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 gram-negative bacteria, such as Salmonella enterica). 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 gram-negative bacteria in a foodstuff, or preventing or treating a disease or disorder in an animal.

Description

Docket No.: 125930-872823NOVEL COMPOUND SEQUENCE LISTING

[0001] This application contains a Sequence Listing that has been submitted in electronically in Extensible Markup Language (.xml) format and is hereby incorporated by reference in its entirety. The XML copy, created on February 6, 2026, is named 125930-872823_SL.xml, and is 1,808,154 bytes in size.FIELD OF THE INVENTION

[0002] The present invention relates to improvements to animal foodstuffs via endolysin enrichment and uses of endolysins for antimicrobial activity.BACKGROUND

[0003] Escherichia coli (E. coli) is a gram -negative, non-acid-fast, uniform staining, non-spore-forming bacillus that grows both aerobically and anaerobically and may be variable in size and shape. Many strains are motile and have peritrichous flagella. Colibacillos is caused by E. coli is a major disease state which affects both humans and animals. In humans pathogenic varieties of E. coli cause serious food poisoning, septic shock, meningitis, or urinary tract infections. The major human infection route is fecal-oral transmission via unhygienic food preparation, farm contamination due to manure fertilization, irrigation of crops with contaminated grey water or raw sewage, or direct consumption of sewage-contaminated water. Enterohemorrhagic E. coli (EHEC), which causes diarrhea and hemolyticuremic syndrome (HUS), in humans is commonly spread from cattle to humans and enterotoxigenic E. coli (ETEC), which causes diarrhea, is commonly spread from pigs and cattle to humans.

[0004] A serious form of E. coli mediated colibacillosis in animals is E. coli Post-Weaning Diarrhea (PWD). This is a disorder that often affects pigs during the first 2 weeks of weaning, although not exceptionally, it can be recorded at 6-8 weeks after weaning, and is characterized by sudden death or diarrhea, and growth retardation in surviving piglets. When ETEC causes PWD, large quantities of watery to a creamy consistency scour are observed, with a distinctive smell, generally lasting one week. Affected pigs are usually depressed with a reduced appetite and a rough sticky wet haircoat. Sudden deaths can occur, particularly at the start of the outbreak and dead pigs are usually dehydrated with sunken eyes. PWD is one of the most serious threats to the swine industry worldwide, with episodes reaching mortality rates of 20 to 30%.

[0005] Outbreaks of neonatal and post-weaning diarrhoea due to ETEC infection, generally affecting a high proportion of pigs, are often recurrent in the same herds and require expensive control measures. Enteric colibacillosis may result in significant economic losses due to mortality, decreased weight gain, cost for treatments, vaccinations and feed supplements. Various approaches have been used to prevent 1108869435.2Docket No.: 125930-872823 ETEC PWD. including passive administration with specific antibodies, dietary supplementation such as prebiotics and probiotics and dietary preventive measures, genetic breeding for ETEC-resistant herds and live oral nontoxigenic E.coli vaccines. The current generation of commercial vaccines have not been shown to be effective against post weaning ETEC infections in pigs. Antimicrobial resistance to several antibiotics such as apramycin, neomycin, trimethoprim-sulphametoxazole and colistin has been increasingly observed in ETEC strains causing PWD. The development of resistance to a wide range of antimicrobial drugs, as well as the demonstrated trend of resistance in ETEC strains to the antibiotics used for the treatment of colibacillosis in pigs, is nowadays a reason for concern. Multidrug resistance among ETEC isolates has been described and recently there has been an increasing tendency for porcine ETEC to express a multidrug-resistant phenotype.

[0006] E. coli is considered as a member of the normal microflora of the poultry intestine, but certain strains, such as those designated as avian pathogenic E. coli (APEC), spread into various internal organs and cause colibacillosis, characterized by systemic fatal disease in poultry.

[0007] E. coli isolates pathogenic for poultry commonly belong to certain serogroups. particularly the serogroups 078, 01, and 02, and to some extent 015 and 055. In domestic poultry, avian colibacillosis is frequently associated with E. coli strains of serotypes O78: K80, O1: K1 and O2: K1. Avian colibacillosis is considered as one of the principal causes of morbidity and mortality, associated with heavy economic losses to the poultry industry by its association with various disease conditions, either as primary pathogen or as a secondary pathogen. It causes a variety of disease manifestations in poultry including yolk sac infection, omphalitis, respiratory tract infection, swollen head syndrome, septicemia, polyserositis, coligranuloma, enteritis, cellulitis and salpingitis. Colibacillosis of poultry is characterized in its acute form by septicemia resulting in death and in its subacute form by pericarditis, airsacculitis and perihepatitis.

[0008] Current methods to prevent avian colibacillosis can lack efficacy. Avian pathogenic E. coli strains are often resistant to antimicrobials approved for poultry, including cephradine, tetracyclines, chloramphenicol, sulfonamides, aminoglycosides and 0-lactam antibiotics. Resistance to drugs develops quickly, for example resistance to fluoroquinolones was reported within several years of the approval of this class of drugs for use in poultry. There is reason for concern that genes conferring resistance to extended-spectrum beta-lactams will emerge in avian pathogenic E. coli strains and reduce the efficacy of ceftiofur, which is currently used on a limited basis in poultry breeding flocks and hatcheries. The great diversity’ among APEC strains limits the possibilities of vaccination, and vaccines are not used on a large scale.

[0009] Salmonella is a facultative anaerobe, rod-shaped, (about 2-3 x 0.4-0.6 pm), Gram-negative, motile, flagellated bacterium (with the exceptions of S. Gallinarum and S. Pullorum), that does not make spores, and tests negative for Gram’s staining and oxidase and which belongs to the Enterobacteriaceae2108869435.2Docket No.: 125930-872823 family. Salmonella spp is an important human pathogen and the primary clinical manifestation of salmonellosis in humans is self-limiting gastroenteritis, characterized by diarrhea, abdominal pain, fever, headache, nausea, and / or vomiting, which typically resolve within 2 to 7 days. However, in certain cases, especially among children and elderly patients, the illness can progress to a severe and lifethreatening condition, accompanied by systemic bacteremia.

[0010] Non-ty phoidal Salmonella (NTS), is one of die leading causes of foodbome disease at a global level, and the primary reservoir of NTS is the intestinal tract of warm-blooded animals, specifically livestock animals destined for food production, with the potential to lead to contaminated food products. Eating contaminated food, especially foodstuffs of animal origin, is believed to be the major transmission source of Salmonella infections to humans, and human salmonellosis is mainly attributed to contaminated food consumption, such as poultry, eggs, pork, and beef. Pork products are one of the main animal-derived foodstuffs involved in Salmonella transmission to humans.

[0011] In pigs, clinical salmonellosis has been mostly associated with two serovars: the swine adapted (but not host-restricted) serovar S. Choleraesuis. especially the Kunzendorf variant, and S. Typhimurium, including the monophasic derivative, 1,4, [5], 12:i-. S. Choleraesuis infections are in decline and conversely S. Typhimurium, including the monophasic derivative, 1,4, [5], 12:i-, infections are increasing. Salmonellosis in pigs causes significant economic losses due to increased mortality, growth retardation, and cost of treatment.

[0012] Clinical disease in pigs may be due to S. Choleraesuis infections, manifested as systemic disease with septicemia (often associated with pneumonia), and S. Typhimurium and S. 1,4, [5],12:i- infections, manifested as enteric disease, characterized by diarrhea, dehydration, decreased feed intake, fever, inanition, and wasting. Salmonella infections in pigs are frequently asymptomatic, even though fecal shedding can occur continuously or intermittently, even over long periods of time, regardless of the presence of clinical signs. Furthermore, after complete clinical recovery from infection, a percentage of pigs can act as carriers and intermittent shedders for at least five months.

[0013] Poultry derived food products, e.g., meat and eggs, are one of the main animal-derived foodstuffs involved in Salmonella transmission to humans. Avian Salmonella infections are important as both a cause of clinical disease in poultry and as a source of food-bome transmission of disease to humans. Salmonella infection is one of the most important bacterial diseases in poultry causing heavy economic losses through mortality and reduced production. The genus Salmonella is a facultative intracellular pathogen causing acute, localized or systemic infections as well as a chronic asymptomatic carrier state.

[0014] Historically the etiological agent of fowl typhoid and pullorum disease is Salmonella enterica subsp. enterica serovar Gallinarum. This serovar is divided into two distinct biovars rmder the serogroup DI. Gallinarum and Pullorum, which are denoted as S. Gallinarum and S. Pullorum, respectively and are responsible for causing fowl typhoid and pullorum disease, respectively. Although chickens are the 3108869435.2Docket No.: 125930-872823 natural hosts for the highly host adapted biovar S. Gallinarum and S. Pullorum and natural outbreaks have also been reported in turkeys, guinea fowl, quail and pheasants, these serovars do not cause disease in humans and other animals. In addition to the host adapted S. Gallinarum and Pullorum, other non-host adapted salmonellae serotypes, such as the Salmonella enterica subsp. enterica serovars Enteritidis and Typhimurium, also cause avian salmonellosis, and importantly, are capable of causing disease in humans and other animals. The success of programs, introduced in mid-20th century, designed to eradicate S. Gallinarum and S. Pullorum has resulted in the spread of the non-host adapted Salmonella serovars in the poultry industry to fill the vacant ecological niche, with adverse consequences for human health.

[0015] The epidemiology of salmonellosis in poultry, particularly with regard to transmission from one generation to the next is known to be closely associated with infected eggs. Crowding, malnutrition, and other stressful conditions as well as unsanitary surroundings can exacerbate mortality and performance losses due to salmonellosis, especially in young birds. A certain percentage of chickens that survive from the initial infection become carriers, with, or without the presence of clinical signs and pathological lesions, and bacteria from these birds can cause human disease via the food-chain, e.g., consumption of contaminated eggs and / or chicken meat.

[0016] Good hygiene and management, together with routine serological tests and slaughter policy, are currently used in efforts to control salmonellosis. Antibiotics have been used to control Salmonella infection through the use of medicated feeds, the practice of dipping hatching eggs in solutions containing antimicrobial agents and the routine inoculation of day-old poults with antibiotics. However, the rise of antibiotic resistance in bacteria means that Salmonella strains of avian origin arc often resistant to a variety of antimicrobials approved for poultry including tetracycline, oxy tetracycline, penicillin, aminoglycosides, sulfisoxazole and fluoroquinolones.

[0017] The use of vaccines in commercial poultry is increasing and due to public health problems associated with S. Enteritidis and 5. Typhimurium, these serovars are the targets of most Salmonella vaccines. Both inactivated (killed) and attenuated (live) vaccines are available; however, neither type of vaccine provides complete protection or cross-protection against all serogroups. Given that vaccines target S. Typhimurium (serogroup B) and S. Enteritidis (serogroup D), the selection pressure for other prevalent serovars, including S. Kentucky (serogroup C), may intensify. Because the efficacy of a vaccine against antigenically dissimilar serovars is reduced or absent, this strategy could enhance new problems caused by emerging serotypes by providing a vacant niche for other serovars to proliferate.

[0018] Vaccines, and antibiotics are generally not used in organic (nonconventional) poultry' production systems which instead often use probiotics and prebiotics. However, these have had limited success for various reasons. When feed is withdrawn prior to slaughter (broilers) and during molting (layers), birds are most susceptible to Salmonella colonization due to large shifts in gut and crop microbial populations. It is during these time periods that probiotics and prebiotics administered in feed either do not protect or4108869435.2Docket No.: 125930-872823 offer only limited protection of the birds. Probiotics applied in the water during this time reduce Salmonella titers but typically do not eliminate the bacteria. A reduction in probiotic effectiveness occurs partially because of the die-off of anaerobic bacteria as a result of their sensitivity to oxygen. An additional problem encountered with probiotic and prebiotic use is the ability of some Salmonella serovars to become invasive. In the initial stages of intestinal infection. Salmonella may cross the intestinal barrier into macrophages. Once inside the macrophages, Salmonella has the ability' to evade lysis, potentially leading to systemic Salmonella infection in birds.

[0019] Thus, there are problems and drawbacks with all of the available avian salmonellosis control methods. Similar problems also affect Salmonella control techniques in pigs. There is thus an unmet need for an effective non-antibiotic antimicrobial agent which targets Salmonella spp.SUMMARY OF THE INVENTION

[0020] In an aspect, provided herein is an endolysin polypeptide having antimicrobial activity, comprising an amino acid sequence of an Escherichia coli 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: 1-1075, or a fragment thereof.

[0021] In an aspect, provided herein is an endolysin polypeptide having antimicrobial activity, comprising an amino acid sequence of a peptidoglycan binding domain, wherein the peptidoglycan binding domain comprises an amino acid sequence of a fragment of the sequence set forth in any one of SEQ IDNOS:598, 602. 631, 634. 663. 677, 684. 689, 697, 698. 727, 739, 832, 860, 911. 925, 1038. 1045, or 1056; and a catalytic domain targeting the cell wall peptidoglycan catalytic domain of a gramnegative bacteria.

[0022] In an aspect, provided herein is an endolysin polypeptide having antimicrobial activity, comprising an amino acid sequence of a signal-anchor-release (SAR) domain, wherein the SAR domain comprises an amino acid sequence of a fragment of the sequence set forth in any one of SEQ ID NOS:7, 23, 288, 291, 292, 294, 306, 313, 315, 317, 318, 321, 322, 332. 333, 336, 337, 339, 340, 344, 348, 353, 357, 358, 359, 360, 362, 365, 366, 367, 368, 369, 372, 389, 396, 402. 409, 410, 413, 416, 417, 494, 527, 573, 574, 579, 582, 583, 587. 591, 593, 1073, 1074, or 1075; and a catalytic domain targeting the cell wall peptidoglycan catalytic domain of a gram-negative bacteria.

[0023] In an aspect, provided herein is an endolysin polypeptide having antimicrobial activity, comprising an amino acid sequence of a Salmonella enterica 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 5108869435.2Docket No.: 125930-872823 least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOS: 1076-1502, or 1505-1517,. or a fragment thereof, optionally any one of SEQ ID NOS:1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1152, 1232, 1506, 1158; 1505, 1416, 1507, 1187, 1364. 1097. 1166.

[0024] In an aspect, provided herein is an endolysin polypeptide having antimicrobial activity, comprising an amino acid sequence of a peptidoglycan binding domain, wherein the peptidoglycan binding domain comprises an amino acid sequence of a fragment of the sequence set forth in any one of SEQ ID NOS:1101, 1106, 1108, 1113, 1152, 1158, 1187, 1232, 1252, 1263, 1308, 1364, 1379, 1380. 1381, 1383, 1384, 1411, 1415, 1416, 1417, 1461, or 1505-1517, optionally any one ofSEQ ID NOS:1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1152, 1232, 1506, 1158; 1505, 1416, 1507, 1187, 1364, 1097, 1166; and a catalytic domain targeting the cell wall peptidoglycan catalytic domain of a gram-negative bacteria.

[0025] The endolysin polypeptide may be purified or isolated. The endolysin polypeptide may have antimicrobial activity that is endolysin activity, optionally wherein the endolysin activity is assessed by the rate of degradation of peptidoglycan (PGN).

[0026] The endolysin polypeptide may have antimicrobial activity that is bacteriolytic activity and / or bacterial growth inhibitory activity.

[0027] The antimicrobial activity of an endolysin may be bacteriolytic activity and / or bacterial growth inhibitory activity. The antimicrobial activity may be against Salmonella enterica. The antimicrobial activity may be against Pseudomonas aeruginosa or Acinetobacter baumannii. The antimicrobial activity may be determined by a cell viability assay, cell turbidity' reduction assay, minimum inhibitory concentration (MIC) assay, minimum bactericidal concentration (MBC) assay; or a focused checkerboard assay, optionally wherein the assay is performed against Escherichia coli. Salmonella enterica.Pseudomonas aeruginosa, or Acinetobacter baumannii.

[0028] In an aspect, provided herein is a composition comprising two or more endolysin polypeptides as described herein. The composition may comprise at least two endolysin domain architectures selected from a PG1 MUR domain, GH24 domain, or an Ami_2 domain. The composition may comprise at least one endolysin comprising a PG1 MUR domain and at least one endolysin comprising an Ami_2 domain: or at least one endolysin comprising GH24 domain and at least one endolysin comprising an Ami_2 domain. The composition may comprise an endolysin comprising an amino acid sequence selected from any one of SEQ ID NOS:1364, 1381, 1187, 1152, 1232, 1416, or 1158; and an endolysin comprising an amino acid sequence selected from any one of SEQ ID NOS: 1288; 1188; 1097; 1094; or 1266; or an endolysin comprising an amino acid sequence selected from any one of SEQ ID NOS: 1139, 1507. 1259. or 1166: and an endolysin comprising an amino acid sequence selected from any one of SEQ ID NOS: 1288; 1188; 1097; 1094; or 1266.6108869435.2Docket No.: 125930-872823

[0029] In an aspect, provided herein is a composition comprising the endolysin peptide as described herein, and one or more outer membrane penetrating (OMP) agent. The OMP agent may be selected from a peptide, metal ion binding agent or chelating agent. cationic steroid, polyamine, or an essential oil. The peptide may be selected from polymyxin B or colistin.

[0030] 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. A recombinant nucleic acid molecule may also comprise a first nucleic acid sequence encoding the endolysin polypeptide as described herein, and a second nucleic acid sequence encoding one or more outer membrane penetrating (OMP) agent. The OMP agent may be selected from a peptide, metal ion binding agent or chelating agent, cationic steroid, polyamine, or an essential oil. The peptide may be selected from polymyxin B or colistin.

[0031] 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, Pichia pastoris, Saccharomyces cerevisiae, or Chlamydomonas reinhardtii.

[0032] 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, Pichia pastoris. Saccharomyces cerevisiae, or Chlamydomonas reinhardtii cell.

[0033] In an aspect, provided herein is a cell lysate comprising the one or more endolysin polypeptides as described herein, optionally wherein the cell lysate further comprises one or more outer membrane penetrating (OMP) agent as described herein. The one or more endolysin polypeptides and / or OMP agent 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.

[0034] 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 further comprises one or more outer membrane penetrating (OMP) agent as described herein. The dried whole culture may be spray dried or freeze dried. The dried whole culture may comprise one or more endolysin polypeptides and / or OMP agents 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.7108869435.2Docket No.: 125930-872823

[0035] 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. The dried material may comprise one or more outer membrane penetrating (OMP) agent as described herein.

[0036] In an aspect, provided herein is a combined material comprising two or more of 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.

[0037] 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 and / or OMP agent 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.

[0038] 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, and optionally one or more outer membrane penetrating (OMP) agent 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 as described herein, a dried material as described

[0039] In an aspect, provided herein is a supplemented water comprising: (a) water; and (b) one or more endolysin polypeptides as described herein, and optionally one or more outer membrane penetrating (OMP) agent 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 as described herein, a dried material as described herein, and / or a combined material as described herein.

[0040] In an aspect, provided herein is a supplemented hydrogel comprising: (a) hydrogel; and (b) one or more endolysin polypeptides as described herein, and optionally one or more outer membrane penetrating (OMP) agent 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 as described herein, a dried material as described herein, and / or a combined material as described herein8108869435.2Docket No.: 125930-872823

[0041] In an aspect, provided herein is a method for reducing a population of gram-negative bacteria in a foodstuff, the method comprising admixing with the foodstuff an endolysin polypeptide as described herein and optionally one or more OMP agent 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 as described herein, a dried material as described herein, and / or a combined material as described herein.

[0042] 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 and optionally one or more OMP agent 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 as 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 a gram-negative bacteria. The gram-negative bacteria may be selected from Escherichia coli, Salmonella enterica. Pseudomonas aeruginosa, or Acinetobacter baumannii. The disease or disorder caused by the gram-negative bacteria may be septicemia, bacteremia, pneumonia, spondylitis, enteric infection, typhoid, or pullorum disease.

[0043] The animal subject for a method described herein may be selected from a swine, optionally a sow, a piglet, a hog, or any other pig, further optionally Sus scrofa domesticus; a ruminant, including cattle, sheep, horses, deer and goats, preferably Bos taunts, and further optionally Bos indicus, a chicken, optionally a broiler chicken or a layer chicken, further optionally Gallus gallus domesticus; a turkey, optionally a domestic turkey, further optionally Meleagris gallopavo domesticus; a duck, optionally a domestic duck, further optionally a domestic duck derived from a mal-lard duck or a domestic duck derived from a Muscovy duck; 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; a pigeon, optionally a domestic pigeon; or a quail, optionally a domestic quail. The animal subject may be a swine, optionally a sow, a piglet, a hog, or any other pig, further optionally Sus scrofa domesticus, and the disease or disorder may be post-weaning diarrhoea (PWD). The animal subject may be a poultry animal, and the disease or disorder may be selected from avian colibacillosis. yolk sac infection, omphalitis, respiratory tract infection, swollen head syndrome, septicemia, polyserositis, coligranuloma. enteritis, cellulitis, or salpingitis.

[0044] 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 and optionally one or more OMP agent 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 described9108869435.2Docket No.: 125930-872823 herein, and / or a combined material as described herein, and instructions for admixing the agent with a foodstuff.BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 depicts a DNA fragment used in the transformation of strain bAL265 for expression of endolysin. The AmyE gene in strain bAL265 was disrupted by the integration of a gene that confers resistance to the antibiotic spectinomycin. The DNA fragment that carries the endolysin is flanked by sequences of homology to the AmyE gene (RHA: right homologous arm; LHA: left homologous arm). The DNA fragment also carried 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 (lad).

[0046] FIG. 2 shows an SDS PAGE gel showing lysates (soluble cell extracts) of IPTG induced Bacillus subtilis transformants comprising various endolysin expression constructs. Samples were loaded are as follows: lane 1 = size standard markers; lane 2 = bAL265 (negative control); lane 3 = bAL526; lane 4 = bAL527; lane 5 = bAL528; lane 6 = bAL529; lane 7 = bAL530; lane 8 = bAL531; lane 9 = bAL532; lane 10 = bAL533; lane 11 = bAL534; lane 12 = bAL535; lane 13 = bAL536; lane 14 = bAL537; lane 15 = bAL265 (negative control). Lanes containing an expressed endolysin band are marked with a “Y" whilst lanes which do not contain an expressed endolysin band are marked “N’‘.

[0047] FIG. 3 shows an SDS PAGE gel showing lysates (soluble cell extracts) of IPTG induced Bacillus subtilis transformants comprising various endolysin expression constructs. Samples loaded are as follows: lane 1 = size standard markers; lane 2 = bAL265 (negative control); lane 3 = bAL443; lane 4 = bAL444; lane 5 = bAL539; lane 6 = bAL540; lane 7 = bAL541; lane 8 = bAL542; lane 9 = bAL543; lane 10 = bAL544; lane 11 = bAL545; lane 12 = bAL547; lane 13 = bAL265 (negative control). Lanes containing an expressed endolysin band are marked with a “Y” whilst lanes which do not contain an expressed endolysin band are marked “N”. Sample 12 (bAL547) is marked “N*” as although it contains an overexpressed band the protein is considerably smaller in size than expected.

[0048] FIG. 4 shows an SDS PAGE gel showing lysates (soluble cell extracts) of IPTG induced Bacillus subtilis transformants comprising various endolysin expression constructs. Samples loaded are as follows: lane 1 = size standard markers; lane 2 = bAL265 (negative control); lane 3 = bAL552; lane 4 = bAL553; lane 5 = bAL554; lane 6 = bAL555; lane 7 = bAL556; lane 8 = bAL557; lane 9 = bAL558; lane 10 = bAL559; lane 11 = bAL560; lane 12 = bAL561; lane 13 = bAL562; lane 14 = bAL563; lane 15 = bAL564; lane 16 = bAL565; lane 17 = bAL566; lane 18 = bAL567; lane 19 = bAL265 (negative control); lane 20 = size standard markers. Lanes containing an expressed endolysin band are marked with a " Y” whilst lanes which do not contain an expressed endolysin band are marked “N”.10108869435.2Docket No.: 125930-872823

[0049] FIG. 5 shows a schematic diagram depicting microplate setup for the Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate validation assay. The values shown in the wells indicate the dilution factor used to dilute the lysate present in each well. Wells 1-10 of each row each comprise 50 µl of diluted lysate and 50 µl of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate. Wells 11-12 of each row each comprise 50 µl of PBS and 50 µl of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate. The plate shows the following lysates tested in rows: Row 1 = bAL265; Row 2 = bAL526; Row 3 = bAL527; Row 4 = bAL529; Row 5 = bAL532; Row 6 = bAL534; Row 7 = bAL535; Row 8 = bAL539.

