Bacteriophages against vancomycin-resistant enterococci

Abstract

Described herein are bacteriophages that infect and lyse vancomycin-resistant Enterococci (VRE), which are useful, for example, for prophylactic or therapeutic treatment of subjects infected with Enterococci such as VRE or at risk for infection by Enterococci such as VRE, and other uses described herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 210,555 filed June 15, 2021, and the entire contents of that specification are incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy (created on 11 June 2021) is named 052209-0383_SL.txt and has a size of 1,952,761 bytes.

[0003] This specification describes a bacteriophage that infects and lyses vancomycin-resistant enterococci (VRE). This bacteriophage is useful, for example, for prophylactic or therapeutic treatment of subjects infected with enterococci such as VRE, or subjects at risk of infection by enterococci such as VRE, and for other uses described herein. [Background technology]

[0004] Bacteriophage Bacteriophages are viruses that infect bacteria and lyse them as part of their replication / lysis cycle. The name bacteriophage (or simply "phage") derives from the Greek word "phago," meaning "eating" or "bacteria-eating," and they were discovered in the first half of the 20th century by Felix D'Herelle. Phages are specific to their target bacterial host and do not infect human or other eukaryotic cells. Bacteriophages have been used therapeutically in humans since 1919 to target pathogenic bacteria. Early enthusiasm led to their use both for the prevention and treatment of bacterial diseases. In the United States, in the 1940s, Eli Lilly commercially manufactured six phage products for human use, including preparations targeting staphylococci, streptococci, and other respiratory pathogens. With the advent of antibiotics, the therapeutic use of phages has gradually declined in the United States and Western Europe, and research has largely ceased. However, bacteriophage therapy continued to be used in Eastern Europe.

[0005] Vancomycin-resistant enterococci (VRE) Enterococci are Gram-positive, facultative anaerobic cocci found in soil, food, water, animals, birds, and insects. In humans, enterococci are common colonizing bacterial species in the human intestinal and urogenital tracts. Enterococci cause a variety of infections, including urinary tract infections, intraperitoneal and pelvic wound infections, endocarditis, and bacteremia. Vancomycin-resistant enterococci (VRE) include strains of several different enterococcal species, and clinically significant VRE infections are known to be caused by Enterococcus faecium and Enterococcus faecalis. Although the classification of enterococci is not yet complete, it is generally accepted that this genus consists of 19 species.

[0006] Antibiotic management of severe enterococcal infections is always difficult due to the organism's inherent resistance to most antimicrobial agents. In the 1970s, enterococcal infections were treated with cell wall activators such as penicillin and aminoglycoside combinations. However, in the 1980s, enterococcal strains with high levels of aminoglycoside resistance and penicillin resistance emerged, mediated by both plasmid-encoded β-lactamase and changes in penicillin-binding proteins. Such organisms are called VREs due to their resistance to vancomycin; these organisms also exhibit resistance to penicillin-aminoglycoside combinations. Despite the availability of two drugs that are susceptible to VRE (quinupristin / dalfopristin and linezolid (see, e.g., Plouffe, Clin. Infect Dis. 31 (Supp. 4): S144-49 (2000)), this microorganism remains a significant cause of morbidity and mortality in immunocompromised patients. Infections caused by VRE present as a particularly serious problem among the elderly, immunocompromised, immunosuppressed, and / or critically ill patients in cancer centers and organ transplant wards. VRE is readily transmitted within hospital settings, and many hospital-acquired outbreaks have been reported. While humans serve as the primary reproductive host, the organism can be readily isolated from the surrounding environment of infected / colonized patients (e.g., bedding, furniture, and personal and household items).

[0007] While attention is focused on the effects of VRE infection on critically ill and immunocompromised patients, intestinal colonization by VRE is becoming relatively common among patients in general hospitals. Once colonies form due to VRE, patients may continue to colonize for life, and those who have colonized are at risk of developing severe blood infections and wound infections due to VRE, for example, due to cancer chemotherapy or immunosuppressive therapy (which may be associated with transplantation), and subsequently becoming immunocompromised. [Overview of the project] [Problems that the invention aims to solve]

[0008] Treatment of VRE infection VRE bacteria are inherently resistant to many antimicrobial agents, and developing novel antibiotics effective against VRE has proven extremely difficult. Treatment of VRE infections with two FDA-approved antibiotics (quinupristin / dalfopristin and linezolid) has been moderately successful, and VRE strains resistant to quinupristin / dalfopristin and linezolid have already been identified. Therefore, there is still an urgent need for effective drugs against VRE, for example, to reduce or prevent the risk of VRE infection and / or to treat patients already infected with VRE. [Means for solving the problem]

[0009] The present invention provides a bacteriophage that infects and lyses VRE. This bacteriophage can be incorporated into compositions such as over-the-counter (OTC) compositions or pharmaceutical compositions for use in reducing, preventing, or treating the risk of VRE infection, can be used to produce vaccine / bacterin compositions, and can be used in assays for detecting VRE.

[0010] Therefore, an isolated bacteriophage is provided that infects and lyses one or more strains of vancomycin-resistant enterococcus (VRE), and this bacteriophage is (i) bacteriophage strain VREML237-2 deposited with ATCC under accession number PTA-126934, or a variant thereof, wherein this variant strain has at least 80% of the genome-wide mean nucleotide identity (gANI) of the bacteriophage strain VREML237-2. (ii) bacteriophage strain VREML237-2 or its variant strain, deposited with ATCC under accession number PTA-126932, wherein the variant strain has at least 80% of the gANI of the bacteriophage strain VREML110-1 or its variant strain; (iii) bacteriophage strain VREML110-1 or its variant strain, deposited with ATCC under accession number PTA-126931 (iv) Bacteriophage strain VREML105 or its variant strain deposited with TCC, wherein the variant strain has at least 80% of the gANI of bacteriophage strain VREML105; (iv) Bacteriophage strain VREML202-1 or its variant strain deposited with ATCC under accession number PTA-126933, wherein the variant strain is the same as bacteriophage (v) bacteriophage strain VREML202-1 or its variant strain having at least 80% of the gANI compared to strain VREML202-1; (v) bacteriophage strain VREML85-2 or its variant strain deposited with ATCC under accession number PTA-126930, wherein the variant strain has at least 80% of the gANI compared to the bacteriophage strain VREML85-2;(vi) Bacteriophage strain VREML110-2 or its variant strain deposited with ATCC under accession number PTA-127012, wherein this variant strain has at least 80% of the gANI of bacteriophage strain VREML110-2; (vii) Bacteriophage strain VREML237-1 or its variant strain deposited with ATCC under accession number PTA-127016, wherein this variant strain (viii) bacteriophage strain VREML237-1 or its variant strain having at least 80% of the gANI compared to bacteriophage strain VREML237-1; (viii) bacteriophage strain VREML85-1 or its variant strain deposited with ATCC under accession number PTA-127011, wherein this variant strain has at least 80% of the gANI compared to bacteriophage strain VREML85-1; (ix (x) Bacteriophage strain VREML137-2 or its variant strain deposited with ATCC under accession number PTA-127013, wherein this variant strain has at least 80% of the gANI of bacteriophage strain VREML137-2; (x) Bacteriophage strain VREML202-2 or its variant strain deposited with ATCC under accession number PTA-127015, wherein this variant strain has at least 80% of the gANI of bacteriophage strain VREML137-2; (xi) Bacteriophage strain VREML202-2 or its variant strain having at least 80% of the gANI of bacteriophage strain VREML202-2; (xi) bacteriophage strain VREML139 or its variant strain deposited with ATCC under accession number PTA-127014, wherein the variant strain is selected from bacteriophage strain VREML139 or its variant strain having at least 80% of the gANI of bacteriophage strain VREML139.

[0011] This isolated bacteriophage may be a variant of one of the deposited bacteriophage strains having at least 90% of the gANI of the deposited bacteriophage strain. This isolated bacteriophage may be a variant of one of the deposited bacteriophage strains having at least 95% of the gANI of the deposited bacteriophage strain. This isolated bacteriophage may be the R of the deposited bacteriophage strain. FL P DNA profile is substantially equivalent to R FL This could be a variant strain of one of the deposited bacteriophage strains that possesses a P DNA profile.

