) to be incorporated into compositions and formulations can be routinely determined by those skilled in the art in accordance with standard practices. Additional Agents

[0264] Any of the above-described compositions, lysates or formulations may further comprise one or more additional agents having antimicrobial activity and wherein the additional agent(s) is not an endolysin polypeptide or fragment. The one or more additional agents may be selected form the group consisting of antibiotic agents, biofilm-degrading agents, biofilm-suppressing agents, sequestering agents such as chitosan, EDTA and citric acid, bacteriostatic agents such as 75 105506204.3Attorney Docket: 125930-848037 glycerol. The one or more additional agents may be selected form the group consisting of a stabilising agent, an anti-clumping agent, a prebiotic agent, a probiotic agent and an edible gel such as congealed water nutrient matrix.

[0265] Any of the formulations and compositions described herein can additionally include, together with the endolysin(s), an antimicrobial agent which is not an endolysin, such as detergents and antibiotics. Suitable antibiotics include aminoglycosides (e.g., gentamicin, kanamycin and streptomycin), beta-lactams (e.g., penicillin, ampicillin, imipenem and cephalosporins such as Ceftazidime), quinolones (e.g., ciprofloxacin), macrolides such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin and telithromycin, oxazolidinones such as linezolid, ansamycins such as rifamycin, sulfonamides, tetracyclines such as Doxycycline. Additional antibiotics include glycopeptides such as vancomycin, sulfisoxazole, trimethoprim, novobiocin, daptomycin and linezolid.

[0266] Generally, the antimicrobial is administered in a microbicidal amount. However, the antimicrobial can also be administered in microbistatic amount.

[0267] Antiparasitic compounds which can be included in the formulations and compositions described herein include the benzazoles (albendazole, mebendazole, thiabendazole, etc.), the azoles (metronidazole, tinidazole, etc.), macrocycles (amphotericin B, rifampin, ivermectin etc.) and others such as pyrantel pamoate, diethylcarbamazine, niclosamide, praziquantel, melarsoprol and eflornithine.

[0268] Antiviral compounds which can be included in the formulations and compositions described herein include the nucleoside analogue reverse transcriptase inhibitors (acyclovir, didanosine, stavudine, zidovudine, lamivudine, abacavir, emtricitabine, entecavir etc.), uncoating inhibitors (amantadine, rimantadine, pleconaril etc.), protease inhibitors (saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, etc.) and others such as zanamivir, oseltamivir, rifampin.

[0269] Antiviral compounds which can be included in the formulations and compositions described herein include an azole, such as miconazole, ketoconazole, clotrimazole, econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, sertaconazole, sulconazole, tioconazole, fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole and albofungin; a macrocycle, such as natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin, hamycin; an allyl amine such as terbinafine, naftifine and butenafine; an echinocandin such as anidulafungin, caspofungin and micafungin; or others such 76 105506204.3Attorney Docket: 125930-848037 as polygodial, ciclopirox, tolnaftate, benzoic acid, undecylenic acid, flucytosine and griseofulvin.

[0270] Antifungal compounds which can be included in the formulations and compositions described herein include an azole, such as miconazole, ketoconazole, clotrimazole, econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, sertaconazole, sulconazole, tioconazole, fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole and albofungin; a macrocycle, such as natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin, hamycin; an allyl amine such as terbinafine, naftifine and butenafine; an echinocandin such as anidulafungin, caspofungin and micafungin; or others such as polygodial, ciclopirox, tolnaftate, benzoic acid, undecylenic acid, flucytosine and griseofulvin. Animal Foodstuffs

[0271] The present invention also provides foodstuffs, preferably animal foodstuffs, comprising one or more foodstuffs and one or more polypeptides of the invention. The foodstuff may also comprise a host cell of the invention which either comprises one or more polypeptides of the invention, or comprises a nucleic acid sequence encoding one or more endolysin polypeptides of the invention. The foodstuff may also comprise a cell lysate of the invention, which may be produced from a host cell of the invention. The foodstuff may also comprise a dried whole culture of the invention. The foodstuff may also comprise a dried whole culture medium of the invention. The foodstuff may also comprise a dried material of the invention. The foodstuff may also comprise a combined material of the invention. The foodstuff may also comprise an antimicrobial formulation as described herein.

[0272] A foodstuff of the invention is suitable for consumption by animals, including poultry, preferably the domestic turkey, Meleagris gallopovo domesticus, or optionally a broiler chicken, preferably Gallus gallus domesticus, or a domestic duck derived from the mallard, Anas platyrhynchos domestica, or a domestic duck derived from the Muscovy duck, Cairina moschata domestica, or a goose, optionally a domestic goose, further optionally a domestic goose derived from the Greylag goose, Anser anser, or a domestic goose derived from the Chinese goose, Anser cygnoides, or a domestic pigeon, Columba livia domestica, or a domestic quail, Coturnix japonica domestica A foodstuff of the invention is suitable for consumption by a pig, preferably Sus scrofa domesticus. Consumption of the foodstuff may have a prophylactic or therapeutic effect on the animal. For example, consumption may reduce populations of C. septicum that thrive within the animal thus treating the animal or an infection caused by C. 77 105506204.3Attorney Docket: 125930-848037 septicum. Consumption by an animal of the foodstuff may also prevent infection of C. septicum, or if the animal previously suffered from infection by C. septicum, may have the effect of preventing further infection as well as curing infection.

[0273] A foodstuff comprising an endolysin of the invention may contain preservatives or any additives normally used in the preparation of foodstuffs for consumption and / or to prolong shelf- life.

[0274] A foodstuff comprising an endolysin of the invention may be provided in any suitable edible form, such as in powdered or ground form, animal feed, animal mash or pelleted form.

[0275] Preferably the foodstuff is a pelleted feed suitable for feeding to poultry. Such feed is commonly manufactured by (i) crushing of the raw materials, typically grains such as wheat or corn, (ii) batching of the raw materials, where the crushed materials / ingredients are measured and mixed in specific proportions to ensure consistency between batches of feed, (iii), mixing, where the ingredients are thoroughly mixed and blended using a blending machine to ensure even distribution of the ingredients throughout the mixture, (iv) conditioning, where the mixture is conditioned using steam and heat, followed by (v) pelleting where the steamed / heated mixture is compressed into compact pellets by extrusion of the mixture through a perforated die plate. Preferably, the endolysin containing material may be added to the other feed ingredients at the batching stage, prior to conditioning and pelleting. Alternatively, the endolysin containing material may be added to the feed as a top dressing after pelleting. Supplemented Water and Supplemented Hydrogel

[0276] The present invention also provides a supplemented water, preferably comprising water and one or more polypeptides of the invention. The supplemented water may also comprise a host cell of the invention which either comprises one or more polypeptides of the invention, or comprises a nucleic acid sequence encoding one or more endolysin polypeptides of the invention. The supplemented water may also comprise a cell lysate of the invention, which may be produced from a host cell of the invention. The supplemented water may also comprise a dried whole culture of the invention. The supplemented water may also comprise a dried whole culture medium of the invention. The supplemented water may also comprise a dried material of the invention. The supplemented water may also comprise a combined material of the invention. The supplemented water may also comprise an antimicrobial formulation as described herein. 78 105506204.3Attorney Docket: 125930-848037