[0050] FIG. 6 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL265. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL265 lysate diluted 200X is indicated by the “triangle” marked line; data for bAL265 lysate diluted 102.400X is indicated by the “circle” marked line.

[0051] FIG. 7 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL526. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL526 lysate diluted 200X is indicated by the “triangle” marked line; data for bAL526 lysate diluted 102,400X is indicated by the “circle” marked line.

[0052] FIG 8 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL527. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL527 lysate diluted 200X is indicated by the “triangle” marked line; data for bAL527 lysate diluted 102,400X is indicated by the “circle” marked line.

[0053] FIG. 9 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL529. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL529 lysate diluted 200X is indicated by the “triangle” marked line; data for bAL529 lysate diluted 102,400X is indicated by the “circle” marked line.

[0054] FIG 10 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL532. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL532 lysate diluted 200X is indicated by the “triangle” marked line; data for bAL532 lysate diluted 102,400X is indicated by the “circle” marked line.

[0055] FIG. 11 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL534. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL534 lysate diluted 200X is indicated by the “triangle” marked line; data for bAL534 lysate diluted 102,400X is indicated by the “circle” marked line.

[0056] FIG 12 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL535. Data for the negative control (PBS) is indicated by the “cross” marked line;11108869435.2Docket No.: 125930-872823 data for bAL535 lysate diluted 200X is indicated by the “triangle” marked line; data for bAL535 lysate diluted 102,400X is indicated by the “circle” marked line.

[0057] FIG. 13 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL539. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL539 lysate diluted 200X is indicated by die “triangle” marked line; data for bAL539 lysate diluted 102,400X is indicated by die “circle” marked line.

[0058] FIG 14 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL541. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL541 lysate diluted 200X is indicated by the “triangle” marked line; data for bAL541 lysate diluted 102.400X is indicated by the “circle” marked line.

[0059] FIG. 15 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL542. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL542 lysate diluted 200X is indicated by die “triangle” marked line; data for bAL542 lysate diluted 102,400X is indicated by the “circle” marked line.

[0060] FIG 16 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL545. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL545 lysate diluted 200X is indicated by the “triangle” marked line; data for bAL545 lysate diluted 102.400X is indicated by the “circle” marked line.

[0061] FIG. 17 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL561. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL561 lysate diluted 200X is indicated by the “triangle” marked line; data for bAL561 lysate diluted 102,400X is indicated by the “circle” marked line.

[0062] FIG 18 shows results of Salmonella enterica subsp. enterica serotype Enteriditis peptidoglycan substrate assay for bAL564. Data for the negative control (PBS) is indicated by the “cross” marked line; data for bAL564 lysate diluted 200X is indicated by the “triangle” marked line; data for bAL564 lysate diluted 102.400X is indicated by the “circle” marked line.

[0063] FIG. 19 shows a schematic diagram depicting microplate setup for the MIC assay. The wells of Block 1 (rows A & B) contain a final concentration of 0.09 pg / ml polymyxin, wells of Block 2 (rows C & D) contain a final concentration of 0.09 pg / ml polymyxin, wells of Block 3 (rows E & F) contain a final concentration of 0.09 pg / ml polymyxin and wells of Block 4 (rows G & H) contain a final concentration of 0.09 pg / ml polymyxin. The values shown in the wells of Block 1 show the final concentrations of lysozyme in each well (mg / ml). The values shown in the wells of Block 2 show the dilution factors of the stock solutions of cell extract / lysate added to each well (the final dilution factors in each well will be 4X higher once the other well components are added). The final three control wells of 12108869435.2Docket No.: 125930-872823 each test block which do not contain lysozyme,or soluble cell extract solution, and which are negative control wells are grey shaded.

[0064] FIG. 20 shows a Clustal alignment of tested endolysin sequences of SEQ ID NO: 1232 (SalEL-5); SEQ ID NO: 1416 (SalEL-6); SEQ ID NO: 1158 (SalEL-7); SEQ ID NO: 1152 (SalEL-4);SEQ ID NO: 1505 (SalEL-2A); and SEQ ID NO: 1381 (SalEL-2) for the purpose of consensus sequence generation.

[0065] FIG. 21 shows a Clustal alignment of tested endolysin sequences of SEQ ID NO: 1232 (SalEL-5); SEQ ID NO: 1506 (SalEL-5A); SEQ ID NO: 1152 (SalEL-4); and SEQ ID NO: 1158 (SalEL-7) for the purpose of consensus sequence generation.DETAILED DESCRIPTION OF THE INVENTIONBacteriophage endolysins

[0066] 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. An endolysin as described herein may comprise a catalytic domain which provides antimicrobial activity (e.g., a cell wall or peptidoglycan cleaving domain). Endolysins as described herein can have antimicrobial effects when applied externally to gramnegative bacteria (e.g., E. coli, S. enterica, etc.). Endolysins that are specific for gram-negative bacteria may comprise one or more catalytic domain. In some embodiments, an endolysin is not modular, or is without other domains. Endolysins specific for gram-negative bacteria may comprise or consist of one or more catalytic domains and not comprise a binding domain. Such endolysins lacking a binding domain can rely on the catalytic domain to serve the purposes of target recognition and catalytic cleavage (e.g., be multifunctional). Endolysins that are specific for gram-negative bacteria may also have a modular organization. Modular-organized endolysins may have a multi-domain structure (e.g., at least a two-domain structure having a catalytic domain and a binding domain (e.g., cell wall binding domain) or signal-anchor-release (SAR) domain, optionally connected by a linker). Endolysins having different domain architectures have also been discovered, and are contemplated for use herein.

[0067] The 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 the13108869435.2Docket No.: 125930-872823 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. The catalytic domain may also have an additional function, such as a means for targeting a specific bacteria or functioning as a binding domain, or a means for functioning as a signal-anchor-release (SAR) domain.

[0068] An endolysin as described herein may have activity specific for a particular bacterial species. Alternatively, an endolysin may have non-specific activity, such as being promiscuous and / or capable of degrading the peptidoglycan cell wall of other bacterial species. For example, an endolysin as described herein may have activity specific for Escherichia coli, but may also target, and have activity against Salmonella enterica. An endolysin as described herein may have activity specific for Salmonella enterica, but may also target, and have activity against Escherichia coli. An endolysin as described herein may possess bioactivity against a large number of Gram-negative bacterial species, e.g..Pseudomonas aeruginosa, Acinetobacter baumannii. etc.

[0069] The binding domain (e.g., cell wall binding domain), if present, may confer specificity of the endolysin against its target species or genera. The cell wall binding 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. Notably, a catalytic domain may also have an influence on the specificity of an endolysin. The peptidoglycan structures differ betw een 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. Multiple binding domains may be present in an endolysin and may be arranged adjacent to one another, in series. An endolysin polypeptide sequence comprising this tandem arrangement of multiple binding domains is described herein as comprising a “peptidoglycan cell wall binding superdomain” or as comprising a “cell wall binding superdomain” or as comprising a “binding superdomain”.

[0070] 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 y, 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 w ide spectrum of action and the latter can be useful for diagnostics. The EAD (enzymatically active domain) is the catalytic domain which degrades peptidoglycan (PG). The catalytic domain can also feature a binding component, or a component for signal anchor release (SAR). In case of single-domain globular endolysins from the phages infecting gram-negative bacteria,14108869435.2Docket No.: 125930-872823 both functions (recognition and hydrolysis) may be carried out by the same domain - meaning that spatially, these functions cannot be differentiated too much. Endolysins targeting gram-negative bacteria may also function as signal anchor release (SAR) domains.

[0071] The inventors have identified and characterised bacteriophage-derived endolysins with high specificity and bacteriolytic activity against a broad spectrum of gram-negative strains, such as Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Acinetobacter baumannii, etc.Endolysin Polypeptides, Fragments and Variants Thereof

[0072] 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 acid sequence set forth in any one of the SEQ ID NOs of the disclosure (such as disclosed in the Sequence Listing), 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 against gram-negative bacteria (e.g., Escherichia coli, Salmonella enterica. Pseudomonas aeruginosa. Acinetobacter baumannii, and other gram-negative species and their different strains). Escherichia coli may be referred to herein as “ coli” or “Ec”. Salmonella enterica may be referred to herein as “S. enterica” or “Se”; Pseudomonas aeruginosa may be referred to herein as “P. aeruginosa” or “Pa”, and Acinetobacter baumannii may be referred to herein as “A. baumannii” or “Ab”.

[0073] 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 domain contained therein, 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 the Sequence Listing) 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 a gram-negative bacteria, such as described herein. The variant endolysin may have antibacterial activity against Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Acinetobacter baumannii, or other gram-negative bacteria, 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.15108869435.2Docket No.: 125930-872823

[0074] 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 the Sequence Listing. 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

[0075] An endolysin as described herein may comprise a cell wall peptidoglycan catalytic domain that targets gram-negative bacteria (e.g., Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Acinetobacter baumannii, or other), also referred to herein as a peptidoglycan catalytic domain or as a catalytic domain. An endolysin may comprise one or more 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: 1-1502, or a variant thereof having at least 80% or greater sequence identity to any one of SEQ ID NOS:1-1502, or a fragment thereof.

[0076] An endolysin as described herein may comprise a binding domain (e.g.. cell wall binding domain), which is a peptidoglycan binding domain. In some embodiments, the binding domain of an endolysin may be distinct from the catalytic domain (e.g., a separate amino acid sequence). In some embodiments, the binding domain of an endolysin is not distinct from the catalytic domain. In some embodiments, the amino acid sequence of the endolysin may be multi-functional, for instance it performs both catalytic activity an confers target recognition and binding functions of a binding domain. A binding domain, whether it be from a multi-functional amino acid sequence or a separate domain, may confer target specificity and / or ensure that the endolysin binds to and degrades the cell wall of a specific bacteria species. The specific degradation and antimicrobial effect of the amino acid sequence may be against a specific bacteria species, or may be promiscuous. Endolysins having an amino acid sequence functioning as a peptidoglycan binding domain, and which targets or binds to E. coli may be selected from any one of SEQ ID NOS:598, 602. 631, 634, 663, 677, 684, 689, 697, 698. 727, 739, 832. 860, 911, 925. 1038. 1045. or 1056. Endolysins having an amino acid sequence functioning as a peptidoglycan binding domain, and which target or binds to Salmonella spp. may be selected from any one of SEQ ID NOS:1101, 1106, 1108, 1113, 1152, 1158, 1187, 1232, 1252, 1263, 1308, 1364. 1379, 1380, 1381, 1383, 1384, 1411, 1415, 1416, 1417, or 1461.

[0077] An endolysin as described herein may comprise a signal-anchor-release (SAR) domain. In some embodiments, an endolysin comprising a SAR domain does not require the presence of additional outer membrane penetrating agents to function. A SAR domain is typically located at the N-terminus of certain endolysin sequences described herein. A SAR domain may comprise a transmembrane region. The SAR 16108869435.2Docket No.: 125930-872823 domain may be rich in glycine and alanine, and / or have a low content of basic amino acids. The SAR domain may enable the endolysin to accumulate in the cytoplasmic membrane of the host bacterium in an inactive form. Upon activation, the SAR domain may facilitate the release of the endolysin into the periplasmic space, where it can degrade the bacterial cell wall. Unlike some endolysins that require holins to disrupt the bacterial membrane, SAR endolysins can be released through a signal-anchor mechanism. This SAR process may involve the endolysin being tethered to the membrane of the bacteria until a specific signal triggers its release. Endolysins having an amino acid sequence functioning as a SAR domain may be selected from any one of SEQ ID NOS:7, 23, 288. 291, 292, 294, 306, 313, 315, 317, 318, 321, 322, 332, 333, 336, 337, 339, 340, 344, 348, 353, 357, 358, 359, 360, 362, 365, 366, 367, 368, 369, 372, 389, 396, 402, 409, 410, 413, 416, 417, 494, 527, 573, 574, 579, 582, 583, 587, 591, 593, 1073, 1074 or 1075. A SAR domain as described herein may be portion of any one of SEQ ID NOS:7, 23, 288, 291, 292, 294, 306, 313, 315, 317, 318, 321, 322, 332, 333, 336, 337, 339, 340, 344, 348, 353, 357, 358, 359, 360, 362, 365. 366, 367, 368, 369, 372, 389, 396, 402, 409, 410, 413, 416, 417, 494, 527, 573, 574, 579, 582, 583, 587, 591, 593, 1073, 1074 or 1075 that imparts SAR functionality.

[0078] An endolysin as described herein may comprise a binding domain, which is a cell wall binding domain (e.g., peptidoglycan binding domain). An endolysin comprising a binding domain, but not a 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 binding domains (e g., two. three, four, or more).

[0079] A “superdomain” is a domain which comprises two domains with similar / idcntical functionality arranged consecutively, or in series, w ith one another. A superdomain may comprise two or more catalytic domains arranged consecutively, or in series, with one another. A superdomain may comprise two or more peptidoglycan binding domains arranged consecutively, or in series, with one another An endolysin as described herein may comprise a superdomain, which is a cell wall binding superdomain (e.g., peptidoglycan binding superdomain). An endolysin comprising a superdomain 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 superdomains (e.g., two. three, four, or more peptidoglycan and / or cell wall binding superdomains (e.g., a mixture of two)).

[0080] An endolysin as described herein may comprise a catalytic domain (e.g., cell wall peptidoglycan catalytic domain) for gram-negative bacteria and either a binding domain or a superdomain as described herein. An endolysin may comprise one or more catalytic domain (e.g.. cell wall peptidoglycan catalytic domains) for gram-negative bacteria (e.g.. two, three, four, or more) and either one or more gramnegative bacteria cell wall peptidoglycan binding domains (e.g., two, three, four, or more), or one or more binding superdomains (e.g., two. three, four, or more cell wall peptidoglycan binding superdomains).17108869435.2Docket No.: 125930-872823

[0081] An endolysin as described herein may further comprise one or more linkers connecting the catalytic domain and the binding domain or SAR domain, if present, and / or one or more linkers connecting multiple catalytic domains, when present, and / or one or more linkers connecting multiple binding domains, when present, and / or one or more linkers connecting multiple SAR domains, when present,.

[0082] 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 tenn “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.Endolysins Comprising Catalytic Domains

[0083] 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: 1-1502. 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. For example, the catalytic domain may also have a binding functionality in addition to its catalytic function. The binding functionality of the catalytic domain can be either specific (e.g.. to a particular species, genera, or strain of bacteria) or nonspecific (e.g., the binding is promiscuous for more than one species, genera, or strain of bacteria). The catalytic domain may have influence on the release of the endolysin into the bacteria (e.g., into the periplasmic space). For example, the catalytic domain may also have a SAR functionality in addition to its catalytic function. The catalytic domain, or a fragment thereof, may have N-acetyl-0-D-glucosaminidase activity, N-acetyl-P-D-muramidase activity’. N-acetylmuramoyl-L-alanine amidase activity and / or endopeptidase activity. SEQ ID NOS:1-1502 represent amino acid sequences having peptidoglycan catalytic functional activity (e.g., catalytic domains) and are contemplated for use as catalytic domains, in whole, or in part (e.g., when a binding domain or SAR domain is present in the sequence).

[0084] In some embodiments, an endolysin polypeptide comprises an Escherichia coli 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: 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, 26, 27. 28, 29, 30, 31. 32, 33, 34, 35, 36. 37, 38, 39, 40. 41, 42, 43, 44, 45, 46. 47, 48, 49, 50. 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, 76, 77, 78. 79. 80, 81, 82, 83. 84, 85, 86, 87. 88, 89, 90, 91, 92. 93, 94, 95, 96. 97, 98,18108869435.2Docket No.: 125930-872823 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119.120. 121, 122, 123. 124, 125, 126, 127, 128. 129, 130, 131. 132, 133, 134. 135, 136, 137, 138, 139. 140, 141. 142, 143, 144. 145, 146, 147, 148, 149. 150, 151, 152. 153, 154, 155. 156, 157, 158, 159, 160. 161, 162. 163, 164, 165, 166, 167, 168, 169, 170. 171, 172, 173. 174, 175, 176, 177, 178, 179, 180, 181. 182, 183. 184, 185, 186, 187, 188, 189, 190, 191. 192, 193, 194. 195, 196, 197, 198, 199, 200, 201, 202. 203, 204. 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223. 224, 225, 226, 227, 228, 229, 230, 231, 232, 233. 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254. 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356. 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377. 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398. 399, 400, 401, 402, 403, 404, 405, 406. 407, 408, 409, 410, 411, 412, 413, 414, 415, 416.417, 418, 419. 420, 421, 422, 423, 424, 425, 426, 427. 428, 429, 430, 431, 432, 433, 434, 435, 436, 437. 438, 439, 440. 441, 442, 443, 444, 445, 446, 447, 448. 449, 450, 451, 452, 453, 454, 455, 456, 457, 458. 459, 460, 461.462, 463, 464, 465, 466. 467, 468, 469. 470, 471, 472, 473, 474, 475, 476, 477, 478, 479. 480, 481, 482. 483, 484, 485, 486, 487. 488, 489, 490. 491, 492, 493, 494, 495, 496, 497, 498, 499, 500. 501, 502, 503. 504, 505. 506, 507, 508. 509, 510, 511. 512, 513, 514, 515, 516. 517, 518, 519, 520, 521.522, 523, 524. 525, 526. 527, 528, 529. 530, 531, 532. 533, 534, 535, 536, 537. 538, 539, 540, 541, 542. 543, 544. 545. 546, 547. 548, 549, 550. 551, 552, 553. 554, 555. 556, 557, 558. 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582. 583, 584, 585. 586, 587, 588, 589, 590. 591, 592, 593. 594, 595, 596, 597, 598, 599, 600, 601. 602, 603. 604, 605, 606. 607, 608, 609, 610, 611. 612, 613, 614. 615, 616, 617. 618, 619, 620. 621, 622. 623, 624. 625, 626, 627. 628, 629, 630, 631, 632. 633, 634, 635. 636, 637, 638. 639, 640, 641, 642, 643. 644, 645. 646, 647, 648. 649, 650, 651, 652, 653. 654, 655, 656. 657, 658, 659. 660, 661, 662, 663, 664. 665, 666. 667, 668, 669, 670, 671, 672, 673, 674. 675, 676, 677. 678, 679, 680, 681, 682, 683, 684, 685. 686, 687. 688, 689, 690, 691, 692, 693, 694, 695. 696, 697, 698. 699, 700, 701, 702, 703, 704, 705, 706. 707, 708. 709, 710, 711, 712, 713, 714, 715, 716. 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727. 728, 729, 730, 731, 732, 733, 734, 735, 736, 737. 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758. 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860. 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881. 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896,19108869435.2Docket No.: 125930-872823 897. 898, 899, 900. 901, 902, 903, 904, 905. 906, 907, 908. 909, 910, 911. 912, 913, 914, 915, 916. 917, 918. 919, 920, 921. 922, 923, 924, 925, 926. 927, 928, 929. 930, 931, 932. 933, 934, 935, 936, 937. 938, 939. 940, 941, 942. 943, 944, 945, 946, 947. 948, 949, 950. 951, 952, 953. 954, 955, 956, 957, 958. 959, 960. 961, 962, 963, 964, 965, 966, 967, 968. 969, 970, 971. 972, 973, 974, 975, 976, 977, 978, 979. 980, 981. 982, 983, 984, 985, 986, 987, 988, 989. 990, 991, 992. 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015. 1016. 1017.1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034.1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051.1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068.1069, 1070, 1071, 1072, 1073, 1074, or 1075. 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, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42. 43, 44, 45, 46, 47, 48, 49, 50, 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, 76, 77, 78, 79. 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91.92. 93. 94. 95.96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113.114, 115, 116. 117, 118, 119. 120, 121, 122, 123, 124. 125, 126, 127. 128, 129, 130, 131, 132, 133, 134, 135, 136, 137. 138, 139, 140. 141, 142, 143, 144, 145. 146, 147, 148. 149, 150, 151, 152, 153. 154, 155, 156, 157, 158. 159, 160, 161. 162, 163. 164, 165, 166. 167, 168, 169. 170, 171, 172, 173, 174. 175, 176, 177, 178, 179. 180, 181, 182. 183, 184. 185, 186, 187. 188, 189, 190. 191, 192, 193, 194, 195. 196, 197, 198, 199, 200. 201, 202. 203. 204, 205. 206, 207, 208. 209, 210, 211. 212, 213. 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240. 241, 242, 243. 244, 245, 246, 247, 248. 249, 250, 251. 252, 253, 254, 255, 256, 257, 258, 259. 260, 261. 262, 263, 264. 265, 266, 267. 268, 269. 270, 271, 272, 273, 274, 275. 276, 277, 278, 279, 280. 281, 282. 283, 284, 285. 286, 287, 288, 289, 290. 291, 292, 293. 294, 295, 296. 297, 298, 299, 300, 301. 302, 303. 304, 305, 306. 307, 308, 309, 310, 311. 312, 313, 314. 315, 316, 317. 318, 319, 320, 321, 322. 323, 324. 325, 326, 327, 328, 329, 330, 331, 332. 333, 334, 335. 336, 337, 338, 339, 340, 341, 342, 343. 344, 345. 346, 347, 348, 349, 350, 351, 352, 353. 354, 355, 356. 357, 358, 359, 360, 361, 362, 363, 364. 365, 366, 367, 368, 369, 370, 371, 372, 373, 374. 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385. 386, 387, 388, 389, 390, 391, 392, 393, 394, 395. 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416. 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518. 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539. 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554,20108869435.2Docket No.: 125930-872823 555. 556, 557, 558. 559, 560, 561, 562, 563. 564, 565, 566. 567, 568, 569. 570, 571, 572, 573, 574. 575, 576. 577, 578, 579. 580, 581, 582, 583, 584. 585, 586, 587. 588, 589, 590. 591, 592, 593, 594, 595. 596, 597. 598, 599, 600. 601, 602, 603, 604, 605. 606, 607, 608. 609, 610, 611. 612, 613, 614, 615, 616. 617, 618. 619, 620, 621, 622, 623, 624, 625, 626. 627, 628, 629. 630, 631, 632, 633, 634, 635, 636, 637. 638, 639. 640, 641, 642, 643, 644, 645, 646, 647. 648, 649, 650. 651, 652, 653, 654, 655, 656, 657, 658. 659, 660. 661, 662, 663, 664, 665, 666, 667, 668. 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679. 680, 681, 682, 683, 684, 685, 686, 687, 688, 689. 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710. 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812. 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833. 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862. 863, 864, 865, 866, 867, 868, 869, 870, 871, 872. 873, 874, 875. 876, 877, 878, 879, 880, 881, 882, 883. 884, 885, 886, 887, 888, 889, 890, 891, 892, 893. 894, 895, 896. 897, 898, 899, 900, 901, 902, 903, 904. 905, 906, 907, 908, 909, 910, 911, 912, 913, 914. 915, 916, 917. 918, 919, 920, 921, 922. 923, 924, 925. 926, 927, 928, 929, 930, 931, 932, 933, 934, 935. 936, 937, 938. 939, 940, 941, 942, 943. 944, 945, 946. 947, 948, 949, 950, 951, 952, 953, 954, 955, 956. 957, 958, 959. 960, 961. 962, 963, 964. 965, 966, 967. 968, 969, 970, 971, 972. 973, 974, 975, 976, 977. 978, 979, 980. 981, 982. 983, 984, 985. 986, 987, 988. 989, 990, 991, 992, 993. 994, 995, 996, 997, 998. 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006. 1007. 1008. 1009. 1010. 1011. 1012. 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042. 1043. 1044. 1045. 1046.1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057. 1058. 1059. 1060. 1061. 1062. 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074. or 1075, or a fragment thereof each.