[0012] Also provided are isolated offspring bacteriophages of the deposited bacteriophage strains disclosed herein, which have 80% or more of the gANI of the deposited bacteriophage strain. These isolated offspring bacteriophages may have 95% or more of the gANI of the deposited bacteriophage strain. These isolated offspring bacteriophages may have 98% or more or 99.9% or more of the gANI of the deposited bacteriophage strain.

[0013] Also provided are compositions comprising one or more bacteriophages from among the bacteriophage strains disclosed herein and pharmaceutically acceptable salts. The one or more bacteriophage strains in the composition are (i) bacteriophage strain VREML237-2 or a variant thereof deposited with ATCC under accession number PTA-126934, wherein the variant has at least 80% of the gANI of bacteriophage strain VREML237-2; (ii) bacteriophage strain VREML237-2 or a variant thereof under accession number PTA-1269 (iii) Bacteriophage strain VREML110-1 or its variant strain deposited with ATCC under accession number PTA-126931, wherein the variant strain has at least 80% of the gANI of the bacteriophage strain VREML110-1; (iii) Bacteriophage strain VREML105 or its variant strain deposited with ATCC under accession number PTA-126931 (iv) bacteriophage strain VREML105 or its variant strain having at least 80% of the gANI compared to bacteriophage strain VREML105; and bacteriophage strain VREML202-1 or its variant strain deposited with ATCC under accession number PTA-126933, wherein this variant strain has at least 80% of the gANI compared to bacteriophage strain VREML202-1. (v) bacteriophage strain VREML202-1 or its variant strain having I; (v) bacteriophage strain VREML85-2 or its variant strain deposited with ATCC under accession number PTA-126930, wherein the variant may include or consist of bacteriophage strain VREML85-2 or its variant strain having at least 80% of the gANI of bacteriophage strain VREML85-2.The bacteriophage strains in this composition may include or consist of (i) bacteriophage strain VREML237-2; (ii) bacteriophage strain VREML110-1; (iii) bacteriophage strain VREML105; (iv) bacteriophage strain VREML202-1; and (v) bacteriophage strain VREML85-2.

[0014] This composition may be provided in an oral dosage form optionally with an enteric coating, and may be provided in a form selected from tablets, hard gel capsules, soft gel capsules, sugar-coated tablets, powders, granules, solutions, suspensions, dispersions, syrups, and microgels. This composition may be provided in a rectal dosage form, and may be provided in a rectal dosage form optionally, as suppositories, enemas, rectal foams, lotions, or gels. This composition may be provided in a vaginal dosage form, and may be provided in a suppository, cream, vaginal foam, lotion, or gel. This composition may be provided in a topical dosage form, and may be provided in a lotion, cream, lotion, gel, or spray. This composition may be provided in a pulmonary dosage form, and may be provided in a powder, aerosol, nebulizer, or inhaler composition. This composition may be provided in an injectable dosage form.

[0015] In any embodiment, the composition optionally further comprises a probiotic. The probiotic may comprise probiotic bacteria, for example, one or more selected from the group consisting of *L. acidophilus*, *L. rhamnosus*, *L. gasseri*, *L. reuteri*, *L. bulgaricus*, *L. plantarum*, *L. johnsonii*, *L. paracasei*, *L. casei*, *L. salivarius*, *L. lactis*, *B. bifidum*, *B. longum*, *B. breve*, *B. infantis*, *B. lactis*, *B. adolescentis*, *Streptococcus thermophilus*, *Bacillus cerus*, *Bacillus subtilis*, and any combination thereof. The probiotic may comprise probiotic yeast, for example, including one or more of *Saccharomyces cerevisiae*, *Saccharomyces boulardii*, *Saccharomyces cerevisiae* var. *boulardii*, *Issatchenkia occidentalis*, *Lachancea thermotolerans*, *Metschnikowia ziziphicola*, *Torulaspora delbrueckii*, and any combination thereof.

[0016] Also provided is a method of reducing the risk of, preventing, or treating VRE colonization or infection in a subject in need thereof, or modulating the microbiome of a human subject, comprising administering to the subject a bacteriophage or composition disclosed herein.

[0017] Also provided are bacteriophages and compositions disclosed herein for use in reducing the risk of, preventing, or treating VRE colonization or infection in a subject in need thereof, or modulating the microbiome of a human subject.

[0018] Also provided is the use of a bacteriophage or composition disclosed herein in the manufacture of a medicament for reducing the risk of, preventing, or treating VRE colonization or infection in a subject in need thereof, or modulating the microbiome of a human subject.

[0019] In any such method, composition for use, or use, the subject may be at risk of, or may be colonized or infected with, VRE, and optionally, the subject may be an immunosuppressed or immunocompromised subject. In any such method, composition for use, or use, the subject may have VRE intestinal colonization, and the method may be effective to reduce or eliminate VRE intestinal colonization.

[0020] In some embodiments of the methods, bacteriophages or compositions for use, or uses disclosed herein, the treatment further comprises administering a probiotic to the subject, and optionally the probiotic is provided in the same composition as the bacteriophage.

[0021] In some embodiments of the methods, bacteriophages or compositions for use disclosed herein, or use, the bacteriophage or composition or pharmaceutical is administered orally.

[0022] In some embodiments of the methods, bacteriophages or compositions for use disclosed herein, or use, the bacteriophages, compositions, or pharmaceuticals are administered rectally.

[0023] In some embodiments of the methods, bacteriophages or compositions for use disclosed herein, or use, the bacteriophage, composition, or pharmaceutical product is administered topically.

[0024] In some embodiments of the methods, bacteriophages or compositions for use disclosed herein, or use, the bacteriophages, compositions, or pharmaceuticals are administered vaginally.

[0025] Also provided is a composition comprising a lytic enzyme produced by a bacteriophage disclosed herein.

[0026] Also provided is a composition comprising a derivative product of a bacteriophage disclosed herein, wherein the derivative product has activity against VRE or encodes a product having activity against VRE, and optionally, the derivative product is one or more selected from DNA, cDNA, mRNA and synthetic polynucleotide sequences, DNA / RNA hybrids, and anti-sigma factor genes and their expression products.

[0027] Also provided are a vaccine comprising a VRE bacterial lysate obtained by lysing a VRE strain with a bacteriophage or its lytic enzyme disclosed herein; a method of vaccinating a subject against VRE infection, comprising administering such a vaccine to the subject; such VRE bacterial lysates for use in the vaccination of a subject against VRE infection; and the use of such VRE bacterial lysates in the preparation of a pharmaceutical for the vaccination of a subject against VRE infection.

[0028] Also provided is a method for detecting VRE in a sample, comprising treating the sample with a bacteriophage or its lytic enzyme disclosed herein to specifically induce the release of a measurable VRE bacterial product, and measuring the released VRE bacterial product, wherein optionally the released VRE bacterial product is one or more of adenosine triphosphate (ATP) and protein kinase (AKT). In some embodiments, the sample is a fecal sample obtained from a subject. [Brief explanation of the drawing]

[0029] [Figure 1] The reference DNA RFLP profiles of the bacteriophages described herein are shown. [Figure 2] The results of evaluating the efficacy of the bacteriophage cocktails described herein in an in vitro human gut simulator model are shown. [Modes for carrying out the invention]

[0030] As described above, the bacteriophages described herein target VRE (i.e., infect and lyse VRE, causing, for example, lytic infection). These bacteriophages may be incorporated into compositions such as over-the-counter (OTC) compositions or pharmaceutical compositions, used to produce vaccine / bacterin compositions, and used in assays for detecting VRE, for use in reducing, preventing, or treating the risk of VRE infection. The bacteriophages and compositions containing them are useful in preventing or reducing VRE infection, such as preventing or reducing intestinal colonization by VRE, thereby preventing or reducing the risk of VRE infection, or treating VRE. While not bound by theory, it is understood that therapies using the bacteriophages and compositions described herein are safe and effective through specific and targeted prevention or reduction of VRE infection, thereby reducing invasive diseases caused by VRE, lowering the incidence, prevalence, and transmission of VRE infection, and furthermore, maintaining a healthy gut microbiome by specifically targeting VRE, and avoiding the side effects associated with broad-spectrum antibiotic therapy.