[0277] A supplemented water of the invention is suitable for consumption by animals, including poultry, preferably the domestic turkey, Meleagris gallopovo domesticus, or optionally a broiler chicken, preferably Gallus gallus domesticus, or a domestic duck derived from the mallard, Anas platyrhynchos domestica, or a domestic duck derived from the Muscovy duck, Cairina moschata domestica, or a goose, optionally a domestic goose, further optionally a domestic goose derived from the Greylag goose, Anser anser, or a domestic goose derived from the Chinese goose, Anser cygnoides, or a domestic pigeon, Columba livia domestica, or a domestic quail, Coturnix japonica domestica A supplemented water of the invention is suitable for consumption by a pig, preferably Sus scrofa domesticus. Consumption of the supplemented water may have a prophylactic or therapeutic effect on the animal. For example, consumption may reduce populations of C. septicum that thrive within the animal thus treating the animal or an infection caused by C. septicum. Consumption by an animal of the supplemented water may also prevent infection of C. septicum or if the animal previously suffered from infection by C. septicum, may have the effect of preventing further infection as well as curing infection.

[0278] The supplemented water of the invention may be supplied to animals / poultry in the standard fashion in place of their usual water or be supplied as a supplement thereto.

[0279] The present invention also provides a supplemented hydrogel, preferably comprising hydrogel and one or more polypeptides of the invention. The supplemented hydrogel may also comprise a host cell of the invention which either comprises one or more polypeptides of the invention, or comprises a nucleic acid sequence encoding one or more endolysin polypeptides of the invention. The supplemented hydrogel may also comprise a cell lysate of the invention, which may be produced from a host cell of the invention. The supplemented hydrogel may also comprise a dried whole culture of the invention. The supplemented hydrogel may also comprise a dried whole culture medium of the invention. The supplemented hydrogel may also comprise a dried material of the invention. The supplemented hydrogel may also comprise a combined material of the invention. The supplemented hydrogel may also comprise an antimicrobial formulation as described herein.

[0280] A supplemented hydrogel of the invention is suitable for consumption by animals, including poultry, preferably the domestic turkey, Meleagris gallopovo domesticus, or optionally a broiler chicken, preferably Gallus gallus domesticus, or a domestic duck derived from the mallard, Anas platyrhynchos domestica, or a domestic duck derived from the Muscovy duck, Cairina moschata domestica, or a goose, optionally a domestic goose, further optionally a domestic goose derived from the Greylag goose, Anser anser, or a domestic goose derived from 79 105506204.3Attorney Docket: 125930-848037 the Chinese goose, Anser cygnoides, or a domestic pigeon, Columba livia domestica, or a domestic quail, Coturnix japonica domestica A supplemented hydrogel of the invention is suitable for consumption by a pig, preferably Sus scrofa domesticus. Consumption of the supplemented hydrogel may have a prophylactic or therapeutic effect on the animal. For example, consumption may reduce populations of C. septicum that thrive within the animal thus treating the animal or an infection caused by C. septicum. Consumption by an animal of the supplemented hydrogel may also prevent infection of C. septicum or if the animal previously suffered from infection by C. septicum, may have the effect of preventing further infection as well as curing infection.

[0281] The hydrogel which is supplemented may be of any composition known in the art. The hydrogel may be comprise or be composed of gum arabic, gelatin, agarose, carrageenan, guar gum, locust bean gum or any other compound capable of forming a three-dimensional polymeric matrix capable of retaining water or other aqueous fluid therein. Alternatively, the hydrogel may comprise or be composed of a mixture of compounds capable of forming a three- dimensional polymeric matrix capable of retaining water or other aqueous fluid therein and the mixture may comprise or consist of two or more selected from the list of: gum arabic, gelatin, agarose, carrageenan, guar gum and locust bean gum. The supplemented hydrogel may additionally comprise a colouring agent, preferably a green colouring agent. The supplemented hydrogel may additionally comprise vitamins, minerals and or other supplements, for example probiotics, or therapeutic agents for example, antibiotics.

[0282] The supplemented hydrogel may be supplied to poultry / animals during their transportation as a convenient source of water. Alternatively, the hydrogel may be supplied to poultry / animals as a supplement to their usual feed. The supplemented hydrogel may be in the form of bars or pellets. Kits Comprising Endolysins

[0283] In an aspect, provided herein is a kit comprising an agent having antimicrobial activity, and instructions for admixing the agent with a foodstuff. In some embodiments, a kit comprises an endolysin polypeptide having antimicrobial activity as described herein, and instructions for admixing the agent with a foodstuff. In some embodiments, a kit comprises a host cell having antimicrobial activity as described herein, and instructions for admixing the agent with a foodstuff. In some embodiments, a kit comprises a cell lysate having antimicrobial activity as described herein, and instructions for admixing the agent with a foodstuff. In some embodiments, a kit comprises a dried whole culture having antimicrobial activity as described 80 105506204.3Attorney Docket: 125930-848037 herein, and instructions for admixing the agent with a foodstuff. In some embodiments, a kit comprises a dried culture medium having antimicrobial activity as described herein, and instructions for admixing the agent with a foodstuff. In some embodiments, a kit comprises a dried material having antimicrobial activity as described herein, and instructions for admixing the agent with a foodstuff. In some embodiments, a kit comprises a combined material having antimicrobial activity as described herein, and instructions for admixing the agent with a foodstuff. Methods of Using the Endolysin Polypeptides of the Invention

[0284] The invention provides for the use of endolysin polypeptides of the invention in various methods. For example, the endolysin polypeptides in accordance with the present invention may also be used in therapy or prophylaxis. In therapeutic applications, endolysin polypeptides or compositions thereof are administered to a subject already suffering from a disorder or condition, in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Accordingly, the present invention may have bacterial static and / or bactericidal properties.

[0285] Such therapeutic treatment may result in a decrease in severity of disease symptoms, or an increase in frequency or duration of symptom-free periods. An amount adequate to accomplish this is defined as “therapeutically effective amount”. In prophylactic applications, polypeptides or compositions are administered to a subject not yet exhibiting symptoms of a disorder or condition, in an amount sufficient to prevent or delay the development of symptoms. Such an amount is defined as a “prophylactically effective amount”. The subject may have been identified as being at risk of developing the disease or condition by any suitable means. Thus the invention also provides a polypeptide of the invention for use in the treatment of the human or animal body.