[0085] For a fragment of said amino acid sequences, 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, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40. 41, 42, 43, 44, 45, 46, 47, 48, 49. 50, 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, 76, 77. 78, 79, 80, 81, 82, 83, 84, 85, 86. 87, 88, 89, 90, 91, 92. 93, 94, 95, 96. 97, 98, 99, 100, 101, 102, 103, 104, 105, 106. 107, 108, 109. 110, 111, 112, 113, 114, 115. 116, 117, 118. 119, 120, 121, 122, 123, 124, 125, 126. 127, 128, 129, 130, 131, 132, 133, 134, 135, 136. 137, 138, 139. 140, 141, 142, 143, 144. 145, 146, 147. 148, 149, 150, 151, 152, 153, 154, 155, 156, 157. 158, 159, 160. 161, 162, 163, 164, 165. 166, 167, 168. 169, 170, 171, 172, 173. 174, 175, 176, 177, 178. 179, 180, 181. 182, 183. 184, 185, 186. 187, 188, 189. 190, 191, 192, 193, 194. 195, 196,21108869435.2Docket No.: 125930-872823 197. 198, 199, 200. 201, 202, 203, 204, 205. 206, 207, 208. 209, 210, 211. 212, 213, 214, 215, 216, 217, 218. 219, 220, 221. 222, 223, 224, 225, 226. 227, 228, 229. 230, 231, 232. 233, 234, 235, 236, 237. 238, 239. 240, 241, 242. 243, 244, 245, 246, 247. 248, 249, 250. 251, 252, 253. 254, 255, 256, 257, 258. 259, 260. 261, 262, 263, 264, 265, 266, 267, 268. 269, 270, 271. 272, 273, 274, 275, 276, 277, 278, 279. 280, 281. 282, 283, 284, 285, 286, 287, 288, 289. 290, 291, 292. 293, 294, 295, 296, 297, 298, 299, 300. 301, 302. 303, 304, 305, 306, 307, 308, 309, 310. 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321. 322, 323, 324, 325, 326, 327, 328, 329, 330, 331. 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352. 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454. 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475. 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496. 497, 498, 499, 500, 501, 502, 503, 504. 505, 506, 507, 508, 509, 510, 511, 512, 513, 514.515, 516, 517. 518, 519, 520, 521, 522, 523, 524, 525. 526, 527, 528, 529, 530, 531, 532, 533, 534, 535. 536, 537, 538. 539, 540, 541, 542, 543, 544, 545, 546. 547, 548, 549, 550, 551, 552, 553, 554, 555, 556. 557, 558, 559. 560, 561, 562, 563, 564. 565, 566, 567. 568, 569, 570, 571, 572, 573, 574, 575, 576, 577. 578, 579, 580. 581, 582, 583, 584, 585. 586, 587, 588. 589, 590, 591, 592, 593, 594, 595, 596, 597, 598. 599, 600, 601. 602, 603. 604, 605, 606. 607, 608, 609. 610, 611, 612, 613, 614. 615, 616, 617, 618, 619. 620, 621, 622. 623, 624. 625, 626, 627. 628, 629, 630. 631, 632, 633, 634, 635. 636, 637, 638, 639, 640. 641, 642. 643. 644, 645. 646, 647, 648. 649, 650, 651. 652, 653. 654, 655, 656. 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680. 681, 682, 683. 684, 685, 686, 687, 688. 689, 690, 691. 692, 693, 694, 695, 696, 697, 698, 699. 700, 701. 702, 703, 704. 705, 706, 707, 708, 709. 710, 711, 712. 713, 714, 715. 716, 717, 718, 719, 720. 721, 722. 723, 724, 725. 726, 727, 728, 729, 730. 731, 732, 733. 734, 735, 736. 737, 738, 739, 740, 741. 742, 743. 744, 745, 746. 747, 748, 749, 750, 751. 752, 753, 754. 755, 756, 757. 758, 759, 760, 761, 762. 763, 764. 765, 766, 767, 768, 769, 770, 771, 772. 773, 774, 775. 776, 777, 778, 779, 780, 781, 782, 783. 784, 785. 786, 787, 788, 789, 790, 791, 792, 793. 794, 795, 796. 797, 798, 799, 800, 801, 802, 803, 804. 805, 806. 807, 808, 809, 810, 811, 812, 813, 814. 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825. 826, 827, 828, 829, 830, 831, 832, 833, 834, 835. 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856. 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958. 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979. 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994,22108869435.2Docket No.: 125930-872823 995. 996, 997, 998. 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006. 1007. 1008. 1009. 1010. 1011. 1012.1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023. 1024. 1025. 1026. 1027. 1028. 1029.1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041. 1042. 1043. 1044. 1045. 1046.1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059. 1060. 1061. 1062. 1063.1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, or 1075, when each sequence is aligned to each other according to their full length.

[0086] In some embodiments, an endolysin polypeptide comprises an Salmonella enterica 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: 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116. 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133. 1134. 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150. 1151. 1152. 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167. 1168. 1169. 1170. 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212. 1213. 1214. 1215. 1216. 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229. 1230. 1231. 1232. 1233. 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245. 1246. 1247. 1248. 1249. 1250. 1251.1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263. 1264. 1265. 1266. 1267. 1268.1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281. 1282. 1283. 1284. 1285.1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299. 1300. 1301. 1302.1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318. 1319.1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335. 1336.1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353.1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370.1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512. 1513, 1514, 1515, 1516, or 1517. 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 23108869435.2Docket No.: 125930-872823 least 98%. at least 99% or 100% identical to the amino acid sequence set forth in any one ofSEQ ID NOS: 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088. 1089. 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101. 1102. 1103. 1104. 1105. 1106.1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120. 1121. 1122. 1123.1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137. 1138. 1139. 1140.1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155. 1156. 1157.1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174.1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191.1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379. 1380. 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396. 1397. 1398. 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413. 1414. 1415. 1416. 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459. 1460. 1461. 1462. 1463.1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473. 1474. 1475. 1476. 1477. 1478. 1479. 1480.1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491. 1492. 1493. 1494. 1495. 1496. 1497.1498, 1499, 1500, 1501, 1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511. 1512. 1513. 1514. 1515. 1516. or 1517. or a fragment thereof each.

[0087] For a fragment of said amino acid sequences, 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 ofSEQ ID NOS:1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089. 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141. 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158. 1159. 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174,24108869435.2Docket No.: 125930-872823 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202. 1203. 1204. 1205. 1206. 1207. 1208.1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220. 1221. 1222. 1223. 1224. 1225.1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238. 1239. 1240. 1241. 1242.1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256. 1257. 1258. 1259.1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274. 1275. 1276.1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293.1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310.1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327.1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498. 1499. 1500, 1501, 1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517, when each sequence is aligned to each other according to their full length.

[0088] In some embodiments, a fragment of any one of SEQ ID NOS:1-1502 comprises the amino acid sequence corresponding to the catalytic domain, binding domain, or SAR domain of any one of SEQ ID NOS:1-1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514. 1515. 1516, 1517. In some embodiments, a fragment of any one of SEQ ID NOS:1-1502 excludes the amino acid sequence corresponding to the catalytic domain, binding domain, or SAR domain of any one of SEQ ID NOS: 1-1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517.

[0089] 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.

[0090] In some embodiments, the endolysin polypeptide having antimicrobial activity comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 1-1075, or a variant thereof each.

[0091] Also provided is a combination of endolysin sequences as described herein, comprising two or more different endolysin polypeptides, or fragments or variants thereof. The different endolysin polypeptides present in a combination may be present as separate polypeptides, e.g.. the combination25108869435.2Docket No.: 125930-872823 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.

[0092] 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 any one of SEQ ID NO:1-1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517, or a fragment thereof each, 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: 1-1502, 1505, 1506, 1507, 1508, 1509. 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517, or a fragment thereof each.Endolysins Comprising Catalytic Domains and Binding Domains

[0093] An endolysin as described herein may comprise a catalytic domain (e.g., a bacterial cell wall peptidoglycan catalytic domain) and either a binding domain (e.g., a bacterial cell wall binding domain) or a binding superdomain, wherein the endolysin comprises the amino acid sequence set forth in any one of SEQ ID NOS: 1-1502. In some embodiments, an amino acid sequence selected from any one of SEQ ID NOS: 1-1502 contains both the catalytic domain and binding domain. Certain catalytic domains described herein can target the cell walls of certain bacteria specifically or non-specifically, and as such the catalytic domains described herein may be utilized for their binding function as part of a larger endolysin molecule. For instance, the amino acid sequences of any one of SEQ ID NOS: 1-1075 may be used to target Escherichia coli specifically, and optionally can be part of a larger endolysin molecule. The amino acid sequences of any one of SEQ ID NOS:1076-1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517, may be used to target Salmonella enterica specifically, and optionally can be part of a larger endolysin molecule. In other words, an endolysin may contain another catalytic mechanism in addition to a targeting or binding domain provided for the amino acid sequence set forth in any one of SEQ ID NOS: 1-1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517.

[0094] A binding domain as described herein may be part of any one of the endolysin amino acid sequences provided described herein. Each of SEQ ID NOS:598, 602, 631, 634, 663, 677. 684, 689, 697, 698, 727, 739, 832, 860, 911. 925, 1038, 1045, and 1056 feature a peptidoglycan binding domain which can bind to the cell wall oi Escherichia colt. Each of SEQ ID NOS: 1101. 1106. 1108, 1113, 1152, 1158, 1187, 1232, 1252, 1263, 1308, 1364, 1379, 1380, 1381, 1383, 1384, 1411, 1415, 1416, 1417, and 1461 feature a peptidoglycan binding domain which can bind to the cell wall of Salmonella enterica. In some embodiments, an endolysin polypeptide comprises a catalytic domain (e.g., peptidoglycan cell wall peptidoglycan) and an amino acid sequence set forth in any one of SEQ ID NOS:598, 602. 631, 634, 663,26108869435.2Docket No.: 125930-872823 677. 684, 689, 697. 698, 727, 739, 832, 860. 911, 925, 1038, 1045, 1056, 1101, 1106, 1108, 1113, 1152, 1158, 1187, 1232, 1252, 1263, 1308, 1364, 1379, 1380, 1381, 1383. 1384. 1411. 1415. 1416. 1417. or 1461. In some embodiments, an endolysin polypeptide comprises a catalytic domain (e.g.. peptidoglycan cell wall peptidoglycan) 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 amino acid sequence set forth in any one of SEQ ID NOS:598, 602, 631, 634, 663, 677, 684, 689, 697, 698, 727. 739, 832, 860, 911, 925, 1038, 1045, 1056, 1101, 1106, 1108, 1113, 1152, 1158, 1187, 1232, 1252, 1263, 1308, 1364, 1379, 1380, 1381, 1383, 1384, 1411, 1415.1416, 1417, or 1461, or a fragment thereof each (e.g., when the fragment is a binding domain). For a fragment of said amino acid sequence, an endolysin polypeptide may comprise a catalytic domain (e.g., peptidoglycan cell wall peptidoglycan) and 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: 598, 602, 631, 634, 663, 677, 684, 689, 697, 698, 727, 739, 832, 860, 911, 925, 1038, 1045, 1056, 1101, 1106, 1108, 1113, 1152, 1158, 1187, 1232, 1252, 1263, 1308, 1364, 1379, 1380, 1381, 1383, 1384, 1411, 1415, 1416, 1417, or 1461, when each sequence is aligned to each other according to their full length.Endolysins Comprising Binding Domain

[0095] 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:598, 602, 631, 634, 663, 677, 684, 689, 697, 698, 727, 739, 832, 860. 911, 925, 1038, 1045, 1056, 1101, 1106, 1108, 1113, 1152, 1158, 1187, 1232, 1252, 1263, 1308, 1364, 1379, 1380, 1381, 1383, 1384, 1411, 1415, 1416, 1417, 1461, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517. or a fragment thereof each (e.g., a fragment corresponding to the binding domain), or optionally a binding superdomain (e.g., a bacterial cell wall binding superdomain) comprising the amino acid sequence set forth in any one of SEQ ID NOS:598, 602, 631. 634, 663, 677.684, 689, 697. 698, 727, 739. 832, 860, 911, 925, 1038, 1045, 1056, 1101, 1106, 1108, 1113, 1152, 1158, 1187. 1232. 1252, 1263, 1308, 1364, 1379, 1380, 1381, 1383, 1384, 1411, 1415, 1416, 1417, 1461, 1505, 1506. 1507. 1508. 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517, or a fragment thereof each (e.g., a fragment corresponding to the binding domain). Two or more cell wall binding domains may be present together arranged adjacent to one another, in series, in an endolysin and an endolysin polypeptide sequence comprising this tandem arrangement of multiple cell wall binding domains is described herein as a “cell wall binding superdomain”. A binding domain or superdomain may provide a means for specificity of the endolysin for its target bacterial species, genera or strain. A binding domain or superdomain 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 or superdomain to the cell wall in the interior of the bacteria cell may prevent the endolysin from lysing other bacteria. The binding domain27108869435.2Docket No.: 125930-872823 or superdomain may enhance the specificity of the endolysin for its target bacterial species, genera or strain but it may not be essential for antimicrobial activity.Endolysins Comprising Catalytic Domains and SAR Domains

[0096] An endolysin as described herein may comprise a catalytic domain (e.g., a bacterial cell wall peptidoglycan catalytic domain) and a signal-anchor-release (SAR) domain, wherein the endolysin comprises the amino acid sequence set forth in any one of SEQ ID NOS:7, 23, 288, 291, 292, 294, 306, 313, 315, 317, 318, 321, 322, 332, 333, 336, 337, 339, 340, 344, 348, 353, 357, 358, 359, 360, 362, 365, 366, 367, 368, 369, 372, 389, 396. 402, 409. 410, 413, 416, 417, 494, 527, 573, 574, 579, 582, 583, 587, 591. 593, 1073, 1074, or 1075. Tn some embodiments, an amino acid sequence selected from any one of SEQ ID NOS:7, 23, 288, 291. 292, 294, 306, 313, 315. 317, 318, 321. 322, 332, 333, 336, 337, 339, 340, 344. 348, 353, 357, 358, 359, 360, 362, 365. 366, 367, 368. 369, 372, 389, 396, 402, 409, 410, 413. 416, 417. 494, 527, 573, 574, 579, 582, 583, 587. 591, 593, 1073, 1074, or 1075 contains both the catalytic domain and SAR domain. The SAR domains present in any one of SEQ ID NOS:7, 23, 288. 291, 292, 294, 306, 313, 315, 317, 318, 321, 322, 332. 333, 336, 337, 339, 340, 344, 348, 353, 357, 358, 359, 360, 362, 365, 366, 367, 368, 369, 372, 389, 396. 402, 409, 410, 413, 416, 417, 494, 527, 573, 574, 579, 582, 583, 587, 591, 593, 1073, 1074, or 1075 may assist penetration of the outer membrane of E. coli.

[0097] In some embodiments, an endolysin polypeptide comprises a catalytic domain (e.g., peptidoglycan cell wall peptidoglycan) 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 amino acid sequence set forth in any one of SEQ ID NOS:7. 23, 288. 291, 292, 294, 306, 313. 315, 317, 318. 321, 322, 332. 333, 336. 337, 339, 340.344, 348, 353. 357, 358. 359. 360, 362. 365, 366, 367. 368, 369, 372. 389, 396. 402, 409, 410. 413, 416, 417, 494, 527, 573, 574, 579, 582, 583, 587, 591, 593, 1073, 1074, or 1075. or a fragment thereof each (e.g., when the fragment is a SAR domain). For a fragment of said amino acid sequence, an endolysin polypeptide may comprise a catalytic domain (e.g.. peptidoglycan cell wall peptidoglycan) and 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:7, 23, 288, 291. 292, 294, 306, 313, 315, 317, 318, 321. 322, 332, 333. 336, 337, 339, 340, 344, 348, 353, 357. 358, 359. 360, 362, 365, 366, 367, 368, 369, 372. 389, 396, 402, 409, 410, 413, 416, 417, 494, 527, 573. 574, 579, 582, 583, 587, 591, 593, 1073, 1074, or 1075, when each sequence is aligned to each other according to their full length.Endolysins Comprising SAR Domain

[0098] An endolysin as described herein may comprise a SAR domain (e.g., a domain for signal-anchor release), optionally wherein the endolysin comprises the amino acid sequence set forth in any one of SEQ ID NOS:7, 23, 288, 291, 292, 294, 306, 313, 315. 317, 318, 321. 322, 332, 333, 336, 337, 339, 340, 344, 348, 353, 357, 358, 359, 360, 362, 365. 366, 367, 368, 369, 372, 389, 396, 402, 409, 410, 413. 416,28108869435.2Docket No.: 125930-872823 417. 494, 527, 573. 574, 579, 582, 583, 587. 591, 593, 1073, 1074, or 1075, or a fragment thereof each (e.g., when the fragment is a SAR domain). A SAR domain may provide a means for enhancing endolysin penetration of the outer membrane of a target bacterial species, genera or strain.Linkers

[0099] An endolysin as described herein may comprise one or more linkers which connect the catalytic domain with a cell wall binding domain or SAR 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, and / or one or more linkers connecting multiple SAR domains. The linker may comprise a polypeptide comprising an amino acid sequence. In certain embodiments, the linker is a polypeptide. The linker may be cleavable or non-cleavable. In certain embodiments, the linker is non-cleavable. A linker may be a linear linker or a branched linker.

[0100] 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: 1503) where n specifies the number of repeats of the unit. The number of repeats of the flexible linker may be from 1-3.

[0101] 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: 1504) where n specifies the number of repeats of the unit. The number of repeats of the non -flexible linker may be from 1-3.

[0102] As a skilled artisan would recognize, any suitable linker may be used to connect the domains of tire 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.

[0103] 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) — ).

[0104] A linker may be selected from PEG, polyacr la ide. poly(2-hydroxyethyl methacrylate), Poly-2-methyl-2-oxazoline (PMOXA), zwitterionic polymers, e.g., poly(carboxybetaine methacrylate) (PCBMA), poly[N-(3-sulfopropyl)-N-mcthacryloxycthyl-N, N dimethyl ammonium bctainc](PSBMA), glycopolymers, or polypeptides.

[0105] 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-phosphate units 29108869435.2Docket No.: 125930-872823 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

[0106] In the present disclosure, gram negative bacteria cell wall peptidoglycan catalytic domains such as described by the entirety or partiality of any one of SEQ ID NOS: 1-1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517, may be defined according to homology search using the Pfam software. Any of the gram negative bacteria cell wall peptidoglycan catalytic domains, such as described by the entirety or partiality of any one of SEQ ID NOS:1-1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517, may be defined according to homology search using HHpred software, InterProScan software or the Structural Classification of Proteins (SCOP) database.

[0107] Endolysins of the invention may comprise more than one Escherichia coli or Salmonella enterica peptidoglycan catalytic domain or binding domain, as described herein. 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 binding superdomain, 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.Endolysin Polypeptides Comprising Fragments and Variants

[0108] 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, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34. 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 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, 76, 77. 78, 19, 80, 81. 82, 83, 84, 85, 86. 87, 88, 89, 90. 91, 92, 93, 94, 95, 96, 97, 98, 99. 100, 101, 102, 103. 104, 105, 106. 107, 108, 109, 110, 111. 112, 113, 114. 115, 116, 117, 118, 119, 120, 121, 122, 123, 124. 125, 126, 127. 128, 129, 130, 131, 132. 133, 134, 135. 136, 137, 138, 139, 140, 141, 142, 143, 144, 145. 146, 147, 148. 149, 150, 151, 152, 153. 154, 155, 156. 157, 158, 159, 160, 161, 162, 163, 164, 165, 166. 167, 168, 169. 170, 171. 172, 173, 174. 175, 176, 177. 178, 179, 180, 181, 182. 183, 184, 185, 186, 187. 188, 189, 190, 191, 192. 193, 194, 195. 196, 197, 198. 199, 200. 201, 202, 203. 204, 205, 206, 207, 208. 209, 210. 211. 212, 213. 214, 215, 216. 217, 218, 219. 220, 221. 222, 223, 224. 225, 226, 227. 228, 229, 230, 231, 232, 233, 234, 235. 236. 237, 238. 239, 240, 241, 242, 243, 244, 245, 246. 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262. 263, 264, 265. 266, 267, 268, 269. 270, 271, 272. 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283. 284, 285, 286, 287, 288. 289, 290. 291, 292, 293. 294, 295, 296, 297, 298. 299, 300, 301. 302, 303, 304. 305, 306, 307, 308, 309. 310, 311. 312, 313, 314. 315, 316, 317, 318, 319. 320, 321, 322. 323, 324, 325. 326, 327, 328, 329, 330. 331, 332. 333, 334, 335, 336, 337, 338, 339, 340. 341, 342, 343. 344, 345, 346, 347, 348, 349, 350, 351. 352, 353, 354, 355, 356, 357, 358, 359, 360, 361. 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372. 373,30108869435.2Docket No.: 125930-872823 374. 375, 376, 377. 378, 379, 380, 381, 382. 383, 384, 385. 386, 387, 388. 389, 390, 391, 392, 393. 394, 395. 396, 397, 398. 399, 400, 401, 402, 403. 404, 405, 406. 407, 408, 409. 410, 411, 412, 413, 414. 415, 416. 417, 418, 419. 420, 421, 422, 423, 424. 425, 426, 427. 428, 429, 430. 431, 432, 433, 434, 435. 436, 437. 438, 439, 440, 441, 442, 443, 444, 445. 446, 447, 448. 449, 450, 451, 452, 453, 454, 455, 456. 457, 458. 459, 460, 461, 462, 463, 464, 465, 466. 467, 468, 469. 470, 471, 472, 473, 474, 475, 476, 477. 478, 479. 480, 481, 482, 483, 484, 485, 486, 487. 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498. 499, 500, 501, 502, 503, 504, 505, 506, 507, 508. 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529. 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631. 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652. 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673. 674, 675, 676, 677, 678, 679, 680, 681. 682, 683, 684, 685, 686, 687, 688, 689, 690, 691. 692, 693, 694. 695, 696, 697, 698, 699, 700, 701, 702. 703, 704, 705, 706, 707, 708, 709, 710, 711, 712. 713, 714, 715. 716, 717, 718, 719, 720, 721, 722, 723. 724, 725, 726, 727, 728, 729, 730, 731, 732, 733. 734, 735, 736. 737, 738, 739, 740, 741. 742, 743, 744. 745, 746, 747, 748, 749, 750, 751, 752, 753, 754. 755, 756, 757. 758, 759, 760, 761, 762. 763, 764, 765. 766, 767, 768, 769, 770, 771, 772, 773, 774, 775. 776, 777, 778. 779, 780. 781, 782, 783. 784, 785, 786. 787, 788, 789, 790, 791. 792, 793, 794, 795, 796. 797, 798, 799. 800, 801. 802, 803, 804. 805, 806, 807. 808, 809, 810, 811, 812. 813, 814, 815, 816, 817. 818, 819. 820. 821, 822. 823, 824, 825. 826, 827, 828. 829, 830. 831, 832, 833. 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857. 858, 859, 860. 861, 862, 863, 864, 865. 866, 867, 868. 869, 870, 871, 872, 873, 874, 875, 876. 877, 878. 879, 880, 881. 882, 883, 884, 885, 886. 887, 888, 889. 890, 891, 892. 893, 894, 895, 896, 897. 898, 899. 900, 901, 902. 903, 904, 905, 906, 907. 908, 909, 910. 911, 912, 913. 914, 915, 916, 917, 918. 919, 920. 921, 922, 923. 924, 925, 926, 927, 928. 929, 930, 931. 932, 933, 934. 935, 936, 937, 938, 939. 940, 941. 942, 943, 944, 945, 946, 947, 948, 949. 950, 951, 952. 953, 954, 955, 956, 957, 958, 959, 960. 961, 962. 963, 964, 965, 966, 967, 968, 969, 970. 971, 972, 973. 974, 975, 976, 977, 978, 979, 980, 981. 982, 983. 984, 985, 986, 987, 988, 989, 990, 991. 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019.1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036.1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138,31108869435.2Docket No.: 125930-872823 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166. 1167. 1168. 1169. 1170. 1171. 1172.1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184. 1185. 1186. 1187. 1188. 1189.1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202. 1203. 1204. 1205. 1206.1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220. 1221. 1222. 1223.1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238. 1239. 1240.1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257.1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274.1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291.1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462. 1463. 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479. 1480. 1481. 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496. 1497. 1498. 1499. 1500, 1501, 1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517, 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.