[0031] definition Technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which the present invention relates, unless otherwise defined. Any suitable materials and / or methods known to those skilled in the art may be used in the practice of the present invention, taking into account the guidance provided herein, but certain materials and methods are described for illustrative purposes only. The materials, reagents, and similar materials mentioned in the following description and examples are available from commercial sources unless otherwise noted.

[0032] As used herein, the singular forms "a," "an," and "it" refer to both singular and plural unless otherwise explicitly stated.

[0033] Where used herein, "about" means the number mentioned, and ±10% of that number, when used with a number. For example, "about 10" should be understood as both "10" and "9 to 11".

[0034] As used herein, the phrases in the form "A / B" or "A and / or B" mean (A), (B), or (A and B), and the phrases in the form "at least one of A, B, and C" mean (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0035] As used herein, the term “contains” means that the composition, method, or kit described contains at least the elements mentioned and may contain other elements not explicitly mentioned.

[0036] As used herein, the terms “effective dose” and “therapeutably effective dose” as used with respect to therapeutic activators (e.g., phages) mean the amount of the agent administered to a subject in need of such treatment to reduce, prevent, or mitigate the risk of VRE infection (or VRE colonization), for example, to produce a specific pharmacological effect. It is emphasized that a therapeutically effective dose, even if considered therapeutically effective by those skilled in the art, is not necessarily effective in preventing or reducing VRE infection (or VRE colonization) in a given subject. The therapeutically effective dose may vary depending on the specific activator, route of administration and dosage form, the subject’s age and weight, and / or the subject’s condition (e.g., type and severity of VRE infection).

[0037] The terms "individual," "subject," and "patient" are used interchangeably in this specification and refer to any individual mammalian subject, such as a human subject (e.g., male and female human subjects).

[0038] When used herein, "prevent" means reducing the risk of VRE infection or VRE colonization in the target population, preventing VRE infection or VRE colonization, or reducing the level of VRE infection or VRE colonization.

[0039] When used herein, "to treat" means to reduce the level of VRE infection in the subject concerned, for example, to reduce the VRE infection to an undetectable level or to eliminate VRE colonization.

[0040] The term VRE "colony formation" is used herein to refer to the presence of VRE in a subject that does not necessarily cause disease in the subject.

[0041] The term VRE "infection" is used herein to refer to VRE infiltration into the tissue of the subject that is causing disease in the subject.

[0042] When used herein, "vancomycin-resistant enterococci (VRE) strains" refer to VRE strains (isolated strains) having a minimum inhibitory concentration of vancomycin of at least 16 μg / ml, which can be evaluated by the assays described in the following examples. For example, see National Committee for Clinical Laboratory Procedures, "Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically." (3rd ed., 1993) (Nat. Committee for Clin. Lab. Standards, Villanova Pa.); National Committee for Clinical Laboratory Standards, "Performance Standards for Antimicrobial Disk Susceptibility Tests." (5th ed., 1993) (Nat. Committee for Clin. Lab. Standards, Villanova Pa.). VREs may belong to the following species: Enterococcus faecium, Enterococcus faecalis, E. gallinarum, E. casseliflavus, E. durans, E. avium, and E. raffinosis. VREs targeted by the phages described herein may typically be present in one or more of the gastrointestinal tract, skin, and genitals, but may also be found in other parts of the body. Non-limiting examples of diseases and conditions that may be caused by VREs include bacteremia (e.g., sepsis), meningitis, pneumonia, endocarditis, urinary tract infections, intra-abdominal infections (e.g., peritonitis), pelvic infections, skin and soft tissue infections (e.g., wound infections), and hospital-acquired infections.

[0043] When used herein, “variant” strains of bacteriophage strains described herein have at least 80% mean nucleotide identity (“gANI”) across the entire genome to the reference bacteriophage strain. gANI is a similarity index between a given pair of genomes. Typically, scientists would identify organisms with a gANI of 95% or higher as belonging to the “same species.” For example, see Olm M., “Are these microbes the 'same'?” microBEnet(2;2017) (available at microbe.net / 2017 / 02 / 15 / are-these-microbes-the-same / ) and Jain et al. (see Jain et al. Nature Comm. 9(1):5114(2018)). Variant strains having at least 90% of the gANI relative to the reference strain are presumed and considered to have the same phenotypic characteristics as the reference bacteriophage strain. The “variant” strain may be obtained independently of the reference strain, may be a descendant of the reference strain, or may be a recombinant derivative of the reference strain (e.g., one or more or all of the reference strain or its genome sequence prepared by recombination using the reference strain or its genome sequence as starting material).

[0044] As used herein, “isolated” with respect to a bacteriophage means that the bacteriophage has been removed from the environment in which it naturally occurs. An “isolated” bacteriophage may be purified from the environment in which it naturally occurs, or it may be cultivated separately from that environment.

[0045] Bacteriophage Provided herein are the following (11) bacteriophage strains and their variant strains, each of which is an isolated bacteriophage having at least 80%, at least 90%, or at least 95% of the gANI compared to the deposited strain. The genome sequences of each of the following strains are listed in the sequence listings incorporated herein by reference: VREML237-2 (Sequence ID 1) deposited with ATCC under accession number PTA-126934 VREML110-1 (SEQ ID NO: 2) deposited with ATCC under accession number PTA-126932 VREML105 (SEQ ID NO: 3) deposited with ATCC under accession number PTA-126931 VREML202-1 (SEQ ID NO: 4) deposited with ATCC under accession number PTA-126933 VREML85-2 (SEQ ID NO: 5) deposited with ATCC under accession number PTA-126930 VREML110-2 (SEQ ID NO: 6) deposited with ATCC under accession number PTA-127012 VREML237-1 (SEQ ID NO: 7) deposited with ATCC under accession number PTA-127016 VREML85-1 (SEQ ID NO: 8) deposited with ATCC under accession number PTA-127011 VREML137-2 (SEQ ID NO: 9) deposited with ATCC under accession number PTA-127013 VREML202-2 (SEQ ID NO: 10) deposited with ATCC under accession number PTA-127015 VREML139 (SEQ ID NO: 11) deposited with ATCC under accession number PTA-127014.

[0046] These isolated depositary strains and their variants having at least 80%, at least 90%, or at least 95% of the gANI compared to these depositary strains are collectively referred to in the discussion herein as the "bacteriophages (or phages) described herein."

[0047] As illustrated in the examples, each of these strains specifically targets (infects and lyses) one or more strains of VRE and is useful for reducing the risk of infection by one or more strains of VRE, preventing infection, and / or treating infection, for example, for reducing or eliminating colonization by one or more strains of VRE, and is also useful for other uses described herein. For example, as illustrated in the examples, each of these strains specifically targets and lyses one or more of the 214 VRE strains isolated from human subjects and one or more of the following five reference enterococcal isolates: E401 (ATCC 51299), E403 (ATCC 19433), E618 (ATCC 700221), E400 (ATCC 29212), and E402 (ATCC 11823).

[0048] As described above, as used herein, “variant” (or “variant strain”) includes offspring obtained by culturing a deposited bacteriophage, as illustrated in the examples below. Restriction fragment length polymorphism (RFLP) can be used to identify bacteriophage strains or their offspring. As defined by Tenover et al., J. Clin. Microbiol. 33(9):2233-39 (1995), offspring strains may have a DNA RFLP profile substantially equivalent to that of the original bacteriophage strain. As reported by Tenover, when analyzing the genomes of similarly grown organisms by electrophoresis after restriction enzyme digestion, some variability in RFLP profiles may be observed. Tenover uses pulsed-field gel electrophoresis (PFGE) to identify chromosomal DNA This document describes a system for interpreting RFLP profiles. Specifically, Tenover describes various categories of genetic and immunological relationships for identifying organisms that are "indistinguishable" or "closely related" to one another. As used herein, bacteriophage strains are R FLA strain is considered "substantially equivalent" to a reference bacteriophage strain if its P profile is "indistinguishable" or "closely related" according to Tenover's criteria, as described above.