[0286] Also provided herein is a method of prevention or treatment of disease or condition in a subject, which method comprises administering a polypeptide of the invention to the subject in a prophylactically or therapeutically effective amount. The polypeptide may be co-administered with another agent. The polypeptide is preferably administered orally but may be administered by any suitable route including, for example, intravenous infusion, intradermal, subcutaneous, percutaneous, intramuscular, intra-arterial, intraperitoneal, intraarticular, intraosseous or other appropriate administration routes. The amount of said polypeptide that is administered may be between 0.01 mg / kg BW and 2 mg / kg BW, between 0.04 and 2 mg / kg BW, between 0.12 mg / kg BW and 2 mg / kg BW, preferably between 0.24 mg / kg and 2 mg / kg BW and most preferably 81 105506204.3Attorney Docket: 125930-848037 between 1 mg / kg and 2 mg / kg BW. The polypeptide may be administered on multiple occasions to the same subject.

[0287] Polypeptides of the invention may be particularly useful in the treatment or prevention of a disease or condition mediated by bacterial infection such as bacterial infection by C. septicum, for example cellulitis, gangrenous dermatitis, clostridial dermatitis, a gut lesion and / or a gastrointestinal infection caused by colonisation of C. septicum in the gastrointestinal tract. Accordingly, the invention provides a polypeptide of the invention for use in the treatment or prevention of a disease or condition characterised by presence of C. septicum.

[0288] The invention also provides a method of treating or preventing a disease or disorder mediated by C. septicum comprising administering to an individual a polypeptide of the invention. The disease or disorder may be a disease or disorder caused by a Clostridium septicum infection, such as cellulitis, gangrenous dermatitis, clostridial dermatitis, a gut lesion and / or a gastrointestinal infection caused by colonisation of C. septicum in the gastrointestinal tract.

[0289] The method may comprise repeat administration of the said polypeptide. The invention also provides a polypeptide of the invention for use in the manufacture of a medicament for the treatment or prevention of a disease or condition mediated by C. septicum.

[0290] The polypeptide of the invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. The route and / or mode of administration may vary depending upon the desired results. Preferred routes of administration include oral administration. Other routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection. Alternatively, a polypeptide can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration. Local administration is also preferred, including peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intra cavity infusion, intravesical administration, and inhalation. In a preferred embodiment, the invention is administered orally in the form of a foodstuff. Other routes that are useful are those that could be used to deliver the endolysin of the invention into the subject, preferably into the gut. 82 105506204.3Attorney Docket: 125930-848037

[0291] Suitable dosage of a peptide of the invention may be determined by a skilled medical practitioner. Actual dosage levels of a peptide may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level may depend upon a variety of pharmacokinetic factors including the activity of the particular antibody employed, the route of administration, the time of administration, the rate of excretion of the antibody, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0292] A suitable dose of a peptide may be in the range of from about 0.1 μg / kg to about 100 mg / kg body weight of the patient to be treated. For example, a suitable dosage may be from about 1 μg / kg to about 10 mg / kg body weight per day or from about 10 μg / kg to about 5 mg / kg body weight per day.

[0293] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, or the method may comprise several divided doses administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation, provided the required interval. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0294] Any of the formulations and compositions described herein can be formulated as a vaccine, to be administered for prophylaxis. Any such vaccine formulation may be administered directly to the animal for prophylaxis. Any such vaccine formulation may be administered to an animal egg for prophylaxis. For example, the vaccine formulation may be administered, e.g., injected, into the egg within the region defined by either the amnion or yolk sac. The vaccine formulation may be administered, e.g., injected, into the amniotic fluid. Any such vaccine formulation may be in the form of a composition comprising the endolysin polypeptide of the invention expressed in a bacterial host cell. 83 105506204.3Attorney Docket: 125930-848037 Methods of Using Endolysin Compositions

[0295] A composition according to the invention can be used to treat animals, including humans, infected with C. septicum, preferably the domestic turkey, Meleagris gallopovo domesticus or broiler chickens such as Gallus gallus domesticus. Any suitable route of administration can be used to administer said composition including but not limited to: oral, aerosol or other device for delivery to the lungs, nasal spray, intravenous, intramuscular, intraperitoneal, intrathecal, vaginal, rectal, topical, lumbar puncture, intrathecal, and direct application to the brain and / or meninges.

[0296] A composition according to the invention, such as comprising a polynucleotide, nucleic acid construct, endolysin polypeptide, vector, or a cell according to the invention is preferably said to be active, functional or therapeutically active or able to treat, prevent and / or delay an infectious disease when it decreases the amount of a C. septicum species present in a patient or in a cell of said patient, a cell line, or a cell free in vitro system, wherein preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of a C. septicum species is still detectable after treatment. In some embodiments, C. septicum species is not detectable after treatment. Assessment of detection as described herein may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 7-14 days, two weeks, or a month or longer after treatment. An amount of C. septicum may be an amount of alive C. septicum. C. septicum species may be detected using standard techniques known by the skilled person such as immunohistochemical techniques using C. septicum-specific antibodies. C. septicum species (alive and / or dead) may be detected using standard techniques known by the skilled person such as microbiological bacterial culture techniques and / or real-time quantitative reverse transcription polymerase chain reaction to assay for bacterial mRNA. Non-limiting examples of how to detect C. septicum include enumeration of C. septicum from the intestinal contents and feces of the subject using standard plate count methodologies, or PCR methods such as real-time PCT or conventional PCR. For PCR-based methods any probes specific for C. septicum may be used. Types of probes may include fluorogenic and hydrolysis-type (5′ nuclease) probes.

[0297] Effectiveness of C. septicum treatment may be assessed in a sample from the subject such as a fecal sample, tissues or cells from a subject by comparison to the amount present in said subject before treatment with said composition or polypeptide according to the invention. Alternatively, the comparison can be made with a sample, tissues or cells of said subject which has not yet been treated with said composition or polypeptide in case the treatment is local. 84 105506204.3Attorney Docket: 125930-848037

[0298] A composition comprising a polynucleotide or a nucleic acid construct or an endolysin polypeptide or a vector or a cell according to the invention may be administered to a subject in need thereof or of a cell, tissue or organ or said patient for a duration of at least one week, one month, six months, one year, or longer.

[0299] Accordingly, the invention provides for a composition according to the invention, for use as a medicament for the treatment of a subject in need thereof. Preferably, said composition is for use as a medicament in the treatment of a condition associated with infection of a subject with C. septicum.

[0300] The invention further provides for a method of treatment, delay and / or prevention of a condition associated with infection of a subject with a C. septicum strain comprising administration an endolysin polypeptide according to the invention, or a polynucleotide according to the invention, or a nucleic acid construct according to the invention, or an expression construct according to the invention, or a host cell according to the invention, or a composition according to the invention. Accordingly, treatment may include the treatment of clostridial dermatitis in a subject.