[0109] An endolysin polypeptide according to the invention may be derived from any one of the E. coli 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 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, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102. 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123. 124, 125, 126, 127, 128, 129, 130, 131. 132, 133, 134, 135, 136, 137, 138, 139, 140, 141. 142, 143, 144. 145, 146, 147, 148, 149, 150, 151, 152. 153, 154, 155, 156, 157, 158, 159, 160, 161, 162. 163, 164, 165. 166, 167, 168, 169, 170, 171, 172, 173. 174, 175, 176, 177, 178, 179, 180, 181, 182, 183. 184, 185, 186. 187, 188, 189, 190, 191. 192, 193, 194. 195, 196, 197, 198, 199, 200, 201, 202, 203, 204. 205, 206, 207. 208, 209, 210, 211, 212. 213, 214, 215. 216, 217, 218, 219, 220. 221, 222, 223, 224, 225. 226, 227, 228. 229, 230. 231, 232, 233. 234, 235, 236. 237, 238, 239, 240, 241. 242, 243,32108869435.2Docket No.: 125930-872823 244. 245, 246, 247. 248, 249, 250, 251, 252. 253, 254, 255. 256, 257, 258. 259, 260, 261, 262, 263. 264, 265. 266, 267, 268. 269, 270, 271, 272, 273. 274, 275, 276. 277, 278, 279. 280, 281, 282, 283, 284. 285, 286. 287, 288, 289. 290, 291, 292, 293, 294. 295, 296, 297. 298, 299, 300. 301, 302, 303, 304, 305. 306, 307. 308, 309, 310, 311, 312, 313, 314, 315. 316, 317, 318. 319, 320, 321, 322, 323, 324, 325, 326. 327, 328. 329, 330, 331, 332, 333, 334, 335, 336. 337, 338, 339. 340, 341, 342, 343, 344, 345, 346, 347. 348, 349. 350, 351, 352, 353, 354, 355, 356, 357. 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368. 369, 370, 371, 372, 373, 374, 375, 376, 377, 378. 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399. 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501. 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522. 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561. 562, 563, 564. 565, 566, 567, 568, 569, 570, 571, 572. 573, 574, 575, 576, 577, 578, 579, 580, 581, 582. 583, 584, 585. 586, 587, 588, 589, 590, 591, 592, 593. 594, 595, 596, 597, 598, 599, 600, 601, 602, 603. 604, 605, 606. 607, 608, 609, 610, 611. 612, 613, 614. 615, 616, 617, 618, 619, 620, 621, 622, 623, 624. 625, 626, 627. 628, 629, 630, 631, 632. 633, 634, 635. 636, 637, 638, 639, 640, 641, 642, 643, 644, 645. 646, 647, 648. 649, 650. 651, 652, 653. 654, 655, 656. 657, 658, 659, 660, 661. 662, 663, 664, 665, 666. 667, 668, 669. 670, 671. 672, 673, 674. 675, 676, 677. 678, 679, 680, 681, 682. 683, 684, 685, 686, 687. 688, 689. 690. 691, 692. 693, 694, 695. 696, 697, 698. 699, 700. 701, 702, 703. 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727. 728, 729, 730. 731, 732, 733, 734, 735. 736, 737, 738. 739, 740, 741, 742, 743, 744, 745, 746. 747, 748. 749, 750, 751. 752, 753, 754, 755, 756, 757, 758, 759. 760, 761, 762. 763, 764, 765, 766, 767. 768, 769. 770, 771, 772. 773, 774, 775, 776, 777. 778, 779, 780. 781, 782, 783. 784, 785, 786, 787, 788. 789, 790. 791, 792, 793. 794, 795, 796, 797, 798. 799, 800, 801. 802, 803, 804. 805, 806, 807, 808, 809. 810, 811. 812, 813, 814, 815, 816, 817, 818, 819. 820, 821, 822. 823, 824, 825, 826, 827, 828, 829, 830. 831, 832. 833, 834, 835, 836, 837, 838, 839, 840. 841, 842, 843. 844, 845, 846, 847, 848, 849, 850, 851. 852, 853. 854, 855, 856, 857, 858, 859, 860, 861. 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872. 873, 874, 875, 876, 877, 878, 879, 880, 881, 882. 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903. 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033,33108869435.2Docket No.: 125930-872823 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061. 1062. 1063. 1064. 1065. 1066. 1067.1068, 1069, 1070, 1071, 1072, 1073, 1074, or 1075 or any one of the S. enterica bacteriophage endolysin polypeptides set forth in any one of SEQ ID NOS: 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097. 1098. 1099. 1100.1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115. 1116. 1117.1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134.1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151.1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168.1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339. 1340. 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356. 1357. 1358. 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373. 1374. 1375. 1376. 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419. 1420. 1421. 1422. 1423.1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434. 1435. 1436. 1437. 1438. 1439. 1440.1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451. 1452. 1453. 1454. 1455. 1456. 1457.1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469. 1470. 1471. 1472. 1473. 1474.1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487. 1488. 1489. 1490. 1491.1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1505, 1506, 1507. 1508. 1509. 1510.1511, 1512, 1513, 1514, 1515, 1516, or 1517, 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.

[0110] An endolysin polypeptide according to the invention may be derived from one of the sequences of SEQ ID NOS: 1-1502. 1505. 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 34108869435.2Docket No.: 125930-872823 1517, by adding one or more additional N- or C-terminal amino acids or chemical moieties to increase stability. solubility and activity.

[0111] 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 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.

[0112] 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:1-1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517. 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: 1-1502. 1505. 1506. 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517, disclosed herein. Site-directed mutagenesis may comprise acid replacement substitution of one or more amino acids.

[0113] 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: 1-1502, 1505, 1506, 1507. 1508. 1509. 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517, as the reference amino acid sequence, or by a sub-sequence set forth in any one of SEQ ID NOS: 1-1502, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, or 1517, 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.

[0114] 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 identify ing 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 the35108869435.2Docket No.: 125930-872823 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, 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 10, M=5, N=4, and a comparison of both strands.

[0115] 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).

[0116] 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.36108869435.2Docket No.: 125930-872823 TABLE 1: Chemical properties of amino acids for conservative substitutionsAmino acid Chemical Properties HydropathyAla (A) aliphatic, hydrophobic, neutral 1.8Cys (C) polar, hydrophobic, neutral 2.5Asp (D) polar, hydrophilic, charged (-) -3.5Glu (E) polar, hydrophilic, charged (-) -3.5Phe (F) aromatic, hydrophobic, neutral 2.8Gly (G) aliphatic, neutral -0.4His (H) aromatic, polar, hydrophilic, charged (+) -3.2Ile (I) aliphatic, hydrophobic, neutral 4.5Lys (K) polar, hydrophilic, charged (+) -3.9Leu (L) aliphatic, hydrophobic, neutral 3.8Met (M) hydrophobic, neutral 1.9Asn (N) polar, hydrophilic, neutral -3.5Pro (P) hydrophobic, neutral -1.6Gln (Q) polar, hydrophilic, neutral -3.5Arg (R) polar, hydrophilic, charged (+) -4.5Ser (S) polar, hy drophilic, neutral -0.8Thr (T) polar, hydrophilic, neutral -0.7Val (V) aliphatic, hydrophobic, neutral 4.2Trp (W) aromatic, hydrophobic, neutral -0.9Tyr (Y) aromatic, polar, hydrophobic -1.3

[0117] 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.

[0118] 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

[0119] 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,37108869435.2Docket No.: 125930-872823 a t50% lysis value (time required for an endolysin to lyse 50% of its initial cell population) and / or a cell viability assay (loglO 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 growth inhibitory activity. Bacteriostatic or growth inhibitory activity may be determined by minimum inhibitory concentration (MIC).

[0120] Endolysins of the invention may provide an antimicrobial effect against Escherichia coli bacteria that is similar to the effect observed against relevant control bacteria (e.g., endolysins may not have specific activity for E. coli but they may be non-specific, and in addition to having activity against E. coli they may also have activity against non-E. coli bacteria). An endolysin of the invention may provide an antimicrobial effect against Escherichia coli bacteria that is higher compared to the effect observed against relevant control bacteria (e.g.. endolysins may have activity specific for Escherichia coli). A relevant control may be any bacterial strain that is not Escherichia coli (herein referred to as a "non-Ec" strain). The non-Ec strain may be selected from a Clostridium colimim, Clostridium leptum. Clostridium cellobioparum, Clostridium septicum, Bifidobacterium adolescentis. E. cecorum, or Salmonella enterica enteric strain.

[0121] A non-Ec strain may be identified by its DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) number (e.g., DSM number (e.g., strain, catalog, or accession number of the database)). The non-Ec strain may be selected from Clostridium colinum strain DSM 6011, Clostridium leptum strain DSM 753, Clostridium cellobioparum strain DSM 1351, Bifidobacterium adolescentis strain DSM 20083, or E. cecorum strains DSM 11364, DSM 20682, DSM 20683, DSM 100908 or DSM 109010. A non-Ec strain may be identified by its NCTC (National Collection of Type Cultures) number (e.g., procured from the Public Health England culture collection, (e g., strain, catalog, or accession number of the database)). The non-Ec strain may be selected from Clostridium septicum strains NCTC 281, NCTC 282, NCTC 283. NCTC 284, NCTC 286, NCTC 501, NCTC 504, NCTC 547, NCTC 549, NCTC 550 or NCTC 551, or E. cecorum strains NCTC 12421 or NCTC 12422. A non-Ec strain may be identified by its ATCC (American Type Culture Collection) number (e.g., strain, catalog, or accession number of the database). The non-Ec strain may be selected from E. cecorum strains ATCC 43198, ATCC 43078, ATCC 35246. or ATCC 39920. A non-Ec strain may be identified by its NCIMB (National Collection of Industrial. Food and Marine Bacteria) number (e.g., strain, catalog, or accession number of the database). The non-Ec strain may be selected from E. cecorum strains NCIMB 702674, NCIMB 702667. NCIMB 702668. NCIMB 702669, NCIMB 702670, NCIMB 702671. or NCIMB 702673. These are non-Ec bacteria strains commonly found in the poultry gut and may be used to determine whether endolysin activity and hence antimicrobial activity is specific to Escherichia coli.

[0122] A non- Ec strain may be a Salmonella enterica enterica reference strain. An Ec specific endolysin polypeptide of the invention may not exhibit antimicrobial effects to one or more of the 38108869435.2Docket No.: 125930-872823 Salmonella enterica enterica reference strains disclosed herein. An endolysin that is not specific for Ec (e.g., a Ec non-specific endolysin polypeptide) may exhibit antimicrobial effects to one or more of the Salmonella enterica enterica reference strains disclosed herein. The Salmonella enterica enterica reference strain may be selected from the Salmonella enterica enterica strains NCTC 14225, NCTC 5721, NCTC 5722. NCTC 6703, NCTC 10679, NCTC 5799, NCTC 4969, NCTC 6756, NCTC 6592, NCTC 7103, NCTC 7401, NCTC 6246, NCTC 6704, NCTC 6247, NCTC 7112, NCTC 7403, NCTC 7407, NCTC 6495, NCTC 7389, NCTC 7394, NCTC 6852, NCTC 6244, NCTC 6850, NCTC 7411, NCTC 7105, NCTC 7390, NCTC 6245, NCTC 7402, NCTC 6588, NCTC 6759, NCTC 7393, NCTC 7400, NCTC 6385, NCTC 6758, NCTC 7102, NCTC 7405, NCTC 7108, NCTC 7408, NCTC 6534, NCTC 6755, NCTC 6586, NCTC 7410, NCTC 7398, NCTC 6248, NCTC 7104, NCTC 6481, NCTC 6948, NCTC 6949, NCTC 6480, NCTC 7101, NCTC 7109, NCTC 7395, NCTC 7406, NCTC 7409, NCTC 7404, NCTC 6705, NCTC 6239, NCTC 6597, NCTC 6757, NCTC 6853, NCTC 6591, NCTC 6946, NCTC 7107, NCTC 7388, NCTC 6482. NCTC 6589, NCTC 6851, NCTC 6388, NCTC 7426, NCTC 6533, NCTC 6849, NCTC 7318, NCTC 14675, NCTC 14674, NCTC 14676, NCTC 12694, NCTC 618, NCTC 14811, NCTC 13349, NCTC 3045, NCTC 5765, NCTC 14253, NCTC 5188, NCTC 6676, NCTC 4444, NCTC 4776, NCTC 14831, NCTC 13665, NCTC 8515, NCTC 4777, NCTC 5694, NCTC 14711, NCTC 14743, NCTC 73, NCTC 74, NCTC 12190, NCTC 14678, NCTC 12115, NCTC 12118, NCTC 9324, NCTC 12117, NCTC 12484, NCTC 12485, NCTC 5715, NCTC 8298, NCTC 5711, NCTC 5714, NCTC 5710, NCTC 10413, NCTC 8391, NCTC 8392, NCTC 12416, NCTC 11881, NCTC 14022, NCTC 14026, NCTC 14011, NCTC 13954, NCTC 14844, NCTC 14017, NCTC 14019, NCTC 14018, or NCTC 14021. The Salmonella enterica enterica reference strain may be selected from the Salmonella enterica enterica strains ATCC 6960, ATCC BAA-1675. ATCC BAA-1675D-5, ATCC 9263, ATCC 51741, ATCC BAA-3136, ATCC BAA-3137, ATCC BAA-3138, ATCC BAA-3141. ATCC BAA-3139, ATCC BAA-3140, ATCC 15480, ATCC 8326, ATCC 51956, ATCC 13076, ATCC 13076DQ, ATCC BAA-3160. ATCC BAA-3243, ATCC 4931. ATCC 31194. ATCC BAA-1045. ATCC 700720D-5, ATCC 29629, ATCC 29631, ATCC 14028DQ, ATCC 13311DQ. ATCC 13311. or ATCC CRM-14028. The Salmonella enterica enterica reference strain may be selected from the Salmonella enterica enterica strains DSM 17420, DSM 4224, DSM 4883, DSM 5569, DSM 9145. DSM 9379, DSM 10062, DSM 554, DSM 11320, DSM 13674, DSM 14221, DSM 14846. DSM 17058, DSM 17420, DSM 10506, or DSM 5569. The Salmonella enterica enterica reference strain may be selected from the Salmonella enterica enterica strains NCIMB 15423, NCIMB 10411, NCIMB 13284, NCIMB 13034. NCIMB 10256, NCIMB 10257, NCIMB 10258, NCIMB 10259, NCIMB 10260, NCIMB 11379, NCIMB 11450, NCIMB 11451, NCIMB 11452, NCIMB 11453, NCIMB 13033, NCIMB 13036, NCIMB 10248, NCIMB 10249, NCIMB 10250, NCIMB 10251, NCIMB 10252, NCIMB 10253, NCIMB 10254, or NCIMB 10255.

[0123] An E. colt strain sensitive to an endolysin of the invention may be identified by its DSMZ number (e.g., DSM number), NCTC number, ATCC number, or NCIMB number, for example a strain.39108869435.2Docket No.: 125930-872823 catalog, or accession number of the database. The E. coli strain sensitive to an endolysin of the invention may be selected from the E. coli strains NCTC 13125, NCTC 12900, NCTC 13128, NCTC 13127, NCTC 13126, NCTC 10650, NCTC 10758, NCTC 9969. NCTC 9043, NCTC 9030, NCTC 9049, NCTC 9967, NCTC 9087. NCTC 9040, NCTC 9106, NCTC 9107, NCTC 9108, NCTC 9123. NCTC 9091, NCTC 9089, NCTC 9038, NCTC 9016, NCTC 11181, NCTC 9013, NCTC 9014, NCTC 10957, NCTC 9009, NCTC 11475, NCTC 8959, NCTC 9041, NCTC 10002, NCTC 9119, NCTC 9102, NCTC 9035, NCTC 9045, NCTC 9065, NCTC 9082, NCTC 9097, NCTC 9100, NCTC 9117, NCTC 9033, NCTC 9120, NCTC 10279, NCTC 9080, NCTC 9084, NCTC 9095, NCTC 6999, NCTC 9060, NCTC 9010, NCTC 40444, NCTC 40664, NCTC 9023, NCTC 9963, NCTC 9011, NCTC 11107, NCTC 9031, NCTC 9098, NCTC 9005, NCTC 9034, NCTC 9068, NCTC 9073, NCTC 9075, NCTC 9085, NCTC 9092, NCTC 9099, NCTC 9104, NCTC 9105, NCTC 9116, NCTC 9036, NCTC 9703, NCTC 9078, NCTC 9022, NCTC 9705, NCTC 10758, NCTC 9047, NCTC 10960, NCTC 9019, NCTC 9070, NCTC 9079, NCTC 11473, NCTC 8960, NCTC 9103, NCTC 9071, NCTC 9081, NCTC 9088, NCTC 9090, NCTC 9109, NCTC 9121, NCTC 9086, NCTC 9083. NCTC 9056, NCTC 9059, NCTC 9062, NCTC 8620, NCTC 9021, NCTC 9044, NCTC 9112, NCTC 9007, NCTC 9048, NCTC 9855, NCTC 10090, NCTC 9006, NCTC 8623, 8009 NCTC, 10959 NCTC, 9053 NCTC. 10087 NCTC. 9004 NCTC, 9002 NCTC, 9032 NCTC 9050, NCTC 9066, NCTC 9064, NCTC 9076, NCTC 1096, NCTC 9101. NCTC 8333, NCTC 9028, NCTC 9114, NCTC 9052, NCTC 9096, NCTC 9061. NCTC 9024, NCTC 9961, NCTC 9012, NCTC 9055, NCTC 9706, NCTC 9003. NCTC 9008, NCTC 10962, NCTC 11022, NCTC 10361, NCTC 9094, or NCTC 9067. The E. coli strain sensitive to an endolysin of the invention may be selected from the E. coli strains ATCC BAA-2196, ATCC 8739. ATCC BAA-2440, ATCC BAA-2192, ATCC 43895. ATCC BAA-2215, ATCC BAA-2193, ATCC BAA-2219. ATCC 15669. ATCC BAA-3337, ATCC 13706, ATCC 11229, ATCC BAA-2649, ATCC BAA-3162, ATCC BAA-3163, ATCC BAA-2452, ATCC BAA-3239, ATCC BAA-3237. ATCC BAA-3236, ATCC BAA-3235, ATCC 29425, ATCC 47107, ATCC 43893, ATCC BAA-2190. ATCC 35150. ATCC BAA-2326, ATCC BAA-1025, ATCC 33694, ATCC 700728, ATCC BAA-1883, ATCC 43889. ATCC 43895, ATCC BAA-3233, ATCC BAA-3234, or ATCC BAA-3162. The E. coli strain sensitive to an endolysin of the invention may be selected from the E. coli strains DSM 27503, DSM 3423, DSM 3950. DSM 6056, DSM 6897, DSM 7140, DSM 1328, DSM 301, DSM 308, DSM 423. DSM 498, DSM 501, DSM 613, DSM 682. DSM 787. DSM 1958, DSM 1077, DSM 1099, DSM 1115, or DSM 1328. The E. coli strain sensitive to an endolysin of the invention may be selected from the E. coli strains NCIMB 2117, NCIMB 86, NCIMB 91, NCIMB 417, NCIMB 2120, NCIMB 4174, NCIMB 8020, NCIMB 8109, NCIMB 8110, NCIMB 8112, NCIMB 8113, NCIMB 8114, NCIMB 8134, NCIMB 8242, NCIMB 8269, NCIMB 8273, NCIMB 8277, NCIMB 8522, NCIMB 8545, NCIMB 8557. These E. coli strains may be referred herein as “E. coli reference strains”.

[0124] An endolysin polypeptide of the invention may exhibit antimicrobial effects to one or more of the E. coli reference strains disclosed herein. An endolysin of the invention may exhibit antimicrobial 40108869435.2Docket No.: 125930-872823 effects against the E. coh strains NCTC 13125, NCTC 12900, NCTC 13128, NCTC 13127, NCTC 13126. NCTC 10650, NCTC 10758, NCTC 9969, NCTC 9043. NCTC 9030, NCTC 9049, NCTC 9967, NCTC 9087. NCTC 9040, NCTC 9106, NCTC 9107, NCTC 9108, NCTC 9123. NCTC 9091, NCTC 9089, NCTC 9038. NCTC 9016, NCTC 11181 NCTC, NCTC 9013, NCTC 9014, NCTC 10957, NCTC 9009, NCTC 11475, NCTC 8959. NCTC 9041, NCTC 10002, NCTC 9119, NCTC 9102, NCTC 9035, NCTC 9045, NCTC 9065, NCTC 9082, NCTC 9097, NCTC 9100, NCTC 9117, NCTC 9033, NCTC 9120, NCTC 10279, NCTC 9080, NCTC 9084, NCTC 9095, NCTC 6999, NCTC 9060, NCTC 9010, NCTC 40444, NCTC 40664, NCTC 9023, NCTC 9963, NCTC 9011, NCTC 11107, NCTC 9031, NCTC 9098, NCTC 9005, NCTC 9034, NCTC 9068, NCTC 9073, NCTC 9075, NCTC 9085, NCTC 9092, NCTC 9099, NCTC 9104, NCTC 9105, NCTC 9116, NCTC 9036, NCTC 9703, NCTC 9078, NCTC 9022, NCTC 9705, NCTC 10758, NCTC 9047, NCTC 10960, NCTC 9019, NCTC 9070, NCTC 9079, NCTC 11473, NCTC 8960, NCTC 9103, NCTC 9071, NCTC 9081, NCTC 9088, NCTC 9090, NCTC 9109, NCTC 9121, NCTC 9086, NCTC 9083. NCTC 9056, NCTC 9059, NCTC 9062, NCTC 8620, NCTC 9021, NCTC 9044, NCTC 9112, NCTC 9007, NCTC 9048, NCTC 9855, NCTC 10090, NCTC 9006, NCTC 8623, NCTC 8009, NCTC 10959, NCTC 9053. NCTC 10087. NCTC 9004, NCTC 9002, NCTC 9032, NCTC 9050, NCTC 9066, NCTC 9064, NCTC 9076, NCTC 1096, NCTC 9101, NCTC 8333, NCTC 9028, NCTC 9114, NCTC 9052. NCTC 9096, NCTC 9061, NCTC 9024, NCTC 9961, NCTC 9012, NCTC 9055, NCTC 9706, NCTC 9003, NCTC 9008. NCTC 10962. NCTC 11022. NCTC 10361, NCTC 9094, NCTC 9067, ATCC BAA-2196, ATCC 8739. ATCC BAA-2440, ATCC BAA-2192. ATCC 43895. ATCC BAA-2215, ATCC BAA-2193, ATCC BAA-2219, ATCC 15669, ATCC BAA-3337, ATCC 13706, ATCC 11229, ATCC BAA-2649. ATCC BAA-3162, ATCC BAA-3163, ATCC BAA-2452, ATCC BAA-3239, ATCC BAA-3237. ATCC BAA-3236. ATCC BAA-3235, ATCC 29425, ATCC 47107, ATCC 43893, ATCC BAA-2190, ATCC 35150, ATCC BAA-2326. ATCC BAA-1025, ATCC 33694, ATCC 700728, ATCC BAA-1883, ATCC 43889. ATCC 43895, ATCC BAA-3233, ATCC BAA-3234. ATCC BAA-3162, DSM 27503, DSM 3423, DSM 3950, DSM 6056, DSM 6897. DSM 7140, DSM 1328, DSM 301, DSM 308, DSM 423. DSM 498. DSM 501, DSM 613, DSM 682. DSM 787. DSM 1958, DSM 1077, DSM 1099, DSM 1115, DSM 1328. NCIMB 2117. NCIMB 86. NCIMB 91, NCIMB 417, NCIMB 2120, NCIMB 4174, NCIMB 8020, NCIMB 8109, NCIMB 8110, NCIMB 8112, NCIMB 8113. NCIMB 8114. NCIMB 8134, NCIMB 8242, NCIMB 8269, NCIMB 8273, NCIMB 8277, NCIMB 8522. NCIMB 8545, or NCIMB 8557.