[0049] Therefore, the "offspring" strain may have a DNA RFLP profile substantially equivalent to that of the parent deposited bacteriophage strain, as illustrated in the examples. The offspring may have 80% or more gANI relative to the parent deposited bacteriophage. The offspring may have 90% or more gANI relative to the parent deposited bacteriophage. The offspring may have 95% or more gANI relative to the parent deposited bacteriophage. The offspring may have 98% or more gANI relative to the parent deposited bacteriophage. The offspring may have 99% or more gANI relative to the parent deposited bacteriophage.

[0050] As stated above, variant strains having at least 90% of the gANI of a reference bacteriophage strain are presumed and considered to have the same phenotypic characteristics as the reference bacteriophage strain. Therefore, this disclosure includes variants of these depositary strains having at least 90% of the gANI of a reference depositary strain, and other variants having the same phenotypic characteristics as the depositary strain.

[0051] Also provided herein are derivative products of the isolated bacteriophages described herein. “Derivative product,” as used herein, refers to a substance constituting a subunit or expression product of the bacteriophage described herein, for example (but not limited to), nucleic acids, partial or complete genes, lytic enzymes, and other gene expression products, and other components of the bacteriophage, such as polyribonucleotides and polydeoxyribonucleotides, for example, modified or unmodified bacteriophage DNA, cDNA, mRNA, and synthetic polynucleotide sequences, and DNA / RNA hybrids, for example, derivative products having activity against VRE, or derivative products encoding products having activity against VRE. Therefore, one or more derivative products of the bacteriophages described herein may be included in the compositions described herein and may be used in the manner described herein.

[0052] Anti-sigma factor genes and their expression products are examples of such derivative products. One or more of the phages described herein may encode an anti-sigma factor gene that inhibits the bacterial transcriptional activity of the host (target) VRE. See, for example, Hughes, et al. Ann. Rev Microbiol 52:231-86 (1998). The anti-sigma factor gene confers an enhanced ability to inhibit transcription within target (VRE) bacterial cells to such phages, thereby enhancing the phage's ability to lyse target bacterial cells. Therefore, anti-sigma factor genes and their expression products are examples of derivative products that may be included in the compositions described herein and may be used in the prophylactic and therapeutic methods described herein (for example, they may be used to enhance the potency of the compositions and / or the effectiveness of the methods).

[0053] Lysolytic enzymes are another example of phage derivative products described herein. The bacteriophages described herein encode one or more lysolytic enzymes involved in the lysis of host (target) VREs. Phage lysolytic enzymes are produced by bacteriophages as part of virions to promote bacterial infection by localized peptidoglycan degradation, or as soluble proteins to induce large-scale cell lysis at the end of the lytic replication cycle. See, for example, Briers, Viruses 11(2):113 (2019). Lysolytic enzymes can cause rapid lysis of target bacteria, resulting in a significant decrease or disappearance of target bacterial levels. In addition to enhancing the potency or effectiveness of the compositions and prophylactic and therapeutic methods described herein, lysolytic enzymes may be used in detection assays for the rapid identification of specific bacteria. For example, see Nelson, et al., “Using bacteriophage lytic enzymes as a diagnostic tool for rapid identification of specific bacteria.” In American Society for Microbiology General Meeting, Salt Lake City, Utah (2002). Lytic enzymes for use in the compositions and methods described herein may be isolated from the phage cultures described herein or prepared by recombinant technology. Thus, lytic enzymes may be included in the compositions described herein and used in the prophylactic and therapeutic methods and detection methods described herein, for example, to enhance the potency of the compositions and / or the effectiveness of the treatment methods, or to detect specific target VRE bacteria.

[0054] composition Also provided herein are compositions comprising one or more of the isolated bacteriophage strains described herein. A composition comprising two or more isolated bacteriophage strains described herein is referred herein to as a bacteriophage “cocktail.” As described above, the composition may be an OTC composition or a pharmaceutical composition. For example, the composition may be a pharmaceutical composition, a health supplement, a functional food composition, a dietary supplement, or a probiotic composition.

[0055] A bacteriophage cocktail may comprise any combination of two or more isolated bacteriophages described herein, for example, any two or more of the following (11) deposited strains (or variants thereof having at least 80%, at least 90%, or at least 95% of the gANI of the deposited strain, e.g., variants having the same phenotypic characteristics as determined by phenotypic assay or variants having at least 90% of the gANI of the deposited bacteriophage strain): VREML237-2 (Sequence ID 1) deposited with ATCC under accession number PTA-126934 VREML110-1 (SEQ ID NO: 2) deposited with ATCC under accession number PTA-126932 VREML105 (SEQ ID NO: 3) deposited with ATCC under accession number PTA-126931 VREML202-1 (SEQ ID NO: 4) deposited with ATCC under accession number PTA-126933 VREML85-2 (SEQ ID NO: 5) deposited with ATCC under accession number PTA-126930 VREML110-2 (SEQ ID NO: 6) deposited with ATCC under accession number PTA-127012 VREML237-1 (SEQ ID NO: 7) deposited with ATCC under accession number PTA-127016 VREML85-1 (SEQ ID NO: 8) deposited with ATCC under accession number PTA-127011 VREML137-2 (SEQ ID NO: 9) deposited with ATCC under accession number PTA-127013 VREML202-2 (SEQ ID NO: 10) deposited with ATCC under accession number PTA-127015 VREML139 (SEQ ID NO: 11) deposited with ATCC under accession number PTA-127014.

[0056] As a specific example, a bacteriophage cocktail may include two or more or all of the following (5) isolated bacteriophage strains (or their variants having at least 80%, at least 90%, or at least 95% of the gANI of the deposited strain, for example, variants having the same phenotypic characteristics as determined by phenotypic assay or variants having at least 90% of the gANI of the deposited bacteriophage strain): VREML237-2 (Sequence ID 1) (Deposited with ATCC under accession number PTA-126934) VREML110-1 (Sequence ID 2) (Deposited with ATCC under accession number PTA-126932) VREML105 (Sequence ID 3) (Deposited with ATCC under accession number PTA-126931) VREML202-1 (Sequence ID 4) (deposited with ATCC under accession number PTA-126933) and VREML85-2 (Sequence ID 5) (deposited with ATCC under accession number PTA-126930).

[0057] As a further example, a bacteriophage cocktail may include each of the following (5) isolated bacteriophage strains (or their variants having at least 80%, at least 90%, or at least 95% of the gANI of the deposited strain, for example, a variant having the same phenotypic characteristics as determined by a phenotypic assay or having at least 90% of the gANI of the deposited bacteriophage strain): VREML237-2 (Sequence ID 1) (Deposited with ATCC under accession number PTA-126934) VREML110-1 (Sequence ID 2) (Deposited with ATCC under accession number PTA-126932) VREML105 (Sequence ID 3) (Deposited with ATCC under accession number PTA-126931) VREML202-1 (Sequence ID 4) (deposited with ATCC under accession number PTA-126933) and VREML85-2 (Sequence ID 5) (deposited with ATCC under accession number PTA-126930).

[0058] As a further example, a bacteriophage cocktail may contain each of the following (5) isolated bacteriophage strains: VREML237-2 (Sequence ID 1) (Deposited with ATCC under accession number PTA-126934) VREML110-1 (Sequence ID 2) (Deposited with ATCC under accession number PTA-126932) VREML105 (Sequence ID 3) (Deposited with ATCC under accession number PTA-126931) VREML202-1 (Sequence ID 4) (deposited with ATCC under accession number PTA-126933) and VREML85-2 (Sequence ID 5) (deposited with ATCC under accession number PTA-126930).

[0059] A specific combination of the isolated bacteriophage strains described herein, to be used in a given bacteriophage cocktail, may be selected based on the target strain of VRE. For example, the isolated bacteriophage strains described herein, or combinations of two or more isolated bacteriophage strains, may be tested in vitro against the target strain of VRE before using the strain or combination in the cocktail to treat a specific subject or group of subjects to confirm their efficacy. Suitable screening methodologies are known in the art and are illustrated in the examples herein. For use in such tests, the target strain of VRE may be obtained from clinical specimens obtained from a specific subject or group of subjects, from the environment of a specific subject or group of subjects, or identified from other sources. Suitable methodologies are known in the art and are illustrated in the examples herein.