[0301] The medical use herein described may be formulated as a product according to the invention for use as a medicament for treatment of the stated diseases but could equally be formulated as a method of treatment of the stated diseases using a product according to the invention, a product according to the invention for use in the preparation of a medicament to treat the stated diseases and use of a product according to the invention for the treatment of the stated diseases. Such medical uses are all envisaged by the present invention. The subject in need of treatment, delay and / or prevention of a condition associated with infection may by any animal subject, preferably a domestic turkey, Meleagris gallopovo domesticus or broiler chickens such as Gallus gallus domesticus. Accordingly, subjects may include livestock such as the domestic duck derived from the mallard duck, Anas platyrhynchos domestica, or a domestic duck derived from the Muscovy duck, Cairina moschata domestica, or a goose, optionally a domestic goose, further optionally a domestic goose derived from the Greylag goose, Anser anser, or a domestic goose derived from the Chinese goose, Anser cygnoides, or a domestic pigeon, Columba livia domestica, or a domestic quail, Coturnix japonica domestica. Examples of subjects also include a pig; Sus scrofa domesticus, a dog or a cat, or a human subject.

[0302] The compound specifically targeting a C. septicum bacterial cell can be an endolysin polypeptide according to the invention, or a polynucleotide according to the invention, or a 85 105506204.3Attorney Docket: 125930-848037 nucleic acid construct according to the invention, or an expression construct according to the invention, or a host cell according to the invention, or a composition according to the invention.

[0303] The endolysin polypeptide according the invention, or a polynucleotide according to the invention, or a nucleic acid construct according to the invention, or an expression construct according to the invention, or a host cell according to the invention, or a composition according to the invention may be used in a method of treatment of an intracellular C. septicum bacterial infection in a subject in need thereof, comprising administration of an effective amount of an agent that increases the intracellular pH of a host cell and / or of an intracellular compartment of a host cell, and administration of an effective amount of a antimicrobial agent.

[0304] The Inventors have surprisingly identified that treatment of certain diseases or disorders in animals may be affected by an antimicrobial formulation comprising a whole-cell composition, wherein whole-cells of the composition are bacterial host cells comprising an endolysin polypeptide having antimicrobial activity, or comprising a fragment or variant of an endolysin polypeptide wherein the fragment or variant has antimicrobial activity. The endolysin polypeptide, or the fragment or the variant of an endolysin polypeptide may be any of the endolysin polypeptides, or the fragments or the variants of an endolysin polypeptide disclosed herein. Alternatively, the endolysin polypeptide, or the fragment or the variant of an endolysin polypeptide may be any endolysin polypeptide, or any fragment or any variant of an endolysin polypeptide which possesses any of the surprisingly advantageous functional antimicrobial activities described and defined herein.

[0305] Provided herein is an antimicrobial formulation for use as a medicament, the formulation comprising a whole-cell composition, wherein whole-cells of the composition are bacterial host cells comprising an endolysin polypeptide having antimicrobial activity, or comprising a fragment or variant of an endolysin polypeptide wherein the fragment or variant has antimicrobial activity.

[0306] Any such antimicrobial formulation may be for use in the treatment of a bacterial infection in a subject (e.g., an animal). Any such antimicrobial formulation may be for use in the treatment of a bacterial infection which is a Clostridium septicum infection. Any such antimicrobial formulation may be for use in the treatment of a disease or disorder caused by a Clostridium septicum infection. The disease or disorder caused by the Clostridium septicum infection may be cellulitis, gangrenous dermatitis, clostridial dermatitis, a gut lesion and / or a gastrointestinal infection. 86 105506204.3Attorney Docket: 125930-848037

[0307] Provided herein is a method for the prevention or treatment of a disease or disorder in a subject (e.g., an animal), the method comprising administering to the subject a prophylactically or therapeutically effective amount of an antimicrobial formulation, the formulation comprising a whole-cell composition, wherein whole-cells of the composition are bacterial host cells comprising an endolysin polypeptide having antimicrobial activity, or comprising a fragment or variant of an endolysin polypeptide wherein the fragment or variant has antimicrobial activity.

[0308] In any such method the disease or disorder may be a bacterial infection in a subject (e.g., an animal). In any such method the bacterial infection may be a Clostridium septicum infection. In any such method the disease or disorder may be caused by a Clostridium septicum infection. The disease or disorder caused by the Clostridium septicum infection may be cellulitis, gangrenous dermatitis, clostridial dermatitis, a gut lesion and / or a gastrointestinal infection.

[0309] In any of the above-described antimicrobial formulations or methods the animal is preferably a domestic turkey, Meleagris gallopovo domesticus or a broiler chicken, Gallus gallus domesticus. The animal may also include livestock such as the domestic duck derived from the mallard duck, Anas platyrhynchos domestica, or a domestic duck derived from the Muscovy duck, Cairina moschata domestica, or a goose, optionally a domestic goose, further optionally a domestic goose derived from the Greylag goose, Anser anser, or a domestic goose derived from the Chinese goose, Anser cygnoides, or the domestic pigeon, Columba livia domestica, or the domestic quail, Coturnix japonica domestica or the pig, Sus scrofa domesticus.

[0310] In any of the above-described antimicrobial formulations or methods the antimicrobial activity of the endolysin polypeptide, fragment or variant may be bacteriolytic activity and / or bacterial growth inhibitory activity. The bacteriolytic activity and / or bacterial growth inhibitory activity of the endolysin polypeptide, fragment or variant may be bacteriolytic activity and / or bacterial growth inhibitory activity against, Clostridium septicum.

[0311] In any of the above-described antimicrobial formulations or methods, when the endolysin polypeptide, fragment or variant is tested in a standard cell viability assay at a concentration of 5 μg / ml against Clostridium septicum strain NCTC 547 it may exhibit a Δ Log 10 value of approximately 0.40 or more, 2.00 or more or 3.70 or more. In any of the above- described antimicrobial formulations or methods, when the endolysin polypeptide, fragment or variant is tested in a standard cell viability assay at a concentration of 5 μg / ml against Clostridium septicum strain NCTC 547 it may exhibit a % reduction in cell viability of approximately 60% or more, 70% or more, 80% or more, 90% or more or 100%. In any of the above-described antimicrobial formulations or methods, when the endolysin polypeptide, 87 105506204.3Attorney Docket: 125930-848037 fragment or variant is tested in a standard cell turbidity reduction assay at a concentration of 5 μg / mL against Clostridium septicum strain NCTC 547 it may exhibit a t50% lysis value of 7 minutes or less; or it may exhibit a t50% lysis value of 3 minutes or less. In any of the above- described antimicrobial formulations or methods, when the endolysin polypeptide, fragment or variant is tested in a standard minimum inhibitory concentration (MIC) / minimum bactericidal concentration (MBC) assay against Clostridium septicum strain NCTC 547 at 2.1×104cells / mL it may exhibit an MIC / MBC value of 17 μg / mL or less, preferably 1.7 μg / mL or less; or it may exhibit an MIC / MBC value of 0.65 μM or less, preferably 0.05 μM or less. EXAMPLES Example 1: Bioinformatics searches