[0125] Endolysins of the invention may provide an antimicrobial effect against Salmonella enterica bacteria that is similar to the effect observed against relevant control bacteria (e g., endolysins may not have activity specific for Salmonella enterica but they may be non-specific, and in addition to having activity against Salmonella enterica they may also have activity against non- Salmonella enterica bacteria). An endolysin of the invention may provide an antimicrobial effect against Salmonella enterica bacteria that is higher compared to the effect observed against relevant control bacteria (e.g.. endolysins may have activity specific for Salmonella enterica). A relevant control may be any bacterial strain that is 41108869435.2Docket No.: 125930-872823 not Salmonella enterica (herein referred to as a “non-Se” strain). The non-Se strain may be selected from a Clostridium colinum, Clostridium leptum, Clostridium cellobioparum. Clostridium septicum, Bifidobacterium adolescentis. or E. cecorum strain.

[0126] A non-Se strain may be identified by its DSMZ number (e.g., DSM number), NCTC number, ATCC number, orNCIMB number, for example a strain, catalog, or accession number of the database. A non-Se strain may be selected from Clostridium colinum strain DSM 6011, Clostridium leptum strain DSM 753, Clostridium cellobioparum strain DSM 1351, Bifidobacterium adolescentis strain DSM 20083, orE. cecorum strains DSM 11364, DSM 20682, DSM 20683. DSM 100908. or DSM 109010. The non-Se strain may be selected from Clostridium septicum strains NCTC 281, NCTC 282, NCTC283. NCTC 284, NCTC 286, NCTC501. NCTC 504, NCTC 547, NCTC 549, NCTC 550 or NCTC 551, orE. cecorum strains NCTC 12421 or NCTC 12422. The non-Se strain may be selected from E. cecorum strains ATCC 43198, ATCC 43078, ATCC 35246, or ATCC 39920. The non-Se strain may be selected from E. cecorum strains NCIMB 702674. NCIMB 702667, NCIMB 702668, NCIMB 702669. NCIMB 702670. NCIMB 702671, or NCIMB 702673. These are non-Se bacteria strains commonly found in the poultry gut and may be used to determine whether endolysin activity and hence antimicrobial activity is specific to Salmonella enterica.

[0127] A non- Se strain may be a E. coli reference strain. A Se specific endolysin polypeptide of the invention may not exhibit antimicrobial effects to one or more of the E. coli reference strains disclosed herein. An endolysin that is not specific for Se (e.g.. a Se non-specific endolysin polypeptide) may exhibit antimicrobial effects to one or more of the E. coli reference strains disclosed herein. The E. coli reference strain may be selected from E. coli strains NCTC 13125, NCTC 12900, NCTC 13128, NCTC 13127, NCTC 13126, NCTC 10650, NCTC 10758, NCTC 9969, NCTC 9043, NCTC 9030, NCTC 9049, NCTC 9967, NCTC 9087, NCTC 9040, NCTC 9106, NCTC 9107, NCTC 9108, NCTC 9123, NCTC 9091, NCTC 9089, NCTC 9038, NCTC 9016. NCTC 11181. NCTC 9013, NCTC 9014, NCTC 10957, NCTC 9009, NCTC 11475, NCTC 8959, NCTC 9041, NCTC 10002, NCTC 9119, NCTC 9102, NCTC 9035, NCTC 9045, NCTC 9065, NCTC 9082. NCTC 9097, NCTC 9100, NCTC 9117, NCTC 9033, NCTC 9120, NCTC 10279, NCTC 9080, NCTC 9084, NCTC 9095, NCTC 6999, NCTC 9060, NCTC 9010, NCTC 40444, NCTC 40664, NCTC 9023, NCTC 9963, NCTC 9011. NCTC 11107. NCTC 9031, NCTC 9098, NCTC 9005, NCTC 9034, NCTC 9068, NCTC 9073. NCTC 9075, NCTC 9085, NCTC 9092. NCTC 9099, NCTC 9104, NCTC 9105. NCTC 9116, NCTC 9036. NCTC 9703, NCTC 9078, NCTC 9022, NCTC 9705, NCTC 10758, NCTC 9047, NCTC 10960, NCTC 9019, NCTC 9070. NCTC 9079. NCTC 11473. NCTC 8960, NCTC 9103, NCTC 9071. NCTC 9081, NCTC 9088. NCTC 9090, NCTC 9109, NCTC 9121, NCTC 9086, NCTC 9083, NCTC 9056, NCTC 9059, NCTC 9062, NCTC 8620, NCTC 9021. NCTC 9044, NCTC 9112, NCTC 9007, NCTC 9048, NCTC 9855. NCTC 10090. NCTC 9006. NCTC 8623, NCTC 8009, NCTC 10959, NCTC 9053, NCTC 10087. NCTC 9004, NCTC 9002, NCTC 9032. NCTC 9050, NCTC 9066, NCTC 9064, NCTC 9076, NCTC 1096. NCTC 9101, NCTC 8333. NCTC 9028, NCTC 9114, NCTC 9052, NCTC 9096, NCTC 9061. NCTC 9024, NCTC 42108869435.2Docket No.: 125930-872823 9961, NCTC 9012, NCTC 9055, NCTC 9706, NCTC 9003, NCTC 9008, NCTC 10962, NCTC 11022, NCTC 10361, NCTC 9094. or NCTC 9067. The E. coli reference strain may be selected from E. coli strains ATCC BAA-2196, ATCC 8739, ATCC BAA-2440. ATCC BAA-2192, ATCC 43895, ATCC BAA-2215, ATCC BAA-2193, ATCC BAA-2219, ATCC 15669, ATCC BAA-3337, ATCC 13706, ATCC 11229, ATCC BAA-2649. ATCC BAA-3162, ATCC BAA-3163, ATCC BAA-2452, ATCC BAA-3239, ATCC BAA-3237, ATCC BAA-3236, ATCC BAA-3235, ATCC 29425, ATCC 47107, ATCC 43893, ATCC BAA-2190. ATCC 35150. ATCC BAA-2326, ATCC BAA-1025, ATCC 33694, ATCC 700728, ATCC BAA-1883, ATCC 43889. ATCC 43895, ATCC BAA-3233, ATCC BAA-3234, or ATCC BAA-3162. The E. coli reference strain may be selected from E. coli strains DSM, DSM 3423, DSM 3950, DSM 6056, DSM 6897, DSM 7140, DSM 1328, DSM 301, DSM 308, DSM 423, DSM 498, DSM 501, DSM 613, DSM 682, DSM 787, DSM 1958, DSM 1077, DSM 1099, DSM 1115, or DSM 1328. The E. coli reference strain may be selected from E. coli strains NCIMB 2117, NCIMB 86, NCIMB 91, NCIMB 417, NCIMB 2120, NCIMB 4174, NCIMB 8020, NCIMB 8109, NCIMB 8110, NCIMB 8112, NCIMB 8113, NCIMB 8114, NCIMB 8134, NCIMB 8242, NCIMB 8269, NCIMB 8273, NCIMB 8277. NCIMB 8522, NCIMB 8545, or NCIMB 8557.

[0128] A Salmonella enterica strain sensitive to an endolysin of the invention may be identified by its DSMZ number (e.g., DSM number). NCTC number. ATCC number, or NCIMB number, for example a strain, catalog, or accession number of the database. The Salmonella enterica strain sensitive to endolysins of the invention may be selected from the Salmonella enterica strains NCTC 14225. NCTC 5721, NCTC 5722. NCTC 6703, NCTC 10679, NCTC 5799, NCTC 4969, NCTC 6756, NCTC 6592. NCTC 7103. NCTC 7401, NCTC 6246, NCTC 6704, NCTC 6247, NCTC 7112, NCTC 7403, NCTC 7407, NCTC 6495. NCTC 7389, NCTC 7394, NCTC 6852, NCTC 6244, NCTC 6850. NCTC 7411, NCTC 7105, NCTC 7390, NCTC 6245, NCTC 7402, NCTC 6588, NCTC 6759, NCTC 7393, NCTC 7400, NCTC 6385, NCTC 6758, NCTC 7102, NCTC 7405, NCTC 7108, NCTC 7408, NCTC 6534, NCTC 6755, NCTC 6586, NCTC 7410, NCTC 7398, NCTC 6248, NCTC 7104, NCTC 6481, NCTC 6948, NCTC 6949, NCTC 6480, NCTC 7101, NCTC 7109, NCTC 7395, NCTC 7406, NCTC 7409, NCTC 7404, NCTC 6705, NCTC 6239, NCTC 6597, NCTC 6757, NCTC 6853, NCTC 6591, NCTC 6946, 7107, NCTC 7388, NCTC 6482, NCTC 6589, NCTC 6851, NCTC 6388, NCTC 7426, NCTC 6533, NCTC 6849, NCTC 7318, NCTC 14675, NCTC 14674, NCTC 14676, NCTC 12694, NCTC 618, NCTC 14811, NCTC 13349, NCTC 3045, NCTC 5765, NCTC 14253, NCTC 5188, NCTC 6676, NCTC 4444, NCTC 4776, NCTC 14831, NCTC 13665, NCTC 8515, NCTC 4777. NCTC 5694, NCTC 14711, NCTC 14743, NCTC 73, NCTC 74, NCTC 12190, NCTC 14678, NCTC 12115, NCTC 12118, NCTC 9324, NCTC 12117, NCTC 12484, NCTC 12485, NCTC 5715, NCTC 8298, NCTC 5711, NCTC 5714, NCTC 5710, NCTC 10413, NCTC 8391, NCTC 8392, NCTC 12416, NCTC 11881, NCTC 14022, NCTC 14026, NCTC 14011, NCTC 13954, NCTC 14844, NCTC 14017, NCTC 14019, NCTC 14018, or NCTC 14021. The Salmonella enterica strain sensitive to an endolysin of the invention may be selected from Salmonella enterica strains ATCC 6960. ATCC BAA-1675, ATCC BAA-1675D-5, ATCC 9263.43108869435.2Docket No.: 125930-872823 ATCC 51741, ATCC BAA-3136, ATCC BAA-3137, ATCC BAA-3138, ATCC BAA-3141, ATCC BAA-3139, ATCC BAA-3140, ATCC 15480, ATCC 8326, ATCC 51956, ATCC 13076, ATCC 13076DQ. ATCC BAA-3160, ATCC BAA-3243, ATCC 4931. ATCC 31194. ATCC BAA-1045, ATCC 700720D-5, ATCC 29629, ATCC 29631, ATCC 14028DQ, ATCC 13311DQ. ATCC 13311. or ATCC CRM-14028. The Salmonella enterica strain sensitive to an endolysin of the invention may be selected from Salmonella enterica strain DSM 17420, DSM 4224, DSM 4883. DSM 5569, DSM 9145, DSM 9379, DSM 10062, DSM 554, DSM 11320, DSM 13674, DSM 14221, DSM 14846, DSM 17058, DSM 17420, DSM 10506, or DSM 5569. The Salmonella enterica strain sensitive to an endolysin of the invention may be selected from NCIMB 15423, NCIMB 10411, NCIMB 13284, NCIMB 13034, NCIMB 10256, NCIMB 10257, NCIMB 10258, NCIMB 10259, NCIMB 10260, NCIMB 11379, NCIMB 11450, NCIMB 11451, NCIMB 11452, NCIMB 11453, NCIMB 13033, NCIMB 13036, NCIMB 10248, NCIMB 10249, NCIMB 10250, NCIMB 10251, NCIMB 10252, NCIMB 10253, NCIMB 10254, or 10255. These Salmonella enterica strains may be referred herein as “S', enterica reference strains”.10129| An endolysin polypeptide of the invention may exhibit antimicrobial effects to one or more of the Salmonella enterica reference strains disclosed herein. An endolysin of the invention may exhibit antimicrobial effects against Salmonella enterica strains NCTC 14225, NCTC 5721, NCTC 5722, NCTC 6703, NCTC 10679, NCTC 5799. NCTC 4969, NCTC 6756, NCTC 6592. NCTC 7103, NCTC 7401. NCTC 6246. NCTC 6704, NCTC 6247, NCTC 7112, NCTC 7403, NCTC 7407. NCTC 6495, NCTC 7389, NCTC 7394. NCTC 6852, NCTC 6244, NCTC 6850, NCTC 7411, NCTC 7105. NCTC 7390, NCTC 6245. NCTC 7402, NCTC 6588, NCTC 6759, NCTC 7393, NCTC 7400. NCTC 6385, NCTC 6758, NCTC 7102. NCTC 7405, NCTC 7108, NCTC 7408, NCTC 6534, NCTC 6755. NCTC 6586, NCTC 7410. NCTC 7398, NCTC 6248, NCTC 7104, NCTC 6481, NCTC 6948, NCTC 6949, NCTC 6480, NCTC 7101. NCTC 7109, NCTC 7395, NCTC 7406, NCTC 7409, NCTC 7404, NCTC 6705, NCTC 6239, NCTC 6597, NCTC 6757, NCTC 6853, NCTC 6591, NCTC 6946, NCTC 7107, NCTC 7388, NCTC 6482, NCTC 6589, NCTC 6851, NCTC 6388, NCTC 7426, NCTC 6533, NCTC 6849, NCTC 7318, NCTC 14675, NCTC 14674, NCTC 14676, NCTC 12694, NCTC 618, NCTC 14811, NCTC 13349, NCTC 3045, NCTC 5765, NCTC 14253, NCTC 5188, NCTC 6676, NCTC 4444, NCTC 4776, NCTC 14831, NCTC 13665, NCTC 8515, NCTC 4777, NCTC 5694, NCTC 14711, NCTC 14743, NCTC 73, NCTC 74, NCTC 12190, NCTC 14678, NCTC 12115, NCTC 12118, NCTC 9324, NCTC 12117, NCTC 12484, NCTC 12485, NCTC 5715, NCTC 8298, NCTC 5711, NCTC 5714. NCTC 5710, NCTC 10413, NCTC 8391, NCTC 8392, NCTC 12416, NCTC 11881, NCTC 14022, NCTC 14026, NCTC 14011, NCTC 13954, NCTC 14844, NCTC 14017, NCTC 14019, NCTC 14018, NCTC 14021, ATCC 6960, ATCC BAA-1675, ATCC BAA-1675D-5, ATCC 9263, ATCC 51741, ATCC BAA-3136, ATCC BAA-3137, ATCC BAA-3138, ATCC BAA-3141, ATCC BAA-3139. ATCC BAA-3140, ATCC 15480, ATCC 8326, ATCC 51956, ATCC 13076, ATCC 13076DQ, ATCC BAA-3160, ATCC BAA-3243. ATCC 4931, ATCC 31194, ATCC BAA-1045, ATCC 700720D-5. ATCC 29629. ATCC 29631. ATCC 14028DQ, ATCC 13311DQ, ATCC 13311, ATCC CRM-14028, DSM 17420, DSM 4224, DSM 44108869435.2Docket No.: 125930-872823 4883, DSM 5569, DSM 9145, DSM 9379, DSM 10062, DSM 554, DSM 11320, DSM 13674, DSM 14221. DSM 14846, DSM 17058. DSM 17420, DSM 10506, DSM 5569, NCIMB 15423, NCIMB 10411, NCIMB 13284. NCIMB 13034, NCIMB 10256, NCIMB 10257. NCIMB 10258, NCIMB 10259, NCIMB 10260, NCIMB 11379, NCIMB 11450, NCIMB 11451, NCIMB 11452, NCIMB 11453.NCIMB 13033, NCIMB 13036, NCIMB 10248, NCIMB 10249, NCIMB 10250, NCIMB 10251.NCIMB 10252, NCIMB 10253, NCIMB 10254, or NCIMB 10255.

[0130] An endolysin of the invention may provide an antimicrobial effect against Pseudomonas aeruginosa bacteria that is higher compared to the effect observed against relevant control bacteria. A relevant control may be any bacterial strain that is not Pseudomonas aeruginosa (herein referred to as a “non-Pa” strain). A non-Pa strain may be selected from a Clostridium colinum, Clostridium leptum, Clostridium cellobiopa, Clostridium septicum, Bifidobacterium adolescentis, or E. cecorum strain.

[0131] A non-Pa strain may be identified by its DSMZ number (e g., DSM number). NCTC number, ATCC number, or NCIMB number, for example a strain, catalog, or accession number of the database. The non-Pa strain may be selected from Clostridium colinum strain DSM 6011, Clostridium leptum strain DSM 753, Clostridium cellobioparum strain DSM 1351, Bifidobacterium adolescentis strain DSM 20083, or / •'. cecorum strains DSM 11364. DSM 20682, DSM 20683, DSM 100908, or DSM 109010. The non-Pa strain may be selected from Clostridium septicum strains NCTC 281, NCTC 282, NCTC283, NCTC 284, NCTC 286, NCTC501, NCTC 504, NCTC 547, NCTC 549, NCTC 550, or NCTC 551, or E. cecorum strains NCTC 12421 or NCTC 12422. The non-Pa strain may be selected from E. cecorum strains ATCC 43198, ATCC 43078, ATCC 35246, or ATCC 39920. The non-Pa strain may be selected from E. cecorum strains NCIMB 702674, NCIMB 702667, NCIMB 702668, NCIMB 702669, NCIMB 702670, NCIMB 702671, or NCIMB 702673. These are non-Pa bacteria strains commonly found in the poultry gut and may be used to determine whether endolysin activity and hence antimicrobial activity is specific to Pseudomonas aeruginosa.

[0132] A Pseudomonas aeruginosa strain sensitive to an endolysin of the invention may be identified by its DSMZ number (e.g., DSM number), NCTC number, ATCC number, or NCIMB number, for example a strain, catalog, or accession number of the database. The Pseudomonas aeruginosa strain sensitive to an endolysin of the invention may be selected from Pseudomonas aeruginosa strains NCTC 12448, NCTC 11839, NCTC 11449, NCTC 11441, NCTC 13415, NCTC 13714, NCTC 13718, NCTC 13618, NCTC 10731, NCTC 13359, NCTC 12447, NCTC 11451, NCTC 11440, NCTC 11453, NCTC 11450, NCTC 11447, NCTC 11446, NCTC 5940. NCTC 7771, or NCTC 11669. A Pseudomonas aeruginosa strain sensitive to an endolysin of the invention may be selected from Pseudomonas aeruginosa strains ATCC 10145, ATCC 27853, ATCC BAA-1744. ATCC BAA-3211, ATCC BAA-2114, ATCC 15442, ATCC BAA-3333. ATCC 15692. ATCC BAA-2110, ATCC BAA-3197, ATCC 47085, ATCC BAA-3312, ATCC BAA-3314. ATCC BAA-3315, ATCC BAA-3316, ATCC BAA-47, ATCC BAA-2109. ATCC BAA-2113, ATCC BAA-2111, or ATCC BAA-2112. A Pseudomonas aeruginosa strain 45108869435.2Docket No.: 125930-872823 sensitive to an endolysin of the invention may be selected from Pseudomonas aeruginosa strains DSM 288. DSM 939, DSM 1117. DSM 1253, DSM 1299, DSM 1707, DSM 3227, DSM 6279. DSM 6195. DSM 7232, DSM 8924, DSM 11148, DSM 11634. DSM 12055, DSM 12736. DSM 13626, DSM 15980, DSM 16074. DSM 16528, or DSM 19880. A Pseudomonas aeruginosa strain sensitive to an endolysins of the invention may be selected from NCIMB 950, NCIMB 5940, NCIMB 6750, NCIMB 6751, NCIMB 8027, NCIMB 8295, NCIMB 8297, NCIMB 8298. NCIMB 8626, NCIMB 8650, NCIMB 8672, NCIMB 8704, NCIMB 9038, NCIMB 9389, NCIMB 9571, NCIMB 9685, NCIMB 9793, NCIMB 9904, NCIMB 10110, or NCIMB 10111. These Pseudomonas aeruginosa strains may be referred herein as “P. aeruginosa reference strains”.

[0133] An endolysin polypeptide of the invention may exhibit antimicrobial effects to one or more of the Pseudomonas aeruginosa reference strains disclosed herein. An endolysin of the invention may exhibit antimicrobial effects against Pseudomonas aeruginosa NCTC 12448, NCTC 11839, NCTC 11449, NCTC 11441, NCTC 13415, NCTC 13714, NCTC 13718, NCTC 13618, NCTC 10731, NCTC 13359, NCTC 12447, NCTC 11451, NCTC 11440, NCTC 11453, NCTC 11450, NCTC 11447, NCTC 11446, NCTC 5940. NCTC 7771, NCTC 11669, ATCC 10145, ATCC 27853, ATCC BAA-1744, ATCC BAA-3211, ATCC BAA-2114, ATCC 15442, ATCC BAA-3333, ATCC 15692, ATCC BAA-2110, ATCC BAA-3197. ATCC 47085. ATCC BAA-3312. ATCC BAA-3314, ATCC BAA-3315, ATCC BAA-3316, ATCC BAA-47, ATCC BAA-2109. ATCC BAA-2113. ATCC BAA-2111, ATCC BAA-2112, DSM 288, DSM 939. DSM 1117, DSM 1253, DSM 1299, DSM 1707, DSM 3227. DSM 6279. DSM 6195, DSM 7232, DSM 8924. DSM 11148, DSM 11634. DSM 12055, DSM 12736, DSM 13626, DSM 15980. DSM 16074, DSM 16528, DSM 19880, NCIMB 950, NCIMB 5940. NCIMB 6750.NCIMB 6751, NCIMB 8027. NCIMB 8295. NCIMB 8297, NCIMB 8298, NCIMB 8626, NCIMB 8650, NCIMB 8672, NCIMB 8704. NCIMB 9038, NCIMB 9389, NCIMB 9571, NCIMB 9685, NCIMB 9793, NCIMB 9904, NCIMB 10110, or NCIMB 10111.

[0134] An endolysin of the invention may provide an antimicrobial effect against A cinetobacler baumannii bacteria that is higher compared to the effect observed against relevant control bacteria. A relevant control may be any bacterial strain that is no Acinetobacter baumannii (herein referred to as a “non-Ab” strain). A non-Ab strain may be identified by its DSMZ number (e.g., DSM number), NCTC number. ATCC number, or NCIMB number, for example a strain, catalog, or accession number of the database. The non-Ab strain may be selected from Clostridium colinum strain DSM 6011. Clostridium leptum strain DSM 753, Clostridium cellobioparum strain DSM 1351, Bifidobacterium adolescentis strain DSM 20083, or E. cecorum strains DSM 11364. DSM 20682, DSM 20683. DSM 100908. or DSM 109010. The non-Ab strain may be selected from Clostridium septicum strains NCTC 281, NCTC 282, NCTC283, NCTC 284. NCTC 286, NCTC501, NCTC 504, NCTC 547. NCTC 549, NCTC 550, or NCTC 551, or E. cecorum strains NCTC 12421 or NCTC 12422. The non-Ab strain may be selected from E. cecorum strains ATCC 43198, ATCC 43078, ATCC 35246, ATCC 39920. The non-Ab strain may be selected from E. cecorum strains NCIMB 702674, NCIMB 702667, NCIMB 702668. NCIMB 46108869435.2Docket No.: 125930-872823 702669, NCIMB 702670, NCIMB 702671, or NCIMB 702673. These are non-Ab bacteria strains commonly found in the poultry gut and may be used to determine whether endolysin activity and hence antimicrobial activity is specific to Acinetobacter baumannii.