[0060] Also provided are compositions comprising isolated bacteriophage strains of one or more bacteriophage strains described herein, and further comprising one or more probiotics. As used herein, “probiotics” includes probiotic bacteria and probiotic yeasts. Suitable probiotic bacteria are known in the art and include: Lactobacillus species (e.g., L. acidophilus, L. rhamnosus, L. gasseri, L. reuteri, L. bulgaricus, L. plantarum, L. johnsonii, L. paracasei, L. casei) *Bacillus casei*, *L. salivarius*, and *L. lactis*, species of the genus *Bifidobacterium* (e.g., *B. bifidum*, *B. longum*, *B. breve*, *B. infantis*, *B. lactis*, and *B. adolescentis*), as well as *Streptococcus thermophilus*, *Bacillus cerus*, and *Bacillus subtilis*.Suitable probiotic yeasts known in the art include: Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces cerevisiae var. boulardii, Issatchenkia occidentalis, Lachancea thermotolerans, Metschnikowia ziziphicola, and Torulaspora delbrueckii.

[0061] Therefore, in some embodiments, the compositions described herein include one or more isolated bacteriophage strains described herein and one or more probiotics. In specific embodiments, the compositions described herein include one or more isolated bacteriophage strains described herein and one or more strains of probiotic bacteria. In other specific embodiments, the compositions described herein include one or more isolated bacteriophage strains described herein and one or more strains of probiotic yeast. In yet another specific embodiment, the compositions described herein include one or more isolated bacteriophage strains described herein, one or more strains of probiotic bacteria, and one or more strains of probiotic yeast. While we do not wish to be bound by theory, the positive health effects of VRE bacteriophages described herein may be further enhanced when combined with probiotics.

[0062] As a specific example, the compositions described herein may include two or more or all of the isolated bacteriophage strains (or their variants described above) and one or more probiotics: VREML237-2 (Sequence ID 1) (Deposited with ATCC under accession number PTA-126934) VREML110-1 (Sequence ID 2) (Deposited with ATCC under accession number PTA-126932) VREML105 (Sequence ID 3) (Deposited with ATCC under accession number PTA-126931) VREML202-1 (Sequence ID 4) (deposited with ATCC under accession number PTA-126933) and VREML85-2 (Sequence ID 5) (deposited with ATCC under accession number PTA-126930).

[0063] These one or more probiotics belong to the Lactobacillus genus (e.g., L. acidophilus, L. rhamnosus, L. gasseri, L. reuteri, L. bulgaricus, L. plantarum, L. johnsonii, L. paracasei, L. casei), L. salivarius and L. lactis, Bifidobacterium species (e.g., B. bifidum, B. longum, B. breve, B. infantis, B. lactis, and B. adolescentis), and Streptococcus thermophilus One or more probiotic bacteria selected from Bacillus thermophilus, Bacillus cereus, and Bacillus subtilis; Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces cerevisiae var. boulardii, Issatchenkia occidentalis, Lachancea thermotolerans, Metschnikowia ziziphicola, and Torulaspora delbruckii. It may be one or more probiotic yeasts selected from (delbrueckii), or any combination thereof.

[0064] As described above, in any embodiment, the composition comprising the isolated bacteriophage and probiotic described herein may be an OTC composition or a pharmaceutical composition.

[0065] In addition, or / or, the compositions described herein may include one or more phage derivative products described herein. In some embodiments, the compositions described herein include one or more enzymes produced by one or more of the bacteriophages incorporated in the composition. In some embodiments, the compositions described herein also include, or / or, one or more anti-sigma factor genes or expression products thereof from one or more of the bacteriophages incorporated in the composition. As described above, in any embodiment, the compositions comprising the bacteriophages and phage derivative products (e.g., lytic enzymes) described herein may be OTC compositions or pharmaceutical compositions.

[0066] The OTC compositions or pharmaceutical compositions described herein comprise an isolated bacteriophage (and, in some embodiments, a probiotic and / or phage derivative product, e.g., a lytic enzyme), one or more pharmaceutically acceptable carriers, and one or more optionally pharmaceutically acceptable excipients. Suitable carriers and excipients for bacteriophages are known in the art. Typically, a pharmaceutically acceptable carrier is a pharmaceutically acceptable, non-toxic carrier, filler, or diluent suitable for use as a vehicle for formulating a pharmaceutical composition for administration to a target patient (animal or human) via an intended route of administration.

[0067] Pharmacovigilant excipients (also called adjuvants or adjuncts) include, but are not limited to, those conventionally used in pharmaceutical compositions for administration to target patients (animals or humans) via the intended route of administration: matrix-forming agents, thickeners, binders, lubricants, pH adjusters, protective agents, viscosity enhancers, wicking agents, disintegrants (e.g., non-foaming and foaming disintegrants), surfactants, antioxidants, wetting agents, colorants, flavoring agents, taste enhancers, sweeteners, preservatives, etc. In addition to being pharmaceutically acceptable, adjuvants are typically selected to be compatible with other components of the composition, including bacteriophages and probiotics (if present).

[0068] The OTC and pharmaceutical compositions described herein may be formulated for any preferred route of administration (which may depend on the site of colonization or infection of VER). Phage compositions for various routes of administration are disclosed. See, for example, Qadir et al., Brazilian J. Pharm. Sci. (2018) 54(1). For example, phages may be formulated for routes of administration including oral, buccal, sublingual, rectal, nasal, topical, ear, vaginal, bronchial, lung, or parenteral (e.g., subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, intrapleural, intravesical, and intrathecal) administration, or for administration via implant, urinary lavage, or catheter. The composition or route of administration may be selected to provide a targeting effect on one or more bacteriophages described herein and may depend on the site of colonization or infection.

[0069] The OTC composition and pharmaceutical composition may be prepared by any suitable method for producing the dosage form in question, such methods being known in the pharmaceutical field. Such methods typically involve associating one or more bacteriophages described herein (and optionally one or more probiotic and / or phage derivative products, e.g., lytic enzymes) with a pharmaceutically acceptable carrier and optionally one or more pharmaceutically acceptable adjuvants.

[0070] In some embodiments, the compositions described herein are formulated for oral administration and administered orally. Dosage forms of OTC compositions and pharmaceutical compositions suitable for oral administration are known in the art and include: individual dosage forms, e.g., tablets (including chewable tablets), hard gel capsules containing a dried phage-containing composition, soft liquid gel capsules containing a liquid phage-containing composition, and sugar-coated tablets; and bulk dosage forms, e.g., powders, granules, solutions, suspensions, dispersions, syrups, microgels, and the like.

[0071] OTC compositions and pharmaceutical compositions formulated for oral administration may be formulated to protect phages from the acidic environment of the stomach. For example, compositions formulated for oral administration may be coated, for example, an enteric coating or a delayed-release coating (e.g., in the form of coated tablets or capsules, or coated granules), or microencapsulated (e.g., alginate-chitosan microspheres) to protect bacteriophages (and optional probiotics and / or phage derivative products, e.g., lytic enzymes) and maintain their viability while passing through the acidic environment of the stomach. Suitable coating and microencapsulation materials are known in the art. In addition, orally, compositions formulated for oral administration may be formulated together with or administered together with gastric acid-reducing agents (e.g., gastric acid reducers), such as antacids (e.g., bicarbonates such as sodium bicarbonate), or proton pump inhibitors (e.g., omeprazole).

[0072] In addition, such compositions may be coated with a delayed-release coating, or may include a delayed-release coating, to release viable phages (and optional probiotics and / or phage derivative products, e.g., lytic enzymes) at a desired release site, such as the small intestine.