[0312] A list of putative endolysin sequences against the gram-positive bacterium Clostridium septicum were compiled from two different bioinformatic searches. In an initial search, fourteen full genome sequences corresponding to C. septicum isolates were selected from public repositories. These genome sequences were searched for prophage regions using “PHASTEST” and similar software. Prophage sequences that were annotated by the software as “endolysin” or “lysin” were selected. A total of ten sequences were selected for further processing. In a follow on search, certain sequences found in the initial search were used to probe for homologous sequences using the “Blast” tool of UNIPROT, filtering for sequences annotated as “virus” and “Clostridium sp”. Fifteen sequences with a similarity percentage between 50 and 90% to the query sequences were selected for further processing, producing a compiled list of 25 putative C. septicum specific endolysin base sequences for further analysis. Example 2: Strain construction

[0313] The 25 sequences from Example 1 were codon optimized for expression in Bacillus subtilis using its most frequently utilized codons. Sequences were domesticated for Golden Gate cloning by removing BsaI and SapI sites while conserving the encoded amino acid sequence of the endolysin, and short sequences were added to either end of the endolysin to facilitate cloning into vectors. The endolysin sequence was also optimized for expression using the software TISIGNER. After sequence optimization, DNA oligonucleotides were chemically synthesized and cloned into the expression vectors using standard Golden Gate methodology (e.g., by means of Type IIS restriction enzymes and DNA ligases). A DNA fragment carrying the endolysin-encoding sequence, flanked by promoter, 5’ UTR, and 3’ UTR sequences, was amplified by PCR. 88 105506204.3Attorney Docket: 125930-848037

[0314] In addition, this DNA fragment contained sequences of homology to the Bacillus subtilis gene amyE for targeting integration into the amyE site of the bacterial chromosome (see FIG.1). The DNA fragment also carried a gene that conferred resistance to erythromycin in addition to the lactose repressor LacI gene. The LacI protein binds to the promoter that drives expression of the endolysin and maintains repression. The DNA fragment amplified by PCR was used to transform Bacillus subtilis strain bAL265, which is derived from strain 168. This strain harbours a spectinomycin resistant gene disruption in the AmyE gene. Transformants were recovered in media supplemented with erythromycin, and confirmed by testing for antibiotic resistance (loss of spectinomycin resistance and gain of erythromycin resistance) and by PCR. Glycerol stocks were prepared for the cryopreservation of the strains at -80°C. Example 3: Endolysin activity screening

[0315] B. subtilis strains were recovered from cryopreservation on LB agar plates and grown at 37ºC. A colony was used to inoculate LB broth and the culture was grown at 37ºC in flasks while shaking until mid-logarithmic growth phase. At this point, 1 mM IPTG was added to the culture to induce the expression of the endolysin gene, which is repressed by the LacI repressor. Upon binding of IPTG, LacI comes off the regulatory region upstream of the endolysin gene, and active expression of the gene is expected. Cells were harvested 4 h after addition of IPTG and processed for SDS-PAGE gels, consisting of sonication to disrupt cellular structures, followed by centrifugation. Supernatants containing the soluble fraction of cells were used to inspect for the recombinant protein in SDS-PAGE gels. Six out of 12 clones analysed presented protein bands in the SDS-PAGE gel that matched the predicted sizes of the recombinant endolysins. The lysates of these clones were used to evaluate the antimicrobial activity of the endolysins against Clostridium septicum. Example 4: Culture growth & induction.

[0316] Bacillus subtilis strains bAL265 (negative control strain), bAL340 (expresses endolysin Sep-EL6 (SEQ ID NO:6)) and bAL379 (expresses endolysin Sep-EL25 (SEQ ID NO:25)) were streaked out from -80°C glycerol stocks onto LB plates which were incubated at 37°C overnight. Cells from each of the plates were used to inoculate 5 ml aliquots of LB medium in 15 ml Falcon tubes which were cultured overnight at 37°C with shaking (200 rpm) in a SciQui Incu- shake MIDI incubator. In the morning 1 ml aliquots from the three cultures were used separately to inoculate 3 x 100 ml portions of LB medium in 250 ml Erlenmeyer flasks. The cultures were cultured at 37°C with shaking (200 rpm) and the optical density (OD) determined using a Jenway 7200 Spectrophotometer set at λ = 600nm. When the OD600of the three cultures 89 105506204.3Attorney Docket: 125930-848037 reached 0.7100 μl of 1M IPTG was added to each flask to induce expression of the endolysins in bAL340 & bAl379. The flasks were cultured for a further 4 hours at 37°C with shaking (200 rpm) before the cultures divided into 2 x 50ml portions, each in a 50 ml centrifuge tube, and the cells were harvested by centrifugation (4°C, 3,900 rpm, 20 min, using an Eppendorf 5810 R centrifuge) whilst the culture supernatants were discarded. Both pellets for each culture were resuspended in 1 ml of LB and the cell suspension transferred to a 1.5 ml microcentrifuge tune and the cells harvested by centrifugation (13,000 rpm, 4°C, 10 min, using an Eppendorf 5430 R centrifuge), whilst the supernatant was discarded. The pellet was frozen at -20°C until required. Lysate preparation

[0317] To make a cell lysate the pellet was resuspended in 0.8 ml of PBS and sonicated for 25 cycles of 5 sec on, 5 sec off at amplitude: 10 μm, using a Soniprep 150 sonicator controlled by an MSE Process Timer controller. The protein concentrations of the three lysate samples was determined using the Bio-Rad DC Protein Quantification kit, with the quantification being done using a Labtech LT-4500 plate reader with the λ set to 720nm. The protein concentrations of all three lysate samples were identical, and were 13 mg / ml. Inoculum preparation

[0318] Clostridium septicum Ramsay (bAL274) was streaked from a -80°C glycerol stock onto a pre-reduced Reinforced Clostridial Medium (RCM) agar plate which was cultured overnight at 37°C in a Don Whitney MACS-MG-500 anaerobic workstation using BOC Anaerobic Mixture (10% CO2; 5% H2; 85% N2). The following day a loopful of cells from the plate was used to inoculate 50 ml of pre-reduced BHI+C (Oxoid: Brain, Heart Infusion, enriched with 50 μg / l vitamin K1, 5 mg / l hemin and 0.5 / l cysteine) medium and this cultured overnight at 37°C in the anaerobic chamber. The following day 3 ml of the culture was diluted in 47 ml of pre-reduced BHI+C to give an OD600of 0.1. The culture was incubated at 37°C in the anaerobic workstation until the OD600= 0.2 at which point it was diluted 1:100 in fresh pre-reduced BHI+C medium to generate a cell suspension containing ~ 1 x 106CFU / ml and this was serially diluted 1:10 in fresh pre-reduced BHI+C medium to generate a cell suspension containing ~ 1 x 105CFU / ml which in turn was diluted 1:10 in fresh pre-reduced BHI+C medium to generate a cell suspension containing ~ 1 x 104CFU / ml. MIC assay

[0319] The MIC assay plate was set up inside the anaerobic chamber as follows: fifty μl of the bAL274 cell suspension containing ~ 1 x 104CFU / ml, prepared as described above, was used to inoculate each of the wells of a 96 well microtitre plate (Starlab), the wells having rounded bases 90 105506204.3Attorney Docket: 125930-848037 and a volume of 280 μl, excepting the wells of column 12 which have 50 μl of BHI+C medium added to them.