[0135] An Acinetobacter baumannii strain sensitive to an endolysin of the invention may be identified by its DSMZ number (e.g., DSM number), NCTC number, ATCC number, or NCIMB number, for example a strain, catalog, or accession number of the database. The Acinetobacter baumannii strain sensitive to an endolysin of the invention may be selected from Acinetobacter baumannii strains NCTC 13422, NCTC 10303, NCTC 13420, NCTC 7250, NCTC 7422, NCTC 7844, NCTC 12156, NCTC 13421, NCTC 13423, NCTC 13486, NCTC 7461, NCTC 13305, NCTC 13424, NCTC 13485, NCTC 10292, NCTC 13303, NCTC 13304, NCTC 7364, NCTC 13487, NCTC 14656, NCTC 10290, NCTC 10391, NCTC 7363, NCTC 13301, or NCTC 13302. The Acinetobacter baumannii strain sensitive to an endolysin of the invention may be selected from Acinetobacter baumannii strains ATCC 19606, ATCC 17978, ATCC BAA-1710, ATCC BAA-3252. ATCC BAA-1605, ATCC BAA-3338, ATCC BAA-3320. ATCC 51432. ATCC BAA-747, ATCC BAA-1790. ATCC BAA-3311, ATCC BAA-3302, ATCC BAA-3301. ATCC BAA-3300, ATCC BAA-3283, ATCC BAA-3282. ATCC BAA-3278. ATCC BAA-3275, ATCC BAA-3276, or ATCC BAA-3257. The Acinetobacter baumannii strain sensitive to an endolysin of the invention may be selected from the Acinetobacter baumannii strains DSM 24110, DSM 25645. DSM 30007. DSM 30008, DSM 30011, DSM 101650, DSM 101993, DSM 102928, DSM 102929, DSM 102930, DSM 102931, DSM 102932, DSM 102933, DSM 130803, DSM 105126, DSM 106838, DSM 111794, DSM 114617, DSM 114760, or DSM 115404. The Acinetobacter baumannii strain sensitive to an endolysin of the invention may be selected from Acinetobacter baumannii strains NCIMB 11517, NCIMB 9689, NCIMB 12457, NCIMB 9300, NCIMB 9299, NCIMB 9293. or NCIMB 9214. These Acinetobacter baumannii strains may be referred herein as “ baumanni reference strains”.

[0136] An endolysin polypeptide of the invention may exhibit antimicrobial effects to one or more of the Acinetobacter baumannii reference strains disclosed herein. An endolysin of the invention may exhibit antimicrobial effects against Acinetobacter baumannii strains NCTC 13422, NCTC 10303, NCTC 13420, NCTC 7250, NCTC 7422, NCTC 7844, NCTC 12156, NCTC 13421, NCTC 13423, NCTC 13486, NCTC 7461, NCTC 13305, NCTC 13424, NCTC 13485, NCTC 10292, NCTC 13303, NCTC 13304. NCTC 7364, NCTC 13487, NCTC 14656, NCTC 10290, NCTC 10391, NCTC 7363, NCTC 13301. NCTC 13302. ATCC 19606. ATCC 17978. ATCC BAA-1710, ATCC BAA-3252, ATCC BAA-1605, ATCC BAA-3338. ATCC BAA-3320, ATCC 51432, ATCC BAA-747. ATCC BAA-1790, ATCC BAA-3311, ATCC BAA-3302, ATCC BAA-3301. ATCC BAA-3300. ATCC BAA-3283, ATCC BAA-3282, ATCC BAA-3278, ATCC BAA-3275, ATCC BAA-3276, ATCC BAA-3257, DSM 24110, DSM 25645. DSM 30007, DSM 30008, DSM 30011, DSM 101650, DSM 101993, DSM 102928, DSM 102929, DSM 102930, DSM 102931, DSM 102932, DSM 102933, DSM 130803, DSM 105126, DSM 106838, DSM 111794, DSM 114617, DSM 114760, DSM 115404, NCIMB 11517, NCIMB 9689, NCIMB 12457, NCIMB 9300, NCIMB 9299, NCIMB 9293, or NCIMB 9214.47108869435.2Docket No.: 125930-872823

[0137] The antimicrobial activity may be determined by using a standard cell viability assay as disclosed herein to calculate a A loglO value. The A loglO value exhibited by an endolysin polypeptide or fragment may be determined using a concentration of 5 pg / ml of an endolysin or fragment of the invention against a reference strain, optionally E. coli 0149 strain NCTC 10650, E. coli 0149 strain DSM 27503, Salmonella enterica enterica Enteritidis strain NCTC 13665, or Salmonella enterica enterica Typhimurium strain NCIMB 11450, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism. The A loglO value may be approximately 0.40 or more, 2.00 or more or 3.70 or more, wherein. Approximately is taken to mean+ / -10% or + / -5%.

[0138] 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 pg / ml of an endolysin or fragment of the invention against a reference strain, such as E. coli 0149 strain NCTC 10650, E. coli 0149 strain DSM 27503. Salmonella enterica enterica Enteritidis strain NCTC 13665, or Salmonella enterica enterica Typhimurium strain NCIMB 11450, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism. The % reduction in cell viability may be approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, or.100%, wherein approximately is + / -!()% or + / -5%.

[0139] 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 pg / ml of an endolysin of the invention or a fragment against a reference strain, such as E. coli 0149 strain NCTC 10650, E. coli 0149 strain DSM 27503, Salmonella enterica enterica Enteritidis strain NCTC 13665, or Salmonella enterica enterica Typhimurium strain NCIMB 11450, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism. The t50 lysis value provided may be approximately 7 minutes or less: or it may provide a t50% lysis value of approximately 3 minutes or less, wherein approximately is + / - 10% or + / -5%.

[0140] The antimicrobial activity may be determined by using a standard minimum inhibitory' concentration (MIC) assay and / or a minimum bactericidal concentration (MBC) assay, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism. A standard minimum inhibitory concentration (MIC) assay or a minimum bactericidal concentration (MBC) assay may be performed against a reference strain at approximately 2.1×104cells / mL, wherein an endolysin of the invention or a fragment thereof exhibits an MIC value or an MBC value of approximately 17 pg / mL or less, preferably approximately 1.7 pg / mL or48108869435.2Docket No.: 125930-872823 less; or it exhibits an MIC value or an MBC value of approximately 0.65 pM or less, preferably approximately 0.05 pM or less, wherein approximately is + / — 10% or + / -5%.

[0141] The antimicrobial activity may be determined by using a focused checkerboard assay as descripted herein. The synergistic effect of the combination of an outer membrane penetrating agent with an endolysin described herein may be determined by using a focused checkerboard assay as described herein.

[0142] 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, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism. 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 Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii strain.

[0143] Any endolysin polypeptide of the invention may be determined to have endolysin hy drolytic activity, and therefore antimicrobial activity, when it promotes a statistically significant increase in the rate of degradation of purified PGN in vitro, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism. 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.

[0144] The ability of a given endolysin polypeptide to promote a statistically significant increase in the rate of degradation of purified PGN in vitro, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism 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 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.

[0145] 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, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B. at a concentration slightly below the MIC for the test organism (e.g., a statistically significant degradation of purified PGN in vitro) at the endpoint (completion) of a PGN degradation assay.49108869435.2Docket No.: 125930-872823

[0146] The ability of a given endolysin polypeptide to promote a statistically significant reduction in the absolute amount of purified PGN in vitro, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism 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.

[0147] The ability" of a given endolysin polypeptide to promote a statistically significant reduction in tire absolute amount of purified PGN in vitro in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism can also be assessed by reference to the reduction in the absolute amount of purified PGN degradation by a reference or control polypeptide, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism. 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, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism. 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.

[0148] Endolysin hydrolytic activity can be assessed in this way for purified endolysins, as well as for endolysins comprised in cell extracts.

[0149] 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, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism. 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, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B. at a concentration slightly below the MIC for the test organism. Preferably the control contains a cell extract 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, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism. A control polypeptide can50108869435.2Docket No.: 125930-872823 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.

[0150] Antimicrobial activity such as bacteriolytic activity and bacteriostatic activity, may be determined according to the assays described in the following section.

[0151] Assays for bacteriolytic activity include spot assays in which endolysin polypeptides of the invention and fragments thereof are added to Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii cell lawns already established on the appropriate medium (e.g., agar plate). The medium must comprise an outer membrane penetrating agent, for example polymyxin B. at a concentration slightly below the MIC for the test organism. 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 Escherichia coli, Salmonella enterica. Pseudomonas aeruginosa, or Acinetobacter baumannii 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.

[0152] Bacteriostatic (growth inhibitory) activity may be determined as described herein. Assays for bacteriostatic activity include spot assays in which Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii 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 medium must comprise an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism. The extent of the bacteriostatic activity can be determined by lack of growth of Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii colonies compared with non-treated controls or controls treated with products that do not affect bacterial growth.

[0153] The relative increase in the bacteriolytic / loglO / t50% lysis value associated with any of the endolysin polypeptides of the invention, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism, 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, optionally in the presence of an outer membrane penetrating agent, for example polymyxin B. at a concentration slightly below the MIC for the test organism 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. A51108869435.2Docket No.: 125930-872823 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 inactive endolysin, or any other suitable polypeptide which is not an endolysin, such as bovine serum albumin.

[0154] The relative increase may be assessed by comparing the endolytic / bacteriolytic / loglO / t50% lysis value / cell viability activity of purified proteins of the present invention (optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism ) against any suitable control protein (optionally in the presence of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism). 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 / loglO / t50% lysis value / cell viability activity in clarified lysates when polypeptides are separately expressed in a host cell, preferably in a Bacillus subtilis or other bacterial host cell, or a fungal host cell (e.g.. Pichia or Saccharomyces). or a microalgal host cell (e.g., Chlamydomonas reinhardtii). More specifically, the antimicrobial activities of the endolysin polypeptides of the invention can be determined using the assays outlined in the following section: Assay for Antimicrobial Effect (A Log 10 and Cell Viability) of Endolysins:

[0155] A Log 10, which is the loglO of the ratio of untreated cell control reactions over endolysin-treated cell reactions, may be calculated according to the following method:1. Inoculate Escherichia coli. Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii strain into BHI+C media and culture at 37° C. overnight to stationary phase;2. Dilute the overnight culture into fresh BHI+C media and culture 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;4. remove supernatant and resuspend cell pellet in 0.9 mL of an appropriate cell diluent (peptone water pH 7.0);5. dilute cell suspension with cell diluent to OD 600 nm 0.6;6. add purified endolysin polypeptide or fragment to a concentration of 5 μg / mL and a suitable amount of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism under anaerobic conditions;7. incubate at 37° C for 1 hour;52108869435.2Docket No.: 125930-872823 8. determine viable cell count by serial dilution, plating cells on RCM solid media and incubating at 37° C until colonies form; and9. 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.

[0156] Cell viability may be calculated according to the following method:1. Inoculate Escherichia coli. Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii strain into BHI+C media and culture at 37° C. overnight to stationary phase;2. Dilute the overnight culture into fresh BHI+C media and culture at 37° C to OD 600 nm approximately 0.6;3. Centrifuge 1.8 m culture at 16,600× g for 5 minutes at room temperature;4. remove supernatant and resuspend cell pellet in 0.9 mL of an appropriate cell diluent (peptone water pH 7.0);5. dilute cell suspension with cell diluent to OD 600 nm 0.6;6. add purified endolysin polypeptide or fragment to a concentration of 5 μg / mL and a suitable amount of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism;7. incubate at 37° C for 1 hour;8. determine viable cell count by serial dilution, plating cells on RCM solid media and incubating at 37° C until colonies form; and9. 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.

[0157] 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 Escherichia coli. Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii strain in BHI+C media and culture at 37° C. overnight to stationary phase;2. inoculate the overnight culture into fresh BHI+C media and culture at 37° C. until the culture reaches exponential phase (OD 620 nm approximately 1.0);53108869435.2Docket No.: 125930-872823 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 and a suitable amount of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism;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:

[0158] 50% = - tes° o / / 1 iys•' ~XOOfosx 1000 / ot=o x

[0159] wherein ODb_t=0 is the optical density of the initial cell suspension at the beginning of the test, prior to addition of the endolysin polypeptide or fragment; and

[0160] wherein ODE_t50% lysis is the optical density' of the cell suspension corresponding to 50% reduction in tire initial cell density following addition of the endolysin polypeptide or fragment.

[0161] Wherein X is the optical density of the blank at the beginning of the test.Assay for Minimum Inhibitory Concentration (MIC) / Minimum Bactericidal Concentration (MBC) of Endolysins:

[0162] The MIC / MBC value, which is defined as the minimum concentration of purified endolysin which leads to cell growth arrest for a given gram-negative bacteria cell density, may be calculated according to the following method:1. Inoculate Escherichia coh. Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumarmii strain in BHI+C media and culture at 37° C. overnight to stationary phase;2. dilute stationary phase culture in fresh BHI+C media;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;54108869435.2Docket No.: 125930-872823 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 pL of diluted stationary phase culture is mixed with 5 pL of the 10x concentrated and purified endolysin to a final cell loading of approximately 104cells / mL and a suitable amount of an outer membrane penetrating agent, for example polymyxin B, at a concentration slightly below the MIC for the test organism;5. incubate cells overnight for 12-20 hrs at 37° C;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 pL of culture from each well into a separate well and make up to 100 pL with BHI+C medium;9. incubate the transferred cultures overnight for 12-20 hrs at 37° C.;10. determine the extent of growth, and therefore the extent of residual viable Ec 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.Focused checkerboard synergy assay

[0163] Air assay which may be used to screen for activity of endolysins of the invention or to assess their relative activities against various gram-negative bacterial targets is as follows. The assay format is a “focused checkerboard” assay, which is designed to look for synergy between an endolysin and an outer membrane penetrating agent (OMP agent). An exemplary OMP agent for use in this assay is polymyxin B, which exhibits synergy with lysozyme and endolysins.

[0164] The focused checkerboard assay is based on the fact that an endolysin may be unable to kill the bacterial target in the absence of OMP agent (e g., polymyxin B). as the endolysin will may have difficulty accessing the bacterial cell wall, which is shielded by the outer membrane. The assay therefore requires the presence of polymyxin B. but it must be at a sublethal concentration (e.g., below the minimum inhibitory concentration (MIC) of polymyxin), otherwise the bacterial cells will be killed by the polymyxin alone. The MIC of polymyxin against E. coli and other gram-negative bacteria may be55108869435.2Docket No.: 125930-872823 determined by using standard protocols well known to those skilled in the art, for example, the MIC determination protocols provided by the Clinical & Laboratory Standards Institute (CLSI).

[0165] Although the concentration of polymyxin B is sublethal in this assay, it is still capable of disrupting the outer membrane to allow the endolysin access to the bacterial cell wall. Therefore, at a particular concentration of endolysin the damage inflicted on the cell wall is sufficient to kill the cell. The assay detects the minimum concentration of endolysin at a particular polymyxin concentration (e.g., a synergistic mixture) effective at killing the cell. This mixture is synergistic as the polymyxin at that concentration will not kill the cell on its own (as it is below the polymyxin MIC) and the endolysin may be unable to kill the cell on its own as it will be unable to access the cell wall.

[0166] In the following example protocol, lysozyme is used in place of endolysin as a positive control, however the protocol is intended for testing endolysins of the invention.Lysozyme control

[0167] Prepare lysozy me solution: weigh out 8 mg lysozyme and dissolve in 1 ml sterile water to make a 8 mg / ml soln. The lysozyme solution is serially diluted using water to create a two-fold dilution series, (e.g., 0.5 ml of the 8 mg / ml lysozyme solution is added to 0.5 ml sterile water, mixed and diluted again etc.) The dilution process is repeated multiple times to create 21 different concentration lysozyme solutions, with each being half as concentrated as the solution preceding it in the dilution series.Endolysin samples

[0168] When assaying endolysins the samples to be assayed may be in the form of whole cell lysates comprising endolysins. cell extracts comprising endolysins, partially purified material comprising endolysins or purified endolysins, or any endolysin composition as described herein. The samples to be assayed should comprise a neat, undiluted sample that is used to prepare a two-fold dilution series of the sample in an appropriate diluent (e.g., buffer or water) to make multiple solutions of the sample, with each being half as concentrated as the solution preceding it in the series. The endolysin samples may be diluted using RO water, PBS, Peptone Water or any other physiologically acceptable buffer well known to those skilled in the art.Polymyxin solution

[0169] A polymyxin B solution is made by weighing out 8 mg of polymyxin B and dissolving it in 1 ml of water to make an 8 mg / ml solution (8,000 pg / ml). The polymyxin 8 mg / ml solution is diluted 1:100 in water (100 pl of 8 mg / ml polymyxin in 10 ml of water) to make a 80 pg / ml solution. This in turn is diluted 1:100 in water (100 pl of 80 pg / ml polymyxin in 10 ml water) to make an 0.8 pg / ml soln. The 0.8 pg / ml solution is serially diluted to create a two-fold dilution series. 5 ml of the 0.8 pg / ml polymyxin B solution is added to 5 ml sterile water, mixed and diluted again etc. The dilution is effected multiple times to create 3 polymyxin stock solutions each half as concentrated as the solution preceding it in the 56108869435.2Docket No.: 125930-872823 series. These three stock solutions comprise polymyxin at the following concentrations: 0.4. 0.2. and 0.1 pg / ml. In the microtitre plate wells, which the assay is set up in, these stock solutions are further diluted 1:4 by other assay components so the final polymyxin concentrations used will be a quarter of the concentration of the three stock solutions, e.g.. the 0.4 pg / ml polymyxin solution will be diluted by other assay components to give a final polymyxin B concentration of 0.1 pg / ml; the 0.2 pg / ml polymyxin solution will be diluted by other assay components to give a final polymyxin B concentration of 0.05 pg / ml; and the 0.1 g / ml polymyxin solution will be diluted by other assay components to give a final polymyxin B concentration of 0.025 pg / ml. The polymyxin solutions are each added as 25 pl in a total assay volume of 100 pl.

[0170] The assay uses three polymyxin concentrations per microtitre plate which span and encompass the MIC. The final polymyxin B concentrations are 0.1, 0.05 & 0.025 pg / ml, plus water (e.g., 0 pg / ml polymyxin) as a negative control. The MIC of polymyxin against E. coli 0149 strain DSM 27503 is 0.122 pg / ml. The most important final polymyxin concentration is 0.05 pg / ml as this is just below the E. coli MIC of polymyxin and synergy between the polymyxin and endolysin / lysozyme should be exhibited at this polymyxin concentration.Plate setup

[0171] 25 pl of each of the 21 endolysin / lysozyme solutions are added separately to 21 wells of a two row 24 well “block” of a standard 96 well microtitre plate. Rows A & B comprise a single 24 well “block” as do rows C & D and E & F and G & H. For example, if the assay uses endolysin / lysozyme 25 pl of the most concentrated endolysin / lysozyme solution 8 mg / ml endolysin / lysozyme (8,000 pg / ml) is added to well Al and the two-fold diluted solution made from this - the 4 mg / ml solution is added to well A2 etc. As the endolysin / lysozyme solutions are added 25 pl in a total volume of 100 pl there is a 4 x dilution factor so, for example, the well to which 25 pl of 8 mg / ml endolysin / lysozy me is added has a final endolysin / lysozyme concentration of only 2 mg / ml (2,000 pg / ml). The last three wells of each two rows set have 25 pl of water instead of endolysin / lysozyme / endolysin added to them. These are the negative controls (no endolysin / lysozyme).

[0172] 25 pl of each of the three polymyxin solutions are added to all of the wells of a two row block of endolysin / lysozyme serial dilutions. For example. 25 pl of 0.4 pg / ml polymyxin is added to all of the wells but one of Rows A & B (final polymyxin concentration: 0.1 pg / ml), 25 pl of 0.2 pg / ml polymyxin is added to all of the wells but one of Row s C & D (final polymyxin concentration: 0.05 pg / ml) and 25 pl of 0.1 pg / ml polymyxin is added to all but one of the wells of Rows E & F (final polymyxin concentration: 0.025 pg / ml) and 25 pl of water is added to all of the wells of Rows G & H. The microtitre plate therefore comprises four two-row blocks with all of the w ells of each block (excepting one well) comprising the same polymyxin concentration but a different endolysin / lysozyme concentration. The last well of each block has 25 pl of water (not polymyxin) added to it.57108869435.2Docket No.: 125930-872823 Controls

[0173] The last block of the plate (Rows G & H) is a endolysin / lysozyme only block (no polymyxin) so the effect of the endolysin / lysozyme solutions in the absence of polymyxin may be assessed. Wells 10 & 11 of each block in Rows B, D. F and H with polymyxin have no endolysin / lysozyme in them so they are the polymyxin / water only controls. Well 12 of each block in Rows B, D. F and H has no polymyxin and no endolysin / lysozyme so these are the double negative (no-polymyxin and no endolysin / lysozyme) controls.

[0174] A schematic diagram illustrating microtiter plate layout and set-up of the focused checkerboard endolysin / lysozyme synergy assay is shown in FIG. 1.Growth of culture

[0175] The previous day a loopful of E. coli 0149 (reference strain DSM 27503) from a Luria-Bertani (LB) agar plate is inoculated into 10 ml of 1 x Mueller-Hinton Broth (MHB) medium in a 50 ml Erlenmeyer flask. The flask is cultured overnight in a shaking incubator set at 37°C and with a shaking speed of 200 rpm. 1.5 hrs before the assay the culture is diluted 1:100 into fresh MHB, e.g., 0.5 ml of the culture is added to 50 ml of fresh MHB in a Erlenmeyer 250 ml flask and grown at 37°C with shaking (200rpm). The growth of the culture is monitored using a spectrophotometer set at λ = 600 nm. The absorbance / optical density of the culture should reach OD600 = 0.45 - 0.5 after 90 minutes which is the right OD600 range and represents mid-exponential phase.Addition of culture

[0176] When it reaches the correct OD600 (e.g., 0.4 - 0.5), the culture is diluted 200 fold in fresh 2 x MHB (double strength MHB medium), for example, 50 pl of the culture is added per 10 ml of 2 x MHB. The assay requires ~ 5 ml of the mix per microtitre plate. The culture and medium are mixed well and 50 pl of the cell / medium mix is added to each well of the plate. The final volume of each well is 100 pl - 25 pl of polymyxin, 25 pl of endolysin / lysozyme and 50 pl of cells / medium and the dilution factor of the other compounds means the 2 x MHB is returned to being its normal 1 x strength. The microtitre plate is covered with a paper breathable membrane (Starlab) and incubated overnight in a microtitre plate shaker in a 37°C incubator. The turbidity / optical density of the wells at 620 nm is determined the next day using a Labtech LT-4500 plate reader. Endolysin activity is indicated by clear / less turbid test well(s) relative to the negative control well(s).

[0177] The protocol can be adapted to screen endolysins for activity against other gram-negative bacteria provided the polymyxin MIC of the target organism is known, or can be determined using standard methods well known in the art to the microbiologist skilled in the art.

[0178] The polymyxin MIC for E. coli 0149 (e.g., strain DSM 27503) is approximately 0.122 pg / ml. The polymyxin MIC for Salmonella enterica enterica Enteritidis (e.g., strain NCTC 13665) is58108869435.2Docket No.: 125930-872823 approximately 3.125 pg / ml and when performing the assay for this organism the three test blocks had polymyxin concentrations of 3.125 pg / ml. 1.56 pg / ml & 0.78 pg / ml. The polymyxin MIC for Salmonella enterica enterica Typhimurium (e.g.. strain NCIMB 11450) is 0.195 pg / ml and when performing the assay for this organism the three test blocks had polymyxin concentrations of 0.19 pg / ml, 0.097 pg / ml & 0.048 pg / ml.

[0179] The protocol detailed in paragraphs

[0163] -

[0178] works whenE coli 0149 is replaced by either of these strains of Salmonella when the concentrations of polymyxin used in setting up the “blocks” are modified to take into account the differences in polymyxin MIC's of the Salmonella strains, relative to the E. coli polymyxin MIC.Methods of Producing Endolysins

[0180] 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.

[0181] 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 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 other bacterial host cell, or a fungal host cell (e.g., Pichia or Saccharomyces cerevisiae), or a microalgal host cell (e.g., Chlamydomonas reinhardtii), 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.