[0073] For example, compositions described herein, formulated for oral administration, may contain one or more of the following components: water, e.g., deionized water, pharmaceutical-grade water, or mineral water; sodium chloride, sodium bicarbonate; buffer (e.g., Tris-HCl at pH 7.0-7.5); sweeteners, e.g., sucrose (e.g., 5% sucrose solution), trehalose, maltodextrin, glycerol, dextran, and sorbitol, cellulose; thickeners, e.g., tapioca, dextrin, gellan gum, and gelatin; and other excipients, e.g., hydroxypropyl methylcellulose, poly(acrylic acid) ("PAA"), poly(ethylene glycol) ("PEG"), and casein, and any combination of two or more of these.

[0074] As a specific and non-limiting example, the compositions described herein may be optionally added to 1 mL of a 0.9% sodium chloride solution containing approximately 1 × 10⁶ units of each composition. 10 PFU can be formulated in a 0.9% sodium chloride solution to obtain phages. Such a composition may be optionally mixed with 15-50 mL of bicarbonate water, etc., immediately before administration for ingestion.

[0075] In alternative embodiments, the compositions described herein are formulated for rectal administration. Dosage forms of OTC compositions and pharmaceutical compositions suitable for rectal administration are known in the art and may include suppositories, rectal foams, lotions, gels, or enemas for rectal administration.

[0076] In alternative embodiments, the compositions described herein are formulated for vaginal administration. Dosage forms of OTC compositions and pharmaceutical compositions suitable for vaginal administration are known in the art and may include suppositories, creams, vaginal foams, lotions, gels, vaginal douches, or catheters for vaginal administration.

[0077] In alternative embodiments, the compositions described herein are formulated for parenteral administration by infusion or injection, such as subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, intrapleural, intravesical, and intrathecal injection. Suitable compositions for parenteral administration include sterile aqueous and non-aqueous compositions known in the art and formulated for infusion or injection. Such compositions may be provided in unit-dose or multi-dose containers (e.g., pre-filled syringes, sealed vials, sealed ampoules, or sealed pouches). Such compositions may be ready for use or may be freeze-dried (lyophilized) or spray-dried compositions that are reconstituted with a sterile liquid carrier (e.g., water) before use.

[0078] In alternative embodiments, the compositions described herein are formulated for topical delivery. Dosage forms of OTC compositions and pharmaceutical compositions suitable for topical administration are known in the art and include solutions, emulsions, creams, lotions, gels, and sprays. For example, the compositions described herein may be formulated as gels for topical administration.

[0079] In alternative embodiments, the compositions described herein are formulated for pulmonary or bronchial administration, for example, by oral or nasal inhalation. Dosage forms of compositions suitable for pulmonary administration include powder compositions and particulate formulations (e.g., dust or mist) that are known in the art and can be produced and administered by quantitatively pressurized aerosols, nebulizers, inhalers, or similar devices.

[0080] treatment The bacteriophages and compositions described herein may be used in prophylactic and therapeutic methods to reduce or prevent the risk of VRE colonization or infection, or to treat VRE colonization or infection, or to modulate a target microbiome (for example, by preventing or reducing VRE colonization), or to reduce or eliminate VRE intestinal colonization, or to treat VRE-related diseases or conditions, or in prophylactic and therapeutic methods for similar effects.

[0081] With such use, the bacteriophages and compositions described herein may be administered according to any effective dosing regimen, which may vary based on the prophylactic or therapeutic effect to be achieved (e.g., prevention of infection versus treatment of colonization or treatment of bacteremia), the specific bacteriophage used, the route of administration and dosage form, and the patient's characteristics (e.g., the patient's age and weight, the patient's condition, and one or more of the type and severity of VRE infection). Exemplary dosages are described below for illustrative purposes only.

[0082] A typical bacteriophage dose for human patients is 10 3 ~10 12 Plaque-forming units (PFUs) (e.g., 10 4 ~10 11 For example, 10 6 ~10 10) is likely to contain one or more bacteriophages, and this dose may be administered once or multiple times per day, for example, 1, 2, 3, 4, or 5 times per day. The dose may be provided as a single discrete dosage form (e.g., one tablet or capsule) or multiple discrete dosage forms (e.g., 2, 3, 4, or more tablets or capsules), or may be provided in a bulk dosage form of a suitable volume (e.g., 1 mL of a liquid composition). Treatment may be continued for one or more days, for example, for one day, one week, two weeks, three weeks, one month, or longer.

[0083] An example of a suitable dosage regimen is 10 each, administered orally 1 to 3 times daily for 1 to 4 weeks 8 to 10 10 PFU bacteriophage-containing 1 to 3 capsules or tablets. Another example of a suitable dosage regimen is 10 8 to 10 11 PFU bacteriophage-containing 1 mL of liquid composition, administered orally 1 to 3 times daily for 1 to 4 weeks.

[0084] As mentioned above, the bacteriophage compositions and methods described herein may be used in combination with an agent that reduces gastric acid. For example, a subject undergoing treatment may also be treated with a proton pump inhibitor (e.g., omeprazole), and / or may also ingest a gastric acid neutralizing agent such as bicarbonate (e.g., sodium bicarbonate) immediately before ingesting a dose of the bacteriophage composition.

[0085] The bacteriophages and compositions described herein may be used prophylactically or therapeutically to treat any subject in need thereof, for example, any subject at risk of VRE colonization or infection (including hospitalized subjects), or any subject colonized or infected with VRE.

[0086] The bacteriophages and compositions described herein may be advantageously used in subjects facing, being treated with, or recovering from one or more of the following: invasive medical procedures, chemotherapy, solid organ transplantation, or immunosuppressive therapy, or in other subjects who are immunosuppressed or immunocompromised. In such embodiments, the bacteriophages and compositions described herein may be administered before, during, and after one or more of the invasive medical procedures, chemotherapy, solid organ transplantation, or immunosuppressive therapy. For example, the course of bacteriophage treatment described herein may be initiated 1 to 4 weeks before other treatment periods (e.g., invasive medical procedures, chemotherapy, solid organ transplantation, or immunosuppressive therapy), and may be continued during and optionally after the other treatment periods.

[0087] VRE detection method The bacteriophages (or their lytic enzymes) described herein can also be used in in vitro assays to detect target bacteria (e.g., VRE) in biological samples obtained from subjects, for example, to diagnose whether a subject is infected with VRE and / or to assess the level of infection (colony formation), for example, to assess the necessity or effectiveness of the treatments described herein. Although not bound by theory, the bacteriophages (or their lytic enzymes) described herein are thought to specifically lyse target bacteria (e.g., VRE) without affecting other prokaryotic or eukaryotic cells that may be present, thereby specifically inducing the release of measurable products of the bacteriophage (e.g., adenosytriphosphate (ATP) and / or protein kinase (ALT)) that are specific to target VRE. Therefore, detection assays can be performed based on the detection of ATP or AKT of the target bacteria.

[0088] An exemplary example of such an assay is as follows: A sample of the clinical material to be analyzed (e.g., a fecal specimen) is obtained and suspended in a suitable buffer. One or more bacteriophages (or their lytic enzymes) are added to this suspension, resulting in the lysis of any target bacterial cells present in the sample and the release of their ATP. To detect ATP (for example), a luciferin + luciferase preparation is added, and the luminescence is measured using a luminometer, for example. A quantitative assay can be developed by creating a calibration curve between the luminometer reading and the number of lysed target bacterial cells (generally, the average number of ATP per bacterial cell is 0.5–1.0 fg). No luminescence indicates the absence of target bacterial cells in the analyzed sample.

[0089] Vaccines and bacterins Also provided are vaccines and bacterins prepared using the bacteriophages (or their lytic enzymes) described herein. For example, a specific strain of a target bacterium (e.g., target VRE) can be lysed using the bacteriophages (including variants of the deposited strains discussed above) or their lytic enzymes to obtain a bacterial lysate containing the immunological epitope of this bacterium, which can then be used to prepare a vaccine / bacterin against the target VRE. The final vaccine / bacterin preparation may be prepared by methods known in the art. When used to obtain a bacterin, the phage can be removed to obtain the final vaccine / bacterin preparation. Alternatively, the phage can be retained in the final preparation in a viable, active state. In such embodiments, the phage present in the vaccine / bacterin formulation may maintain activity against the target bacterium (e.g., target VRE), providing another mechanism of action for the efficacy of the preparation, for example, by lysing the target bacterium (VRE) present in the vaccinated subject. For example, the final vaccine / bacterin preparation, 10 3 ~10 12 The preparation can be made so that phages are present at levels in the range of PFU / ml. For example, the final vaccine / bacterin preparation can be prepared at 10 6~10 10 The solution can be prepared to contain phages at levels in the PFU / ml range.