[0320] The bAL265, bAL340 and bAL379 cell lysates were each used to prepare a twofold dilution series comprising eight samples where the first sample in the series represents undiluted lysate and the second a two-fold dilution, the third a four-fold dilution and the fourth a six-fold dilution, etc., where the diluent was PBS. Additionally, a two-fold dilution series of a 10 mg / ml solution of lysozyme was generated in a similar fashion, as well as a two-fold dilution series of a 0.2 μg / ml solution of penicillin. 50 μl of each of the lysate samples from the three dilution series were used to inoculate two adjacent wells in different columns in the microtitre plate. Samples comprising bAL265 was added to columns 1 & 2, bAL340 was added to columns 3 & 4 and bAL379 was added to columns 5 & 6. The most concentrated lysate samples, e.g., the undiluted lysates, were added to the wells of the topmost row, e.g., row A, and the diluted lysates from the two-fold dilution series being added to subsequent rows.

[0321] 50 μl of the lysozyme solutions from the lysozyme dilution series were used to inoculate two adjacent wells in two columns (columns 7 & 8) in the microtitre plate. The most concentrated lysozyme sample, e.g., the undiluted lysozyme (10 mg / ml) being added to the wells of the topmost row, e.g., row A, and the dilutions being added to subsequent rows.

[0322] 50 μl of the penicillin solutions from the penicillin dilution series were used to inoculate two adjacent wells in two columns (columns 9 & 10) in the microtitre plate. The most concentrated penicillin sample, e.g., the undiluted penicillin (0.2 μg / ml) being added to the wells of the topmost row, e.g., row A and the dilutions being added to subsequent rows.

[0323] 50 μl of PBS were used to inoculate all of the wells of columns 11 & 12.

[0324] Columns 3 & 4 and 5 & 6 represented the test samples that contain lysate comprising endolysins. Columns 1 & 2 represented a control lysate which does not comprise endolysins. Columns 7 & 8 represented a positive control in the form of lysozyme that degrades C. septicum cell wall material, and columns 9 &10 represented a second positive control (penicillin), which inhibits enzymes involved in cell wall synthesis / maintenance and causes cell death due to cell wall failure. Column 11 was a cell-only control and column 12 represented a blank (no cells).

[0325] The microtitre plate was covered in a “Breathable Seal, self adhesive” film (Starlab) and incubated overnight at 37°C in the anaerobic chamber, with shaking using a SciQuip Microplate Mixer. The following day the OD620nmof the wells of the MIC assay plate were determined, 91 105506204.3Attorney Docket: 125930-848037 using the Labtech 4500 plate reader, and the averaged readings for the two replicates for each sample, and the single replicate readings for the “cells only” and “medium only” columns were determined. Results

[0326] Results of the MIC assay are provided in TABLE 3. TABLE 3: MIC assay results Lysate bAL265 bAL340 bAL379 Lysozyme Penicillin Cells Medium

[0327] The results indicate that there is a reduction in turbidity in the replicate columns comprising bAL340 and bAL379 lysates, relative to the replicate columns comprising negative control bAL265 lysate. The OD620of samples of bAL340 lysate (comprising endolysin SepEL6 (SEQ ID NO:6)) and bAL379 lysate (comprising endolysin SepEL25 (SEQ ID NO:25)) were lower than those of similarly diluted bAL265 lysate (negative control with no endolysins). Thus, it may be concluded that endolysins SepEL6 (SEQ ID NO:6) and SepEL25 (SEQ ID NO 25) exert an antibacterial effect against Clostridium septicum. Example 5: Culture growth & induction

[0328] Bacillus subtilis strains bAL265 (negative control strain), bAL340 (expressing endolysin Sep-EL6 (SEQ ID NO:6)), bAL371 (expressing endolysin Sep-EL2 (SEQ ID NO:2)), bAL373 (expressing endolysin Sep-EL3 (SEQ ID NO:3)), bAL375 (expressing endolysin Sep-EL9 (SEQ ID NO:10)), bAL377 (expressing endolysin Sep-EL4v2 (SEQ ID NO:7)), bAL379 (expressing endolysin Sep-EL25 (SEQ ID NO:25)), bAL381 (expressing endolysin Sep-EL7 (SEQ ID NO:8)), bAL383 (expressing endolysin Sep-EL8 (SEQ ID NO:9)) and bAL385 (expressing endolysin Sep-EL18 (SEQ ID NO:18)) were generated as described above, and streaked out from -80°C glycerol stocks onto LB plates which were incubated at 37°C overnight. Cells from each of the plates were used to inoculate 5 ml aliquots of LB medium in 15 ml 92 105506204.3Attorney Docket: 125930-848037 Falcon tubes which were cultured overnight at 37°C with shaking (200 rpm) in a SciQui Incu- shake MIDI incubator. In the morning, 1 ml aliquots from each of the cultures were used separately to inoculate 100 ml portions of LB medium in 250 ml Erlenmeyer flasks. The cultures were cultured at 37°C with shaking (200 rpm) and the optical density(OD) determined using a Jenway 7200 Spectrophotometer set at λ = 600nm. When the OD600of the cultures reached 0.7, 100 μl of 1M IPTG was added to each flask to induce expression of the endolysins. The flasks were cultured for a further 4 hours at 37°C with shaking (200 rpm) before the cultures were divided into 2 x 50 ml portions, each in a 50 ml centrifuge tube, and the cells were harvested by centrifugation (4°C, 3,900 rpm, 20 min, using an Eppendorf 5810 R centrifuge), whilst the culture supernatants were discarded. Both pellets for each culture were resuspended in 1 ml of LB and the cell suspension transferred to a 1.5 ml microcentrifuge tube and the cells harvested by centrifugation (13,000 rpm, 4°C, 10 min, using an Eppendorf 5430 R centrifuge) whilst the supernatant was discarded. The pellets were frozen at -20°C until required. Lysate preparation

[0329] To make a cell lysate for each particular strain, their pellets were resuspended in 0.8 ml of PBS and sonicated for 25 cycles (5 sec on, 5 sec off at amplitude: 10 μm, using a Soniprep 150 sonicator controlled by an MSE Process Timer controller). The protein concentrations of the lysate samples were determined using the Bio-Rad DC Protein Quantification kit, with the quantification being done using a Labtech LT-4500 plate reader with the λ set to 720nm. The protein concentrations of all lysate samples are as shown in TABLE 4, below. TABLE 4: Soluble protein concentrations of tested endolysins Strain Endolysin SEQ ID No [soluble protein] mg / ml93 105506204.3Attorney Docket: 125930-848037 TSC plate bioassay method