[0182] 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), fungal cells (e.g.. yeast cells (e.g.. Pichia or Saccharomyces). microalgal cells (e.g., Chlamydomonas reinhardtii) 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 or59108869435.2Docket No.: 125930-872823 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.

[0183] 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.

[0184] 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 other bacterial host cell. Such cells typically include eukaryotic cells such as fungal host cells (e.g., Pichia or Saccharomyces), or microalgal host cells (e.g., Chlamydomonas reinhardtii). 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.

[0185] 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 tenninus to improve expression.

[0186] 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 other bacterial host cell, a fungal host cell (e.g., Pichia or Saccharomyces), or a microalgal host cell (e.g., Chlamydomonas reinhardtii). 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 ty pically 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.60108869435.2Docket No.: 125930-872823

[0187] 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.

[0188] 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.

[0189] 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 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

[0190] The invention further provides nucleic acid molecules and vectors which encode an endolysin polypeptide of the invention. The invention further provides nucleic acid molecules and vectors which encode an outer membrane penetrating (OMP) agent as described herein. Separate nucleic acids and / or vectors may encode the endolysin polypeptide and OMP agent, or they may be encoded by the same nucleic acid and / or vector. The nucleic acid may encode multiple endolysin polypeptides and / or OMP agents. 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.

[0191] 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 61108869435.2Docket No.: 125930-872823 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.

[0192] 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 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.

[0193] 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.).

[0194] 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 62108869435.2Docket No.: 125930-872823 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.

[0195] 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 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 ability level of the skilled person. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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 for63108869435.2Docket No.: 125930-872823 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.

[0200] 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: pXTl, 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.

[0201] 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

[0202] 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.

[0203] 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.

[0204] Each endolysin of the invention may further comprise the endolysin sequence together with an expression-enhancing peptide sequence to further assist with expression. When 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 or 64108869435.2Docket No.: 125930-872823 other bacterial host cell, a fungal host cell (e.g., Pichia or Saccharomyces, or a microalgal host cell (e.g., Chlamydomonas reinhardtii). 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, sufl, 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.

[0205] Any of tire 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. The purification may remove contamination due to the lysate of the host cell, for example the cellular components derived from either the bacterial host cell (e.g., Bacillus subtilis), fungal host cell (e.g., Pichia or Saccharomyces), or microalgal host cell (e.g., Chlamydomonas remhardtii).Host Cells and Expression Systems

[0206] 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 or other bacterial host cell, a fungal host cell (e.g., Pichia or Saccharomyces, or a microalgal host cell (e.g., Chlamydomonas reinhardtii).

[0207] The Bacillus subtilis cell may be from the expression strain, 168, or from another strain. Nonlimiting 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, or SCK6.

[0208] 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 other bacterial host cell, a fungal host cell (e.g., Pichia or Saccharomyces), or a microalgal host cell (e g., Chlamydomonas reinhardtii). Non-limiting examples of the Pichia pastoris host cell of the invention are those of the following strains: GS115, KM71, X-33 and SMD1168. Non-limiting examples of the Saccharomyces cerevisiae host cell of the invention are those of the following strains: S288C, CEN. PK2-1C, BY4741 / BY4742, W303 and YVH10. Non-limiting examples of the Chlamydomonas reinhardtii host cell of the invention are those of the following strains: CC-125, CC-400, UVM4 and cw!5. 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 65108869435.2Docket No.: 125930-872823 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.

[0209] 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.

[0210] 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).

[0211] Non-limiting examples of green algae, which are especially well-suited for use include members of the Chlamydomonas species, particularly Chlamydomonas reinhardtii'. the Chlorella 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. SpeciesChlorophytaChlamydomonas reinhardtiiVolvox carteriDunaliella salinaDunaliella viridisHaematococcus pluvialisChlorella sorokinianaChlorella kessleri (Parachlorella kessleri)66108869435.2Docket No.: 125930-872823 SpeciesChlorella ellipsoideaChlorclla vulgarisUlva lactucaOstreococcus tauriRhodophytaCyanidioschyzon merolaePorphyra yezoensisPorphyra miniataKappaphycus alvareziiGracilaria changiiPorphyridium spGracilariaHeterokontophytaLaminaria j aponicaUndaria pinnatifidaPhaeodactylum tricomutumNavicula saprophila (Fistulifera sapr ophila)Cylindrotheca fusifonnisThalassiosira weissflogiiDinoflagellatesAmphidinium sp.Symbiodinium microadriaticumCyanobacteriaSpirulina platensis (Arthrospira platensis)Anabaena spSynechocystis sp.SynechococcusNosctoc muscorumEuglenidsEuglena gracilis67108869435.2Docket No.: 125930-872823

[0212] 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 / bl9464. 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.

[0213] 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.

[0214] 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 68108869435.2Docket No.: 125930-872823 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.

[0215] 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 or Saccharomyces cerevisiae, 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.

[0216] 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 or Saccharomyces cerevisiae, 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 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.

[0217] 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 69108869435.2Docket No.: 125930-872823 be a cell of a unicellular microorganism, optionally a yeast cell, such as Pichia pastoris or Saccharomyces cerevisiae, 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.

[0218] 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 or Saccharomyces cerevisiae. an algal cell such as Chlamydomonas reinhardtii, a fungal cell, or a bacterial cell, preferably a 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 ly sate 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 or Saccharomyces cerevisiae, 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.

[0219] 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 70108869435.2Docket No.: 125930-872823 dried material of the invention. The combined material may be dried, preferably by spray drying or freeze drying.

[0220] 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.

[0221] 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.Outer membrane penetrating agents

[0222] Gram-negative bacteria such as Escherichia coli and Salmonella enterica possess an outer membrane (OM) which shields and protects the cell wall by preventing endolysins from accessing and degrading it. An outer membrane penetrating (OMP) agent as described herein may facilitate an endolysin’s penetration (e.g., traversion) of the outer membrane. OMP agents are known to function through covalent and / or noncovalent binding. An OMP agent may disrupt the OM sufficiently for an endolysin to access the peptidoglycan cell wall of a bacteria. An OMP may improve the antimicrobial effect of an endolysin as described herein. An endolysin as described herein may have improved antimicrobial effect when combined with at least one OMP agent. Administration of an endolysin may have improved antimicrobial activity when a combination of an endolysin and at least one OMP are administered.

[0223] Many OMP agents are recognized in the art. and are contemplated for use with an endolysin of the invention. OMP agents can be broadly classified based on structural feature, for example, at least as peptide or peptide-based OMP agents; metal ion binding or chelating OMP agents; cationic steroid OMP agents; polyamine OMP agents. An OMP agent may be an antimicrobial peptide (AMP) and / or cell-penetrating peptide (CPP) as recognized in the art.

[0224] A peptide or peptide-based OMP agent may comprise from 5-10 amino acid residues, 11-15 amino acid residues, 16-20 amino acid residues, 21-25 amino acid residues, 26-30 amino acid residues, 31-35 amino acid residues, 36-40 amino acid residues, 41-45 amino acid residues, 46-50 amino acid residues, 51-55 amino acid residues, 56-60 amino acid residues, 61-65 amino acid residues, 66-70 amino71108869435.2Docket No.: 125930-872823 acid residues, 71-75 amino acid residues, or 76-80 amino acid residues. The peptide or peptide-based OMP agent may be chemically modified.

[0225] In an aspect, an OMP agent is peptide or peptide-based OMP agent, which is a a-helical OMP agent. The α-helical OMP agent may be rich in positive-charged residues (e.g., Lys, Arg, or His) and / or lack a Cys residue. These positively charged residues may interact with negatively charged regions of the cell wall or cell membrane. The α-helical OMP agent may also comprise hydrophilic residues (e g., polar residues) and / or may be amphipathic. The a-helical OMP agent may also have conformational changes when interacting with a cell wall or cell membrane. In some embodiments, the OMP agent is an a-helical OMP agent selected from a cathelidicin, lactoferrin (e.g., lactoferricin), cecropin, magainin, or spiderine family of peptides.

[0226] In an aspect, an OMP agent is peptide or peptide-based OMP agent, which is a β-sheet OMP agent. The β-sheet OMP agent may comprise one or more (e.g., at least two) β-strands. The β-strands may be stabilized by hydrogen bonds, disulfide linkages, homodetic cyclic structures, or any combination thereof. In some embodiments, the OMP agent is a β-sheet OMP agent selected from lactoferricin B. protegrin-1, polymephusin, tachyplesin, or gomesin.

[0227] In an aspect, an OMP agent is peptide or peptide-based OMP agent, which is an extended OMP agent. The extended OMP agent may comprise a predominance of a few amino acids (e.g., Gly, Arg, Pro, Trp, or His). Extended OMPs may have poorly defined structure and / or lack typical secondary structure. However, due to the presence of a high proportion of a few amino acids, these peptides may adopt novel folds. Such OMP agents may adopt extended structures when they interact with a cell wall or cell membrane. An extend OMP agent may be stabilized by hydrogen bonding and / or van der Waals forces with lipids rather than the intrapeptide interactions commonly observed in other peptides, such as α-helical OMP agent or β-sheet OMP agent. In some embodiments, an extended OMP agent may an indolicidin family peptide. Indolicidin family peptides may be dominated by Pro. Trp, and Arg residues. In some embodiments, an extended OMP agent is a histatin family peptide. Histatin family peptides may be His-rich. In some embodiments, an extended OMP agent is a Pro- and Arg-rich OMP agent, for example a drosocin, pyrrhocoricin, or apidaecin family peptide. Some OMP agents belong to other families, such as mammalian PR-39, which is rich in Pro-Arg residues, but also belongs to the cathelicidin family of peptides.

[0228] In an aspect, an OMP agent is a peptide or peptide-based OMP agent comprising a membrane penetrating side-chain, for example, an acyl or hydrophobic chain. In some embodiments, the peptide-based OMP agent comprises one or more polymyxins (e.g., polymyxin types A, B, E, or a combination thereof). After binding to lipopolysaccharide in the outer membrane of gram-negative bacteria, polymyxins can disrupt the outer and / or inner membranes. In some embodiments, the OMP agent is polymyxin B. In some embodiments, the OMP agent comprises a mixture of polymyxins Bl, Bl-I, B2,72108869435.2Docket No.: 125930-872823 B3, and / or B6. In some embodiments, the OMP agent is colistin. The colistin may be colistin A or colistin B. In some embodiments, the OMP agent comprises polymyxins El, E2, or both

[0229] In an aspect, an OMP agent is a metal ion binding or a chelating agent. In some embodiments, the metal ion binding or a chelating agent is selected from an organic acid, optionally citric acid; EDTA; sodium bicarbonate; chitosan; or a charged molecule, optionally sodium polyphosphate or sodium hexametaphosphate.

[0230] In an aspect, an OMP agent is a cationic steroid. In some embodiments, the cationic steroid is selected from squalamine or SM-7.

[0231] In an aspect, an OMP agent is a polyamine. In some embodiments, the poly amine is selected from spermidine or pentamidine.

[0232] In an aspect, an OMP agent is an essential oil. In some embodiments, the essential oil is selected from carvacrol, thymol, eugenol, citral, geraniol, linalool, citronellol, limonene, or cinnamaldehyde. 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. A composition as described herein may further comprise one or more outer membrane penetrating agent, as described herein.

[0234] The composition may comprise a population of any of the endolysin polypeptides or fragments described 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. A composition comprising a population of any of the endolysin polypeptides or fragments as described herein may further comprise one or more outer membrane penetrating agent.

[0235] The composition may comprise two or more endolysin polypeptides. The two or more endolysin polypeptides may comprise at least two different endolysin domain architectures. The endolysin domain architecture may be selected from a PG1 MUR domain. GH24 domain, or an Ami_2 domain. The composition may comprise at least one endolysin comprising a PG1 MUR domain and at least one endolysin comprising an Ami_2 domain. The composition may comprise at least one endolysin comprising GH24 domain and at least one endolysin comprising an Ami_2 domain. In certain embodiments, the composition may comprise an endolysin comprising an amino acid sequence selected from any one of SEQ ID NOS: 1364. 1381. 1187. 1152. 1232. 1416. or 1158; and an endolysin73108869435.2Docket No.: 125930-872823 comprising an amino acid sequence selected from any one of SEQ ID NOS:1288; 1188; 1097; 1094; or 1266. In certain embodiments, the composition may comprise an endolysin comprising an amino acid sequence selected from any one of SEQ ID NOS: 1139. 1507. 1259, or 1166; and an endolysin comprising an amino acid sequence selected from any one of SEQ ID NOS:1288; 1188; 1097; 1094; or 1266.

[0236] 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. A composition comprising a population of host cells as described herein may further comprise one or more outer membrane penetrating agent.

[0237] 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 or Saccharomyces cerevisiae, 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 or Saccharomyces cerevisiae, 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. A wholecell or lysate composition as described herein may further comprise one or more outer membrane penetrating agent.

[0238] 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 or Saccharomyces cerevisiae, an algal cell such as 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. A dried culture medium composition as described herein may further comprise one or more outer membrane penetrating agent.74108869435.2Docket No.: 125930-872823

[0239] 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 physiologically acceptable buffer, or one or more OMP agent as described herein.

[0240] Any of the above-described compositions may be encapsulated in a material which surrounds and protects the endolysin and / or OMP agent 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). For a coating, 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.

[0241] 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.

[0242] 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 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. Such formulations may further comprise one or more outer membrane penetrating agent, as described herein.

[0243] 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 75108869435.2Docket No.: 125930-872823 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.

[0244] 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).

[0245] 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 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 diglycerides.

[0246] 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 a76108869435.2Docket No.: 125930-872823 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.

[0247] 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.

[0248] Pharmaceutical formulations suitable for oral administration may be provided in convenient unit forms including capsules, tablets, gels, pastes, ointments etc.

[0249] Formulations and compositions may comprise a thickener. Suitable thickeners include synthetic hectorite, Irish moss, iota carrageenan, gum tragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose, hydroxy butyl methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, and colloidal silica.

[0250] 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.

[0251] 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, com starch, or; 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.

[0252] 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.77108869435.2Docket No.: 125930-872823

[0253] 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 heptadecaethyleneoxy cetanol, 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.

[0254] 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.

[0255] 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 paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cety l alcohol. Sweetening agents and flavouring agents may’ be added. These compositions may be preserved by’ the addition of an antioxidant such as ascorbic acid.

[0256] 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 fonn in ampules, pre-filled syringes, small volume infusion or in multi-dose containers, e.g., with an added preservative.

[0257] 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.

[0258] 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 78108869435.2Docket No.: 125930-872823 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.

[0259] 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.

[0260] 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 may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilising agents, or suspending agents.

[0261] 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.

[0262] Any of the fonnulations and compositions described herein can take the fonn 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.

[0263] 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.

[0264] 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, appropriate79108869435.2Docket No.: 125930-872823 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

[0265] 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 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.

[0266] 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.

[0267] Generally, the antimicrobial is administered in a microbicidal amount. However, the antimicrobial can also be administered in a microbiostatic amount.

[0268] 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.

[0269] 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.

[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 80108869435.2Docket No.: 125930-872823 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 chicken, Gallus gallus domesticus, optionally a broiler or a layer chicken, or a domestic turkey, Meleagris gallopovo 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, Coturnixjaponica domestica. A foodstuff of the invention is suitable for consumption by a swine, optionally a sow, a piglet, a hog, or any other pig, further optionally Sus scrofa domesticus. A foodstuff of the invention is suitable for consumption by a ruminant, including cattle, sheep, horses, deer and goats, preferably Bos taurus, and further optionally Bos indicus. Consumption of the foodstuff may have a prophylactic or therapeutic effect on the animal. For example, consumption may reduce populations of gram-negative bacteria that thrive within the animal thus treating the animal or an infection caused by gram-negative bacteria.Consumption by an animal of the foodstuff may also prevent infection of gram-negative bacteria, or if the animal previously suffered from infection gram-negative bacteria, may have tire effect of preventing further infection as well as curing infection.

[0273] A foodstuff comprising an endolysin of the invention (in any form) may further comprise one or more outer membrane penetrating agent, as described herein.

[0274] 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.81108869435.2Docket No.: 125930-872823

[0275] 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.

[0276] 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 com, (ii) batching of die 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

[0277] 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. The supplemented water may also comprise one or more outer membrane penetrating agent, as described herein.

[0278] A supplemented water of the invention is suitable for consumption by animals, including poultry, preferably the domestic chicken, Gallus gallus domesticus, optionally a broiler or a layer chicken, or a domestic turkey. Meleagris gallopovo 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 swine, optionally a sow, a piglet, a hog, or any other pig, further optionally Sus scrofa domesticus. A supplemented water of the invention is suitable for consumption by a ruminant, including cattle, sheep, horses, deer and goats, preferably Bos taurus, and further optionally Bos indicus. Consumption of the supplemented water may have a82108869435.2Docket No.: 125930-872823 prophylactic or therapeutic effect on the animal. For example, consumption may reduce populations of gram-negative bacteria that thrive within the animal thus treating the animal or an infection caused by gram-negative bacteria. Consumption by an animal of the supplemented water may also prevent infection of gram-negative bacteria or if the animal previously suffered from infection by gram-negative bacteria, may have the effect of preventing further infection as well as curing infection.

[0279] 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.

[0280] 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. The supplemented hydrogel may also comprise one or more outer membrane penetrating agent, as described herein.

[0281] A supplemented hydrogel of the invention is suitable for consumption by animals, including poultry, preferably the domestic chicken, Gallus gallus domesticus, optionally a broiler or a layer chicken, or a domestic turkey, Meleagris gallopovo 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 hydrogel of the invention is suitable for consumption by a swine, optionally a sow, a piglet, a hog, or any other pig, further optionally Sus scrofa domesticus. A supplemented hydrogel of the invention is suitable for consumption by a raminant, including cattle, sheep, horses, deer and goats, preferably Bos taurus, and further optionally Bos indicus. Consumption of the supplemented hydrogel may have a prophylactic or therapeutic effect on the animal. For example, consumption may reduce populations of gram-negative bacteria that thrive within the animal thus treating the animal or an infection caused by gram-negative bacteria. Consumption by an animal of the supplemented hydrogel may also prevent infection of gram-negative bacteria or if the animal previously suffered from infection by gram-negative bacteria, may have the effect of preventing further infection as well as curing infection.83108869435.2Docket No.: 125930-872823

[0282] The hydrogel which is supplemented may be of any composition known in the art. The hydrogel may be comprised or be composed of. gum arable, 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.

[0283] 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

[0284] 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 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. In some embodiments, a kit comprises one or more outer membrane penetrating agent, as described herein.Methods of Using the Endolysin Polypeptides of the Invention

[0285] 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 arc 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 bacteriostatic and / or bactericidal properties.84108869435.2Docket No.: 125930-872823

[0286] 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.

[0287] An endolysin as described herein (e.g., an endolysin polypeptide) may be administered in combination with one or more outer membrane penetrating (OMP) agent, for use in any method as described herein. The OMP agent may enhance the antimicrobial (e.g., bacteriostatic and / or bactericidal properties) of an endolysin.

[0288] Also provided herein is a method of prevention or treatment of disease or condition in a subject, which method comprises administering an endolysin polypeptide of the invention to the subject in a prophylactically or therapeutically effective amount. The endolysin polypeptide may be co-administered with another agent. For example the other agent may be an antimicrobial agent, or an OMP agent. The endolysin polypeptide is preferably administered orally but may be administered by any suitable route including, for example, intravenous infusion, intradermal, subcutaneous, percutaneous, intramuscular, mtra-arterial, intraperitoneal, intraarticular, intraosseous or other appropriate administration routes. The amount of said endolysin 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 between 1 mg / kg and 2 mg / kg BW. The endolysin polypeptide may be administered on multiple occasions to the same subject.102891 Endolysin polypeptides of the invention are particularly useful in the treatment or prevention of a disease or condition mediated by bacterial infection such as bacterial infection by a gram-negative bacteria. The gram-negative bacteria of the disease or condition may be selected from Escherichia coli, Salmonella enterica. Pseudomonas aeruginosa, or Acinetobacter baumannii. The gram-negative bacteria of the disease or condition may be selected based on the specificity of the endolysin polypeptide (e.g., a catalytic domain, binding domain, or SAR domain). The disease or condition may be septicemia, bacteremia, pneumonia, spondylitis, enteric (e.g., gastrointestinal) infection, typhoid, orpullorum disease. Accordingly, the invention provides a polypeptide of the invention (e.g., endolysin polypeptide) for use in the treatment or prevention of a disease or condition characterised by presence of a gramnegative bacteria, optionally w herein the gram-negative bacteria is selected from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii.85108869435.2Docket No.: 125930-872823

[0290] 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 a gram-negative bacteria, optionally wherein the gramnegative bacteria is selected from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii.

[0291] 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.

[0292] Suitable dosage of an endolysin polypeptide of the invention may be determined by a skilled medical practitioner. Actual dosage levels of an endolysin polypeptide 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.

[0293] A suitable dose of an endolysin polypeptide may be in the range of from about 0.1 pg / kg to about 100 mg / kg body weight of the patient to be treated. For example, a suitable dosage may be from about 1 pg / kg to about 10 mg / kg body weight per day or from about 10 pg / kg to about 5 mg / kg body weight per day.

[0294] 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 86108869435.2Docket No.: 125930-872823 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.

[0295] 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 tire 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.Methods of Using Endolysin Compositions

[0296] A composition according to the invention, such as comprising an endolysin as described herein, optionally in combination with one or more OMP agent, polynucleotide, nucleic acid construct, endolysin polypeptide, vector, or a cell according to the invention can be used to treat animals, including humans, infected with a gram-negative bacteria, preferably a swine, optionally a sow, a piglet, a hog, or any other pig. further optionally Sus scrofa domesticus', a bovine animal; a ruminant, including cattle, sheep, horses, deer and goats, preferably Bos taurus, and further optionally Bos mdicus', a poultry animal, for example a domestic chicken, optionally a broiler or a layer chicken, optionally Gallus gallus domesticus'. a domestic turkey, optionally Meleagris gallopovo domesticus; a domestic duck, optionally derived from the mallard, further optionally Anas platyrhynchos domestica; a domestic duck, optionally derived from the Muscovy duck, further optionally Cairina moschata domestica; a goose, optionally a domestic goose, further optionally a domestic goose derived from the Greylag goose, further optionally Anser anser; a domestic goose derived from the Chinese goose, further optionally Anser cygnoides; a domestic pigeon, optionally Columba livia domestica; or a domestic quail, optionally Coturnix japonica domestica. 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.

[0297] A composition according to the invention, such as comprising an endolysin as described herein, optionally in combination with one or more OMP agent, 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 an gram-negative bacteria species present in a patient or in a cell of said patient, a cell line, or a cell free in vitro system, wherein preferably 99%, 90%, 80%. 70%, 60%, 50%, 40%. 30%, 20%, 10%,87108869435.2Docket No.: 125930-872823 5% or less of the initial amount of the gram-negative species is still detectable after treatment. In some embodiments, the gram-negative bacterial 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 gram-negative bacteria species may be an amount of alive bacteria. Gram-negative bacteria species may be detected using standard techniques known by the skilled person such as immunohistochemical techniques antibodies specific for the species of bacteria. Gram -negative bacteria species (alive and / or dead) may be detected using standard techniques known by the skilled person such as microbiological bacterial culture techniques and / or realtime quantitative reverse transcription polymerase chain reaction to assay for bacterial mRNA. Nonlimiting examples of how to detect gram-negative bacteria include enumeration of the bacteria 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 the bacteria species may be used. Types of probes may include fluorogenic and hydrolysis-type (5' nuclease) probes.|0298| Effectiveness of gram-negative bacteria 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.

[0299] 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.

[0300] 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 a gram-negative bacteria.

[0301] The invention further provides for a method of treatment, delay and / or prevention of a condition associated with infection of a subject with a gram-negative bacteria comprising administration an endolysin polypeptide according to the invention, or a composition 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 the disease or condition in a subject in need thereof.