[0090] In specific embodiments, a vaccine / bacterin preparation is prepared for a prevalent strain of the target bacterium to obtain a vaccine / bacterin preparation containing the immunological epitope most relevant to protecting the target patient population from infection. In more specific embodiments, the bacteriophage is retained unchanged in the final vaccine / bacterin preparation, as discussed above.

[0091] Bacteriophage-based vaccines and bacterins can also be prepared using recombinant constructs expressing genes associated with the bacteriophage (e.g., genes related to lytic activity, e.g., those encoding container enzymes), or isolated or recombinantly produced lytic enzymes, which can be used in place of the phage itself in the protocols outlined above. An example of this general methodology is outlined in Panthel et al., Infect Immun. 71(1):109-16 (2003).

[0092] According to any of these embodiments, the use of a vaccine containing the bacterin described above for immunizing a required target, such as a subject who has VRE or is at risk of VRE infection. Based on the guidance provided herein, a person skilled in the art may develop a suitable vaccination protocol.

[0093] kit Also provided are kits for carrying out the various embodiments described herein.

[0094] Kits for the prophylactic or therapeutic use of bacteriophages as described herein may include the compositions described herein, along with instructions for the prophylactic or therapeutic use of the compositions described herein, and optionally together with packaging materials.

[0095] Kits for the prophylactic or therapeutic use of vaccines / bacterins described herein may include the vaccines / bacterins described herein, along with instructions for the prophylactic or therapeutic use of vaccines / bacterins described herein, and optionally together with packaging materials.

[0096] A kit for a VRE detection assay may include one or more bacteriophages (or one or more lytic enzymes) as described herein, along with instructions for using the bacteriophage (or enzyme) in the VRE detection assay as described herein, and optionally with packaging materials. [Examples]

[0097] The present invention will be further illustrated by the following embodiments, which are not intended to limit the scope of the invention.

[0098] Example 1: VRE isolated strain Obtaining VRE isolates VRE was isolated from patients in the surgical intensive care unit and intermediate intensive care unit of the University of Maryland VA Medical Center in Baltimore, Maryland. Enterococci were isolated from urine, wounds, and sterile body fluids using Trypticase Soy Agar (BBL, Cockeysville Md.) supplemented with Maryland 5% sheep blood. VRE was isolated from stool samples on Colistin Nalidixic Acid (CNA) agar (Difco Labs, Detroit, Mich.) supplemented with defibrous sheep blood (5%), vancomycin (10 μg / ml), and amphotericin (1 μg / ml). See Facklam, et al., Enterococcus. In Manual of Clinical Microbiology (6th ed. 1995) (Am. Soc. Microbiol., Washington, DC), pp. 308-312.

[0099] Identification of VRE Enterococci were identified by esculin hydrolysis and growth in 6.5% NaCl at 45°C. Species-level identification was performed according to Facklam and Collins, J. Clin. Microbiol., 27:731-34 (1989).

[0100] VRE antimicrobial susceptibility testing Antimicrobial susceptibility to ampicillin, vancomycin, streptomycin, and gentamicin was determined using the E test quantitative minimum inhibitory concentration procedure (AB Biodisk, Solna Sweden). The efficacy of each tested antimicrobial agent was confirmed using quality control strains of E. faecium (ATCC 29212, ATCC 51299). Except for vancomycin, susceptibility interpretations according to the National Committee for Clinical Laboratory Standards were followed. See National Committee for Clinical Laboratory Procedures, “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically.” (3rd ed., 1993) (Nat. Committee for Clin. Lab. Standards, Villanova Pa.); and National Committee for Clinical Laboratory Standards, “Performance Standards for Antimicrobial Disk Susceptibility Tests.” (5th ed., 1993) (Nat. Committee for Clin. Lab. Standards, Villanova Pa.). VRE isolates were defined as those with a minimum inhibitory concentration of vancomycin of at least 16 μg / ml.

[0101] Generally, different definitions of VRE stocks Separate VRE isolates were characterized by contour-clamped homogeneous electric field electrophoresis after digestion of chromosomal DNA with Smal. See Verma, P. et al., “Epidemiologic characterization of vancomycin-resistant enterococci recovered from a University Hospital,” In Abstracts of the 94th General Meeting of the American Society for Microbiology, Las Vegas, New. (1994); Dean, et al., “Vancomycin-resistant enterococci (VRE) of the vanB genotype demonstrating glycoprotein (G) resistance inducible by vancomycin (V) or teicoplanin (T),” In Abstracts of the 94th General Meeting of the American Society for Microbiology, Las Vegas, New. (1994). For VRE strains that differed by only 1-3 bands after the initial analysis, electrophoresis using Apal digestion was also performed. See Donabedian, J. Clin. Microbiol., 30;2757-61 (1992). Vancomycin resistance genotypes (vanA, vanB, or vanC) were defined by polymerase chain reaction analysis using specific primers selected from publicly available gene sequences.See Goering, RV and the Molecular Epidemiological Study Group, “Guidelines for evaluating pulsed field restriction fragment patterns in the epidemiological analysis of nosocomial infections.” Abstract of the Third International Meeting of Bacterial Epidemiological Markers; Cambridge, England (1994).

[0102] Using these methodologies, we identified 214 distinct VRE strains from those isolated at VA Medical Center.

[0103] Example 2: Bacteriophage Isolation of VRE phage 500 ml of water from Chesapeake Bay (a potential source of phages) was mixed with 100 ml of 10-fold concentrated LB broth (Difco Laboratories) (containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl). 1 ml of LB broth culture of a VRE strain was inoculated into this water-broth mixture and incubated at 37°C for 24 hours to enrich the mixture with any bacteriophages capable of infecting the added VRE strain. After incubation, the mixture was centrifuged at 5,000 g for 15 minutes to remove any substances that might interfere with subsequent filtration. The supernatant was filtered through a 0.45 μm Millipore filter. The filtrate was evaluated by the streak plate method and Appelman tube turbidity test, and its solubility activity against various VRE strains was assessed using the methodologies outlined below.

[0104] Plaque assay: Plaque assays can be used to assess whether a given VRE strain is susceptible to infection by a given bacteriophage preparation. Plaque picking can be used to obtain a single-strain bacteriophage preparation.

[0105] Mix 0.1 ml of an 18-24 hour nutrient broth culture of the VRE strain to be tested with 0.1 ml of a diluted VRE bacteriophage preparation, then add it to 4.5 ml of 0.7% molten agar in the nutrient broth at 45°C. Pour this mixture entirely into a petri dish containing 25 ml of nutrient broth solidified with 2% agar, and incubate overnight at 37°C. During the overnight incubation at 37°C, VRE will grow in the agar, and some VRE cells infected with the bacteriophage will form a confluent turf. The phage will replicate and lyse the first infected cells, and then infect and lyse adjacent bacteria. This agar restricts the physical spread of the phage across the plate, resulting in the formation of small, visible, transparent areas called plaques on the plate, where the bacteriophage disrupts VREW within the confluent turf of VRE growth.

[0106] The number of plaques formed from a given amount of a given dilution of a bacteriophage preparation reflects the titer of the bacteriophage preparation. A single morphologically distinct plaque represents a single phage particle replicated in the VRE within that region of the bacterial lawn. Therefore, a pure bacteriophage preparation can be obtained by extracting the material from the plaque (referred to as a "plaque pick") using a pipette such as a Pasteur pipette and using this material as an inoculum for further proliferation cycles of this phage. By performing further plaque assays on phage preparations grown from the same plaque pick, all plaques will have the same single appearance (plaque morphology) as the extracted plaque, which is a further indicator of purity. Thus, the efficacy of bacteriophages against VRE can be evaluated using plaque assay techniques, and the bacteriophage purity of the bacteriophage preparation can also be assessed.