[0330] Clostridium septicum strains bAL274 (Clostridium septicum Ramsay) and bAL275 Clostridium septicum were streaked from -80°C glycerol stocks onto non-reduced Brucella agar enriched with hemin (5 mg / l) and vitamin K1 (50 μg / l). The plates were incubated overnight at 37°C in an anaerobic chamber. The following day cotton swabs were used to collect samples of bAL274 and bAL275 from the plates and transfer them into 5 ml samples of pre-reduced MHA broth (Oxoid), to create cell suspensions with an OD600of 0.2. Using cotton swabs the bacterial cell suspensions were streaked over the entire surfaces of tryptose sulfite cycloserine (TSC) agar plates and left to dry. When the plates were dry, 30 μl samples of each of the lysates were separately pipetted onto the surface of the plates inoculated with both bAL274 and bAL275 to compare the sensitivities of the two C. septicum strains. Controls including a positive control (30 μl of 10 mg / ml lysozyme, a second positive control comprising a Bio-Rad Susceptibility Disc comprising 10 μg of ampicillin and a negative control (30 μl of PBS) were also added to the surfaces of the plates inoculated with bAL274 and bAL275. The test samples and control solutions were spaced apart on the plates and two replicates (A1 & A2 and B1 and B2) were done for each C. septicum strain, whilst the discs containing ampicillin were placed on separate plates and only a single replicate was performed per C. septicum strain. The plates were incubated overnight at 37°C in an anaerobic chamber and the results (See FIGS.2A-2C) were recorded by photographing the plates the following day. Results and Discussion

[0331] TSC is an indicator agar which turns black in the presence of Clostridium septicum, whilst bacterial free areas are indicated by white / pale zones. The results (FIGS.2A-2C) indicated that the tested samples produced from bAL340 (containing endolysin SepEL6 (SEQ ID NO:6)) and bAL379 (containing endolysin SepEL25 (SEQ ID NO:25)) are most capable of inhibiting the growth of C. septicum at the tested concentration and produced visible white / pale zones in this assay. The positive controls, lysozyme and ampicillin, also produced white bacteria-free zones, whilst the negative controls, PBS and bAL265 did not.

[0332] The endolysins exhibiting the highest antibacterial activity against Clostridium septicum in this assay were endolysin SepEL6 (SEQ ID NO:6) and endolysin SepEL25 (SEQ ID NO:25). Both of these endolysins are 325 amino acids long and share considerable sequence identity (97.85 % identical), with differences being seven amino acids in the cell-wall binding portion (highlighted in TABLE 5 below). 94 105506204.3Attorney Docket: 125930-848037 TABLE 5: Comparison of amino acid differences between SepEL6 and SepEL25 endolysins 5

[0333] In view of the substantial sequence similarity (e.g., identical catalytic domain) and excellent antimicrobial performance, consensus sequences SEQ ID NO:79 (endolysin comprising catalytic domain and cell binding domain); SEQ ID NO:80 (cell wall binding domain) and SEQ ID NO:82 (cell wall binding domain) were further developed. 95 105506204.3Attorney Docket: 125930-848037 SEQUENCE TABLE TABLE 6: Endolysin polypeptide sequences MAYSLNDLISKASNHEKMKGVHPLVKEKFLQVVTRAYNELGYKLVLAEGYRSVARQNELYAQGRTK K A F II G W K V K V K L T M L T N R L T GF A V R K L Q P WAttorney Docket: 125930-848037 P S KI I R LI K 3) A M D A L V QI E C N F F N T E Y R K R S A IP G V D P T W P T WAttorney Docket: 125930-848037 P T W K V M L A A F K K K M N R L A V K Q P P SAttorney Docket: 125930-848037RLI AM A LQIENF NR KIP GP T P T P T K MDKF F IF SAttorney Docket: 125930-848037 K AGKEE EL AKIY KRKTFIKFY E MLR F E X7Attorney Docket: 125930-848037 F K N r I, Y K101 105506204.3

Claims

Attorney Docket: 125930-848037 CLAIMS What is claimed is:

1. An endolysin polypeptide having antimicrobial activity, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:50, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49, or a fragment thereof, optionally SEQ ID NO:

50.

2. The endolysin polypeptide of claim 1, further comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof, optionally SEQ ID NO:

80.

3. The endolysin polypeptide of claim 2, further comprising a linker between (a) the amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof, optionally SEQ ID NO:50, and (b) the amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof, optionally SEQ ID NO:

80.

4. The endolysin polypeptide of claim 1 or 2, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of 102 105506204.3Attorney Docket: 125930-848037 SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof.

5. An endolysin polypeptide having antimicrobial activity, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:80, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 81, 82, or 83, or a fragment thereof, optionally SEQ ID NO:

80.

6. The endolysin polypeptide of claim 5, further comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof, optionally SEQ ID NO:

50.

7. The endolysin polypeptide of claim 6, further comprising a linker between (a) the amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82, or 83, or a fragment thereof, optionally SEQ ID NO:80, and (b) the amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or a fragment thereof, optionally SEQ ID NO:

50.

8. The endolysin polypeptide of claim 5 or 6, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 79, or a fragment thereof. 103 105506204.3Attorney Docket: 125930-848037 9. The endolysin polypeptide of any one of claims 1-8, wherein the endolysin polypeptide is purified or isolated.

10. The endolysin polypeptide of any one of claims 1-9, wherein the antimicrobial activity is bacteriolytic activity and / or bacterial growth inhibitory activity.

11. The endolysin polypeptide of any one claims 1-10, wherein the antimicrobial activity is against Clostridium septicum.

12. The endolysin polypeptide of any one of claims 1-11, wherein the antimicrobial activity is determined by a (a) cell viability assay; (b) cell turbidity reduction assay; or (c) minimum inhibitory concentration (MIC) assay or minimum bactericidal concentration (MBC) assay, optionally wherein the assay is performed against Clostridium septicum.

13. The endolysin polypeptide of any one of claims 1-11, wherein the antimicrobial activity is determined by endolysin activity, optionally wherein the endolysin activity is assessed by the rate of degradation of peptidoglycan (PGN).

14. A recombinant nucleic acid molecule comprising a first nucleic acid sequence encoding the endolysin polypeptide of any one of claims 1-13, optionally wherein the recombinant nucleic acid molecule further comprises a second nucleic acid sequence encoding a promoter and wherein the first nucleic acid sequence is operably linked to the second nucleic acid sequence; further optionally wherein the first nucleic acid sequence of the recombinant nucleic acid molecule encodes a cDNA.

15. The recombinant nucleic acid molecule of claim 14, which comprises the first and second nucleic acid sequences.

16. An expression vector comprising the nucleic acid molecule of claim 14.

17. An expression vector comprising the nucleic acid molecule of claim 15.

18. The recombinant nucleic acid molecule of claim 15 or the expression vector of claim 17, wherein the promoter is capable of promoting expression of the endolysin polypeptide in a cell of a unicellular microorganism. 104 105506204.3Attorney Docket: 125930-848037 19. The recombinant nucleic acid molecule of claim 15 or the expression vector of claim 17, wherein the promoter is capable of promoting expression of the endolysin polypeptide in Bacillus subtilis.