[0302] 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 88108869435.2Docket No.: 125930-872823 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 swine, optionally a sow, a piglet, a hog. or any other pig, further optionally Sus scrofa domesticus, a ruminant, including cattle, sheep, horses, deer and goats, preferably Bos taunts, and further optionally Bos indicus, a domestic chicken, such as a broiler or a layer chicken, optionally Gallus gallus domesticus. Accordingly, subjects may include livestock such as the domestic turkey. Meleagris gallopovo domesticus, or a domestic duck derived from die 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 a dog or a cat, or a human subject.

[0303] In some embodiments, the animal is a swine, optionally a sow. a piglet, a hog. or any other pig, further optionally Sus scrofa domesticus, and the disease or disorder is post-weaning diarrhoea (PWD). In some embodiments, the animal is a poultry animal, and the disease or disorder is selected from avian colibacillosis, yolk sac infection, omphalitis, respiratory tract infection, swollen head syndrome, septicemia, polyserositis, coligranuloma, enteritis, cellulitis, or salpingitis.

[0304] The compound specifically targeting a gram-negative bacterial cell can be 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.|0305| The endolysin polypeptide according the invention, or composition according to the invention (e.g., further comprising an OMP agent), 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 gram-negative 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.

[0306] 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 tire 89108869435.2Docket No.: 125930-872823 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. It is contemplated an combining an endolysin of the invention, or a fragment or variant thereof with one or more OMP agent as described herein may increase antimicrobial activity.

[0307] 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. The whole-cell composition may further comprise one or more OMP agent.

[0308] 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 gram-negative bacterial infection. Any such antimicrobial formulation may be for use in the treatment of a disease or disorder caused by a gram-negative bacterial infection. The disease or disorder caused by the gram-negative bacterial infection may be septicemia, bacteremia, pneumonia, spondylitis, enteric infection, typhoid, pullorum disease, post-weaning diarrhoea (PWD). avian colibacillosis, yolk sac infection, omphalitis, respiratory’ tract infection, swollen head syndrome, septicemia, polyserositis, coligranuloma, enteritis, cellulitis, or salpingitis. The formulation may further comprise one or more OMP agent as described herein.

[0309] 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. One or more OMP agents as described herein may be administered in combination with any formulation as described herein.

[0310] In any of the above-described antimicrobial formulations or methods the antimicrobial activity of die endolysin polypeptide, fragment or variant may be bacterioly tic 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 a gram-negative bacteria.90108869435.2Docket No.: 125930-872823

[0311] In any of the above-described methods, the gram-negative bacteria may be selected from Escherichia coli. Salmonella enterica. Pseudomonas aeruginosa, or Acinetobacter baumanni.EXAMPLESExample 1: Endolysin selection from Bioinformatics Searches

[0312] Twenty sequences from the putative endolysin sequences targeting Salmonella were selected for further investigation. The list of the endolysin sequences is provided in TABLE 3.

[0313] TABLE 3: Selected Endolysin SequencesEndolysin SEQ ID No Protein Size, amino acids Architecture Laboratory IDSalEL-1 SEQ ID 1364 264 PG1 MURSalEL-2 SEQ ID 1381 264 PG1 MURSalEL-3 SEQ ID 1187 263 PG1 MURSalEL-4 SEQ ID 1152 264 PG1 MURSalEL-5 SEQ ID 1232 264 PG1 MURSalEL-6 SEQ ID 1416 264 PG1 MURSalEL-7 SEQ ID 1158 264 PG1 MURSalEL-8 SEQ ID 1411 199 NDSalEL-9 SEQ ID 1263 205 NDSalEL-10 SEQ ID 1308 205 NDSalEL-11 SEQ ID 1139 163 GH24SalEL-12 SEQ ID 1307 114 PET M15SalEL-13 SEQ ID 1156 205 SLT relatedSalEL-14 SEQ ID 1259 182 GH24SalEL-15 SEQ ID 1288 151 Ami 2SalEL-16 SEQ ID 1188 151 Ami 2SalEL-17 SEQ ID 1097 151 Ami 2SalEL-18 SEQ ID 1094 151 Ami 2SalEL-19 SEQ ID 1266 151 Ami-2SalEL-20 SEQ ID 1166 166 GH24Example 2: Strain construction

[0314] The 20 sequences selected from the bioinformatics searches (Table Y) were codon optimized for expression in Bacillus subtilis, using its most frequently used codons. Sequences were domesticated for Golden Gate cloning by removing Bsal and SapI sites, while conserving the original amino acid sequence, and short sequences were added to either end of the endolysin to facilitate cloning into vectors. After sequence optimization, sequences were synthesized and the DNA fragment containing the sequence was 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.91108869435.2Docket No.: 125930-872823

[0315] 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. Typically two transformants (one transformant, in the case of SalEL-7) for each endolysin, which exhibited the correct resistance profiles were selected, issued with a Laboratory Strain ID, and used to prepare glycerol stocks for the cryopreservation of the strains at -80°C. The Laboratory Strain IDs which correspond to the Endolysin Laboratory ID No (and SEQ ID No) are listed in TABLE 4, below. Of the 20 endolysins initially selected (see TABLE 3) we obtained at least one transformant was obtained for 19 endolysins (see TABLE 4).

[0316] Genomic DNA was prepared from each of the strains listed in TABLE 4 and the DNA sequences of the endolysin genes inserted into them was determined by Sanger. The DNA sequences of the endolysin genes obtained was in most cases was expected, however, in three cases the endolysin genes in the successful strains comprised point mutations that altered the amino acid sequences.

[0317] Production of endolysin of strain bAL529 (SalEL-2A) resulted in an amino acid sequence of MAILKLGNRGTEVKALQDSLNKIGFTLVADGIFGKATENAVKTVQAGAGLVIDGIVGPKTSYAI RNAGDAHQEHLTEADLVDAARELGVELASMKAVNQVESRGTGFTKTGKIKTLFERHIMYKKV AAKFGQARANALYQLYPTLVNPNSGGYIGGDAELERLQGVIALDEDCAYESASYGLFQIMGFNC QICGYSNAKEMFTDFLTGERAHLLAFVKFIKADANMWKALKNKNWAEFARRYNGPAYAKNQ YDTKLAAAYKSFC (SEQ ID NO: 1505), a variant of SEQ ID NO: 1381 having a valine at position 166 instead of an alanine.

[0318] Production of endolysin of strain bAL535 (SalEL-5A) resulted in an amino acid sequence of MAILKLGNRGSEVKDLQQSLNKIGFSLTADGIFGKATENAVKSVQAGAGLVIDGIAGPKTFYAIR NAGDAHQEHLTEADLVDAARELGIELASMKAVNQVESRGTGFTKTGKIKTLFERHIMYKKVTA KFGQ ARAN ALYQLYPTLVNPNSGGYIGGD AELERLQGAIALD EDC AYES ASYGLFQIMGFNCQI CGYSNAKEMFTDFLTGERAHLLAFVKFIKADANMWKALKNKNWAEFARRYNGPAYAKNQYD TKLAVAYKSFC (SEQ ID NO: 1506), a variant of SEQ ID NO: 1232 having an aspartic acid at position 15 instead of an alanine.

[0319] Production of endolysin of strain bAL544 (SalEL-11 A) resulted in an amino acid sequence of MKRTISNNGLKFTAAWEQLKTKAYRATSQEKYLTIGFGHYGPDVKPGDTITEDGTYNLLSQDM DEAVKLADSIASYKFNQAQFDAICDLIFNVGPKAVAAGTGTGQAIRTGDVETLRTKLPQFRNQL92108869435.2Docket No.: 125930-872823 GKPYLGIYRRAIGRLALFDGKTWSEAERIGREVKSL (SEQ ID NO: 1507), a variant ofSEQ ID NO: 1139 having a threonine at position 55 instead of an alanine.

[0320] TABLE 4: Tested Endolysin SequencesEndolysin SEQ ID NO Laboratory Molecular Protein of Figure Laboratory strain ID weight, correct size no of ID Da observed sample SalEL-1 SEQ ID NO: 1364 bAL526 28681.63 YES 2 SalEL-1 SEQ ID NO: 1364 bAL527 28681.63 YES 2 SalEL-2 SEQ ID NO: 1381 bAL528 28780.8 NO 2 SalEL-2A SEQ ID NO: 1505 bAL529 28780.8 YES 2 SalEL-3 SEQ ID NO: 1187 bAL530 28697.68 NO 2 SalEL-3 SEQ ID NO: 1187 bAL531 28697.68 NO 2 SalEL-4 SEQ ID NO: 1152 bAL532 28823.86 YES 2 SalEL-4 SEQ ID NO: 1152 bAL533 28823.86 YES 2 SalEL-5 SEQ ID NO: 1232 bAL534 28857.87 YES 2 SalEL-5A SEQ ID NO: 1506 bAL535 28857.87 YES 2 SalEL-6 SEQ ID NO: 1416 bAL536 28831.79 NO 2 SalEL-6 SEQ ID NO: 1416 bAL537 28831.79 NO 2 SalEL-7 SEQ ID NO: 1158 bAL539 28795.82 YES 3 SalEL-8 SEQ ID NO: 1411 bAL566 22575.28 NO 4 SalEL-8 SEQ ID NO: 1411 bAL567 22575.28 NO 4 SalEL-9 SEQ ID NO: 1263 bAL540 23363.34 NO 3 SalEL-9 SEQ ID NO: 1263 bAL541 23363.34 YES 3 SalEL-10 SEQ ID NO: 1308 bAL542 23513.49 YES 3 SalEL-10 SEQ ID NO: 1308 bAL543 23513.49 YES 3 SalEL-11A SEQ ID NO: 1507 bAL544 18028.98 YES 3 SalEL-11 SEQ ID NO: 1139 bAL545 18028.98 YES 3 SalEL-12 SEQ ID NO: 1307 bAL546 12568.8 NDSalEL-12 SEQ ID NO: 1307 bAL547 12568.8 NO* 3 SalEL-13 SEQ ID NO: 1156 bAL552 22891.88 NO 4 SalEL-13 SEQ ID NO: 1156 bAL5...

Claims

Docket No.: 125930-872823CLAIMSWhat is claimed is:An endolysin polypeptide, comprisingan 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:1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1152, 1232, 1506, 1158; 1505, 1416, 1507, 1187. 1364, 1097, 1166, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087. 1088. 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107. 1108, 1109, 1110, 1111, 1112. 1113. 1114. 1115. 1116, 1117. 1118, 1119, 1120, 1121, 1122, 1123, 1124. 1125. 1126. 1127. 1128. 1129. 1130. 1131. 1132. 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141. 1142. 1143. 1144. 1145. 1146. 1147. 1148, 1149, 1150, 1151, 1153, 1154, 1155, 1156, 1157. 1159. 1160. 1161. 1162. 1163. 1164. 1165, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175. 1176. 1177. 1178. 1179. 1180. 1181, 1182, 1183, 1184, 1185, 1186, 1188, 1189, 1190, 1191, 1192, 1193. 1194. 1195. 1196. 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208. 1209. 1210. 1211. 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224. 1225. 1226. 1227, 1228, 1229, 1230, 1231, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241. 1242. 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256. 1257. 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272. 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440. 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474. 1475, 1476, 1477, 1478, 1479. 1480. 1481. 1482. 1483. 1484. 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, or 1502, or a fragment thereof.102108869435.2Docket No.: 125930-872823 2. An endolysin polypeptide, comprisingan 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 SEQ ID NO: 1097, or a fragment thereof.

3. An endolysin polypeptide, comprisingan 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 SEQ ID NO: 1166, or a fragment thereof.

4. An endolysin polypeptide, comprising(a) the amino acid sequence of a peptidoglycan binding domain, wherein the peptidoglycan binding domain comprises an amino acid sequence of a fragment of the sequence set forth in any one of SEQ ID NOS:1101, 1106, 1108. 1113. 1152. 1158. 1187. 1232. 1252.1263, 1308, 1364, 1379, 1380, 1381, 1383. 1384. 1411. 1415. 1416. 1417. or 1461; and (b) a catalytic domain targeting die cell wall peptidoglycan catalytic domain of a gramnegative bacteria.

5. The endolysin polypeptide of any one of claims 1-4, wherein the endolysin polypeptide is purified or isolated.

6. The endolysin polypeptide of any one of claims 1-5, wherein the endolysin polypeptide has antimicrobial activity that is endolysin activity, optionally wherein the endolysin activity is assessed by the rate of degradation of peptidoglycan (PGN).

7. The endolysin polypeptide of any one of claims 1-6, wherein the endolysin polypeptide has antimicrobial activity that is bacteriolytic activity and / or bacterial growth inhibitory activity.

8. The endolysin polypeptide of claim 7. wherein the antimicrobial activity is against Salmonella enterica.

9. The endolysin polypeptide of claim 7, wherein the antimicrobial activity is against Pseudomonas aeruginosa or Acinetobacter baumannii.

10. The endolysin polypeptide of any one of claims 7-9, wherein the antimicrobial activity is determined by a(a) cell viability assay:(b) cell turbidity reduction assay;(c) minimum inhibitory concentration (MIC) assay or minimum bactericidal concentration (MBC) assay; or103108869435.2Docket No.: 125930-872823 (d) a focused checkerboard assay.optionally wherein the assay is performed against Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii.

11. A composition, comprising the endolysin peptide of any one of claims 1-10 and one or more outer membrane penetrating (OMP) agent, optionally wherein the OMP agent is selected from a peptide, metal ion binding agent or chelating agent, cationic steroid, polyamine, or an essential oil, further optionally wherein the peptide is selected from polymyxin B or colistin.

12. A recombinant nucleic acid molecule comprising a first nucleic acid sequence encoding the endolysin polypeptide of any one of claims 1-10, 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.

13. The recombinant nucleic acid molecule of claim 12, which comprises the first and second nucleic acid sequences.

14. A recombinant nucleic acid molecule comprising a first nucleic acid sequence encoding the endolysin polypeptide of any one of claims 1-10, and a second nucleic acid sequence encoding one or more outer membrane penetrating (OMP) agent, optionally wherein the OMP agent is selected from a peptide, metal ion binding agent or chelating agent, cationic steroid, polyamine, or an essential oil, further optionally wherein the peptide is selected from polymyxin B or colistin.

15. An expression vector comprising the recombinant nucleic acid molecule of claim 12.

16. An expression vector comprising the recombinant nucleic acid molecule of claim 13.

17. An expression vector comprising the recombinant nucleic acid molecule of claim 1418. The recombinant nucleic acid molecule of claim 13 or the expression vector of claim 16, wherein the promoter is capable of promoting expression of the endolysin polypeptide in a cell of a unicellular microorganism.

19. The recombinant nucleic acid molecule of claim 13 or the expression vector of claim 16. wherein the promoter is capable of promoting expression of the endolysin polypeptide in Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Chlamydomonas reinhardtii.

20. A host cell comprising the recombinant nucleic acid molecule or expression vector of any one of claims 12-19.104108869435.2Docket No.: 125930-872823 21. The host cell of claim 20. which is a Bacillus subtilis. Pichia pastoris. Saccharomyces cerevisiae.or Chlamydomonas reinhardtii cell.

22. A composition, comprising two or more endolysin polypeptides according to any one of claims 1-12, optionally wherein the two or more endolysin polypeptides comprises at least two endolysin domain architectures selected from a PG1 MUR domain, GH24 domain, or an Ami_2 domain, further optionally wherein the composition comprises at least one endolysin comprising a PG1 MUR domain and at least one endolysin comprising an Ami_2 domain; or at least one endolysin comprising GH24 domain and at least one endolysin comprising an Ami_2 domain.

23. The composition of claim 22. comprising(a) an endolysin comprising an amino acid sequence selected from any one ofSEQ ID NOS: 1364. 1381. 1187. 1152. 1232. 1416. or 1158; and an endolysin comprising an amino acid sequence selected from any one of SEQ ID NOS: 1288; 1188; 1097; 1094; or 1266; or(b) an endolysin comprising an amino acid sequence selected from any one ofSEQ ID NOS: 1139, 1507, 1259. or 1166; and an endolysin comprising an amino acid sequence selected from any one of SEQ ID NOS: 1288; 1188; 1097; 1094; or 1266.

24. A cell lysate comprising the one or more endolysin polypeptides according to any one of claims 1-10, optionally wherein the cell lysate further comprises one or more outer membrane penetrating (OMP) agent, further optionally wherein the OMP agent is selected from a peptide, metal ion binding agent or chelating agent, cationic steroid, polyamine, or an essential oil, further optionally wherein the peptide is selected from polymyxin B or colistin.

25. The cell lysate of claim 24, wherein the one or more endolysin polypeptides is expressed from the recombinant nucleic acid molecule or expression vector of any one of claims 12-19, or is expressed by a population of one or more host cells according to claim 20 or 21.

26. A dried whole culture comprising the one or more endolysin polypeptides according to any one of claims 1-10, wherein the dried whole culture is spray dried or freeze dried, optionally wherein the dried whole culture further comprises one or more outer membrane penetrating (OMP) agent, further optionally wherein the OMP agent is selected from a peptide, metal ion binding agent or chelating agent, cationic steroid, poly amine, or an essential oil, further optionally wherein the peptide is selected from polymyxin B or colistin.

27. A dried whole culture 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 12-19, or is expressed by a population of one or more host cells according to claim 20 or 21.105108869435.2Docket No.: 125930-872823 28. A dried culture medium comprising the one or more endolysin polypeptides according to any one of claims 1-10. wherein the dried culture medium is spray dried or freeze dried, optionally wherein the dried culture medium further comprises one or more outer membrane penetrating (OMP) agent, further optionally wherein the OMP agent is selected from a peptide, metal ion binding agent or chelating agent, cationic steroid, polyamine, or an essential oil, further optionally wherein the peptide is selected from polymyxin B or colistin..

29. The dried culture medium of claim 28, wherein the one or more endolysin polypeptides is expressed from the nucleic acid molecule or expression vector of any one of claims 12-19, or is expressed by a population of one or more host cells according to claim 20 or 21.

30. A dried material comprising the one or more endolysin polypeptides according to any one of claims 1-10, the population of one or more host cells according to claim 20 or 21, and / or the cell lysate according to claim 24 or 25, optionally wherein the dried material is spray dried or freeze dried, further optionally wherein the dried whole culture further comprises one or more outer membrane penetrating (OMP) agent, further optionally wherein the OMP agent is selected from a peptide, metal ion binding agent or chelating agent, cationic steroid, polyamine, or an essential oil, further optionally wherein the peptide is selected from polymyxin B or colistin.

31. A combined material comprising two or more of(i) the one or more endolysin polypeptides according to any one of claims 1-10; (ii) the composition of any one of claims 11, 22. or 23;(iii) the population of one or more host cells according to claim 20 or 21; (iv) the cell lysate according to claim 24 or 25;(v) the dried whole culture of claim 26 or 27;(vi) the dried culture medium of claim 28 or 29;(vii) the dried material of claim 30,optionally wherein the combined material is dried, further optionally wherein the combined material is spray dried or freeze dried.

32. A method for producing an endolysin polypeptide having antimicrobial activity, the method comprising culturing a host cell comprising the recombinant nucleic acid or expression vector of any one of claims 12-19 under conditions in which the endolysin polypeptide is expressed.

33. The method of claim 32, further comprising lysing the host cell to produce a cell lysate comprising the endolysin polypeptide.

34. The method of claim 32 or 33, further comprising purifying or isolating the endolysin polypeptide.106108869435.2Docket No.: 125930-872823 35. The method of claim 32 or 33, which does not comprise further purification or isolation of the endolysin polypeptide.

36. An animal foodstuff comprising: (a) one or more foodstuffs; and (b) one or more endolysin polypeptides according to any one of claims 1-10 or the composition according to any one of claims 11, 22, or 23.

37. 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 24 or 25; and / or(iii) the dried whole culture of claim 26 or 27; and / or(iv) the dried culture medium of claim 28 or 29; and / or(v) the dried material of claim 30; and / or(vi) the combined material of claim 31.

38. A supplemented water comprising: (a) water; and (b) one or more endolysin polypeptides according to any one of claims 1-10, or the composition according to any one of claims 11, 22, or 23.

39. A supplemented water comprising:(a) water; and(i) the host cell of claim 20 or 21; and / or(ii) the cell lysate of claim 24 or 25; and / or(iii) the dried whole culture of claim 26 or 27; and / or(iv) the dried culture medium of claim 28 or 29; and / or(v) the dried material of claim 30; and / or(vi) the combined material of claim 31.

40. A supplemented hydrogel comprising: (a) hydrogel; and (b) one or more endolysin polypeptides according to any one of claims 1-10 or the composition according to any one of claims 11, 22, or 23.

41. A supplemented hydrogel comprising:(a) hydrogel; and(i) the host cell of claim 20 or 21; and / or(ii) the cell lysate of claim 24 or 25; and / or(iii) the dried whole culture of claim 26 or IT, and / or(iv) the dried culture medium of claim 28 or 29; and / or(v) the dried material of claim 30; and / or107108869435.2Docket No.: 125930-872823 (vi) the combined material of claim 31.

42. A method for reducing a population of gram-negative bacteria in a foodstuff, the method comprising admixing with the foodstuff(i) the endolysin polypeptide of any one of claims 1-10; and / or(ii) the composition of any one of claims 11, 22, or 23; and / or(iii) the host cell of claim 20 or 21; and / or(iv) the cell lysate of claim 24 or 25; and / or(v) the dried whole culture of claim 26 or 27; and / or(vi) the dried culture medium of claim 28 or 29; and / or(vii) the dried material of claim 30; and / or(viii) the combined material of claim 31.

43. 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 1-10; and / or(ii) the composition of any one of claims 11. 22, or 23; and / or(iii) the host cell of claim 20 or 21; and / or(iv) the cell lysate of claim 24 or 25; and / or(v) the dried whole culture of claim 26 or IT, and / or(vi) the dried culture medium of claim 28 or 29; and / or(vii) the dried material of claim 30; and / or(viii) the combined material of claim 31.wherein the disease or disorder is caused by a gram-negative bacteria, optionally wherein the gram-negative bacteria is selected from Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii.

44. 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 36 or 37, and / or the supplemented water of claim 38 or 39 and / or the supplemented hydrogel of claim 40 or 41 wherein the disease or disorder is caused by a gram-negative bacteria, optionally wherein the gram-negative bacteria is selected from Salmonella enterica, Pseudomonas aeruginosa, or Acinetobacter baumannii.

45. The method of claim 43 or 44, wherein the disease or disorder caused by a gram-negative bacteria is septicemia, bacteremia, pneumonia, spondylitis, enteric infection, typhoid, or pullorum disease.

46. The method of any one of claims 43-45. wherein the animal is selected from108108869435.2Docket No.: 125930-872823 (a) a swine, optionally a sow, a piglet, a hog, or any other pig, further optionally Sus scrofa domesticus;(b) a ruminant, including cattle, sheep, horses, deer and goats, preferably Bos taurus, and further optionally Bos indicus;(c) a chicken, optionally a broiler chicken or a layer chicken, further optionally Gallus gallus domesticus',(d) a turkey, optionally a domestic turkey, further optionally Meleagris gallopavo domesticus',(e) 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;(f) 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;(g) a pigeon, optionally a domestic pigeon; or(h) a quail, optionally a domestic quail.

47. The method of any one of claims 43-46. wherein(a) the animal is a swine, optionally a sow, a piglet, a hog, or any other pig, further optionally Sus scrofa domesticus, and the disease or disorder is post-weaning diarrhea (PWD);(b) the animal is a poultry animal, and the disease or disorder is selected from avian colibacillosis, yolk sac infection, omphalitis, respiratory tract infection, swollen head syndrome, septicemia, polyserositis, coligranuloma, enteritis, cellulitis, or salpingitis.

48. A kit comprising(a) an agent having antimicrobial activity, wherein the agent is selected from(i) the endolysin polypeptide of any one of claims 1-10; and / or(ii) the composition of any one of claims 11, 22, or 23; and / or(iii) the host cell of claim 20 or 21; and / or(iv) the cell lysate of claim 24 or 25; and / or(v) the dried whole culture of claim 26 or 27; and / or(vi) the dried culture medium of claim 28 or 29; and / or(vii) the dried material of claim 30; and / or(viii) the combined material of claim 31; and(b) instructions for admixing the agent with a foodstuff.109108869435.2

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