20. A host cell comprising the nucleic acid molecule or expression vector of any one of claims 14-19.

21. The host cell of claim 20, which is a Bacillus subtilis cell.

22. A cell lysate comprising the one or more endolysin polypeptides according to any one of claims 1-13.

23. The cell lysate of claim 22, wherein the one or more endolysin polypeptides is expressed from the nucleic acid molecule or expression vector of any one of claims 14-19, or is expressed by a population of one or more host cells according to claim 20 or 21.

24. A dried whole culture comprising the one or more endolysin polypeptides according to any one of claims 1-13, wherein the dried whole culture is spray dried or freeze dried.

25. A dried whole culture of claim 24, wherein the one or more endolysin polypeptides is expressed from the nucleic acid molecule or expression vector of any one of claims 14- 19, or is expressed by a population of one or more host cells according to claim 20 or 21.

26. A dried culture medium comprising the one or more endolysin polypeptides according to any one of claims 1-13, wherein the dried culture medium is spray dried or freeze dried.

27. The dried culture medium of claim 26, wherein the one or more endolysin polypeptides is expressed from the nucleic acid molecule or expression vector of any one of claims 14-19, or is expressed by a population of one or more host cells according to claim 20 or 21.

28. dried material comprising the one or more endolysin polypeptides according to any one of claims 1-13, the population of one or more host cells according to claim 20 or 21, and / or the cell lysate accordin g to claim 22 or 23, optionally wherein the dried material is spray dried or freeze dried.

29. A combined material comprising two or more of (i) the one or more endolysin polypeptides according to any one of claims 1- 13; 105 105506204.3Attorney Docket: 125930-848037 (ii) the population of one or more host cells according to claim 20 or 21; (iii) the cell lysate according to claim 22 or 23; (iv) the dried whole culture of claim 24 or 25; (v) the dried culture medium of claim 26 or 27; (vi) the dried material of claim 28, optionally wherein the combined material is dried, further optionally wherein the combined material is spray dried or freeze dried.

30. A method for producing an endolysin polypeptide having antimicrobial activity, the method comprising culturing a host cell comprising the nucleic acid or expression vector of any one of claims 14-19 under conditions in which the endolysin polypeptide is expressed.

31. The method of claim 30, further comprising lysing the host cell to produce a cell lysate comprising the endolysin polypeptide.

32. The method of claim 30 or 31, further comprising purifying or isolating the endolysin polypeptide.

33. The method of claim 30 or 31, which does not comprise further purification or isolation of the endolysin polypeptide.

34. An animal foodstuff comprising: (a) one or more foodstuffs; and (b) one or more endolysin polypeptides according to any one of claims 1-13.

35. An animal foodstuff comprising: (a) one or more foodstuffs; and (i) the host cell of claim 20 or 21; and / or (ii) the cell lysate of claim 22 or 23; and / or (iii) the dried whole culture of claim 24 or 25; and / or (iv) the dried culture medium of claim 26 or 27; and / or (v) the dried material of claim 28; and / or (vi) the combined material of claim 29.

36. A supplemented water comprising: (a) water; and (b) one or more endolysin polypeptides according to any one of claims 1-13.

37. A supplemented water comprising: 106 105506204.3Attorney Docket: 125930-848037 (a) water; and (i) the host cell of claim 20 or 21; and / or (ii) the cell lysate of claim 22 or 23; and / or (iii) the dried whole culture of claim 24 or 25; and / or (iv) the dried culture medium of claim 26 or 27; and / or (v) the dried material of claim 28; and / or (vi) the combined material of claim 29.

38. A supplemented hydrogel comprising: (a) hydrogel; and (b) one or more endolysin polypeptides according to any one of claims 1-13.

39. A supplemented hydrogel comprising: (a) hydrogel; and (i) the host cell of claim 20 or 21; and / or (ii) the cell lysate of claim 22 or 23; and / or (iii) the dried whole culture of claim 24 or 25; and / or (iv) the dried culture medium of claim 26 or 27; and / or (v) the dried material of claim 28; and / or (vi) the combined material of claim 29.

40. A method for reducing a population of Clostridium septicum bacteria in a foodstuff, the method comprising admixing with the foodstuff (i) the endolysin polypeptide of any one of claims 1-13; and / or (ii) the host cell of claim 20 or 21; and / or (iii) the cell lysate of claim 22 or 23; and / or (iv) the dried whole culture of claim 24 or 25; and / or (v) the dried culture medium of claim 26 or 27; and / or (vi) the dried material of claim 28; and / or (vii) the combined material of claim 29.

41. A method for the prevention or treatment of a disease or disorder in an animal, the method comprising administering to the animal (i) the endolysin polypeptide of any one of claims 1-13; and / or (ii) the host cell of claim 20 or 21; and / or (iii) the cell lysate of claim 22 or 23; and / or (iv) the dried whole culture of claim 24 or 25; and / or (v) the dried culture medium of claim 26 or 27; and / or 107 105506204.3Attorney Docket: 125930-848037 (vi) the dried material of claim 28; and / or (vii) the combined material of claim 29. wherein the disease or disorder is caused by Clostridium septicum.

42. A method for the prevention or treatment of a disease or disorder in an animal, the method comprising administering to the animal the animal foodstuff of claim 34 or 35, and / or the supplemented water of claim 36 or 37 and / or the supplemented hydrogel of claim 38 or 39 wherein the disease or disorder is caused by Clostridium septicum.

43. The method of claim 41 or 42, wherein the disease or disorder caused by Clostridium septicum is cellulitis or gangrenous dermatitis.

44. The method of any one of claims 41-43, wherein the animal is selected from (a) a turkey, optionally a domestic turkey; (b) a chicken, optionally a broiler chicken; (c) a duck, optionally a domestic duck, further optionally a domestic duck derived from a mallard duck or a domestic duck derived from a Muscovy duck; (d) a goose, optionally a domestic goose, further optionally a domestic goose derived from the Greylag goose, or a domestic goose derived from the Chinese goose; (e) a pigeon, optionally a domestic pigeon; (f) a quail, optionally a domestic quail; (g) a pig, optionally a domestic pig.

45. A kit comprising (a) an agent having antimicrobial activity, wherein the agent is selected from (i) the endolysin polypeptide of any one of claims 1-13; and / or (ii) the host cell of claim 20 or 21; and / or (iii) the cell lysate of claim 22 or 23; and / or (iv) the dried whole culture of claim 24 or 25; and / or (v) the dried culture medium of claim 26 or 27; and / or (vi) the dried material of claim 28; and / or (vii) the combined material of claim 29; and (b) instructions for admixing the agent with a foodstuff. 108 105506204.3

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