[0107] Streak plate method: The streak plate method can be used to screen for one or more phages against one or more VRE strains.

[0108] The 18-hour LB broth cultures of one or more enterococcal strains to be tested are grown at 37°C (approximately 10 9 The amount of each culture (CPU / ml) in a platinum loop is streaked onto a nutrient agar plate in a single row. As a result, on each plate, numerous different VRE strains streaked onto the plate grow in a single straight line.

[0109] A drop of the bacteriophage filtrate to be tested is applied to each VRE streak, and the plate is incubated at 37°C for 6 hours. At this point, the streaks of various VRE strains are investigated for the phage's ability to form clear areas without bacterial growth, indicating lysis of a particular VRE strain by a specific phage. By determining which VRE streaks produce clear areas without growth and which do not, the VRE host range of a given phage filtrate can be determined.

[0110] Appelman turbidity test Phage preparations can be screened against one or more VRE strains using the Appleman turbidity test (see Adams, Bacteriophages. (Interscience Publ. New York NY) (1959)).

[0111] Prepare 18-hour LB broth cultures of one or more VRE strains. Add 0.1 ml of phage filtrate or its dilution to 4.5 ml of the VRE broth culture and incubate at 37°C for 4 hours (single-phage preparation containing a single phage) or incubate at 37°C for 4 to 18 hours (multivalent preparation containing at least two phages). Phage-free VRE broth cultures were used as a control. The ability of phage preparations to lyse VRE strains was investigated, as demonstrated by clearing the turbidity of the broth culture, which is normally turbid due to bacterial growth. The host range of a given phage preparation can be determined by which VRE broth cultures clear the phage preparation and which do not.

[0112] These methodologies were used to isolate and identify the bacteriophages described herein.

[0113] Example 3: Phage Characterization Restricted Fragment Length Polymorphism (RFLP) Profile The RFLP profiles of the bacteriophages described herein are shown in Figure 1. DNA was isolated from the bacteriophages using the Qiagen Plasmid Miniprep or Midiprep kit (Valencia, CA) according to the manufacturer's instructions. This DNA was quantified by measuring absorbance at 260 nm. Approximately 0.5–1 μg of DNA was digested with an appropriate restriction enzyme (Hind III digestion), stained with ethidium bromide, and the RFLP profile was determined on a 1% agarose gel.

[0114] Genome analysis and mean nucleotide identity of VRE phage Whole-genome sequencing and sequence analysis can be used to identify the bacteriophages or their offspring described herein. Offspring with mean nucleotide identity (ANI) of 95% or higher are considered to be of the “same species” as defined by Olm M., “Are these microbes the 'same'?” microBEnet(2;2017) (available at microbe.net / 2017 / 02 / 15 / are-these-microbes-the-same / ) and Jain et al. (Nature Comm. 9(1):5114(2018)). Phages with a gANI of 95% or higher relative to a reference phage are considered to be “substantially equivalent” to the reference bacteriophage.

[0115] VRE bacteriophages were sequenced using MiSeq with a read length of 2 × 250 bp. Reads were sorted for Illumina adapter, length (≧50 bp), and quality (q≧20), and mapped to enterococcal reference sequences available in GenBank. Unmapped reads were collected and assembled using Unicycler assembler. Strain descriptions were evaluated by calculating mean nucleotide identity (gANI) across the entire genome. See, for example, Jain et al., above; Varghese et al., Nucleic Acids Res. 43(14):6761-71 (2015).

[0116] Example 4: Production of phage preparations The VRE bacteriophages described herein can be propagated as illustrated herein.

[0117] A strain of a target bacteriophage capable of growing (e.g., VRE) or another closely related bacterial species is batch cultured using a suitable growth medium (e.g., BHI broth) and inoculated with the bacteriophage at a predetermined multiple of infection (MOI). After incubation and bacterial lysis, the bacteriophage is recovered, purified, and / or concentrated to obtain phage progeny suitable for the applications described herein.

[0118] Suitable purification and concentration procedures include filtration, centrifugation (including continuous flow centrifugation), size exclusion chromatography, ion exchange chromatography, and one or more of other known bacteriophage purification and concentration techniques. See, for example, Adams MH. Bacteriophages. 443-519 (Interscience Publishers, Ltd, London) (1959). The purity of the phage preparation can be evaluated by one or more of electron microscopy, SDS-PAGE, DNA restriction digestion, and analytical ultracentrifugation.

[0119] The bacteriophage concentration of the preparation can be adjusted using a phage titer measurement protocol. For example, determine the bacteriophage concentration of the preparation. If a more concentrated phage preparation is desired, increase the concentration by filtration, centrifugation, or one or more of the other means. If a lower concentration phage preparation is desired, decrease the concentration by diluting with water or other pharmaceutically acceptable diluents (e.g., pharmaceutically acceptable buffers). Typical pharmaceutical or OTC compositions have a phage titer of 10. 6 ~10 12 It is PFU / mL, for example, if the phage titer is 10 9 ~10 11 The concentration is PFU / mL.

[0120] Bacteriophage preparations can be stored at 2–8°C. Alternatively, phage preparations can be freeze-dried or spray-dried for storage, or stabilized by encapsulation using an approach known in the art (e.g., one or more of proteins, lipids, and polysaccharides). During reconstitution, phage titer can be verified using phage titration protocols, host bacterial assays, or other known bacteriophage assay techniques. See, for example, Adams, above.

[0121] Example 5: Phage activity The bacteriophages of the bacteriophage strains described herein efficiently lyse VRE strains, thereby demonstrating their effectiveness against VRE strains.

[0122] The VRE phages described herein can be used individually or in various combinations to target specific VRE strains or subgroups of strains. For example, the following five strains of phages can be combined into a cocktail to efficiently target all 214 VRE strains isolated at VA Medical Center and five reference enterococcal isolates: E401 (ATCC 51299), E403 (ATCC 19433), E618 (ATCC 700221), E400 (ATCC 29212), and E402 (ATCC 11823): VREML237-2 (Sequence ID 1) (Deposited with ATCC under accession number PTA-126934) VREML110-1 (Sequence ID 2) (Deposited with ATCC under accession number PTA-126932) VREML105 (Sequence ID 3) (Deposited with ATCC under accession number PTA-126931) VREML202-1 (Sequence ID 4) (Deposited with ATCC under accession number PTA-126933) VREML85-2 (Sequence ID 5) (deposited with ATCC under accession number PTA-126930).

[0123] As shown in the table below, this five-phage cocktail lyses 94% of the 214 VRE strains isolated at VA Medical Center, as well as 5 reference strains.

[0124] [Table 1]

[0125] [Table 2]

[0126] The PhageSelector™ software used in this analysis is described in Cieplak, et al., Gut Microbes 9(5):01-19(2018).

[0127] Example 6: Efficacy in an in vivo mouse model The efficacy of the bacteriophages described was demonstrated in the in vivo mouse model described herein.

[0128] We attempted to establish sustainable VRE colony formation in an animal model as follows.

[0129] ICR non-inbred mice (6 weeks old) were decolonized over 3 days by subcutaneous injection of 1 mg / ml gentamicin and clindamycin (2.4 mg / day / mouse) in drinking water, thereby reducing the normal intestinal microbiome. Antibiotics were discontinued and the mice were flushed 24 hours before VRE administration. VRE diluted with physiological saline (approximately 2 × 10⁻⁶) 3 Each mouse was given a single forced oral dose of CFU. Starting 1 hour after VRE administration, the animals were subjected to forced oral administration every 8 hours for 7 days, totaling 21 doses, resulting in approximately 1 × 10⁶ doses. 9At a PFU / dose (n=10), mice were treated with 0.2 mL of the five phage cocktail described herein, or with PBS (n=5). Fresh fecal pellets (1 pellet / mouse) were collected daily, and the bacterial levels of the feces were quantified. The fecal pellets were weighed in pre-weighed sterile Eppendorf tubes, serially diluted with 0.9% physiological saline, and spread on Enterococcosel® agar (EA), a selective medium for enterococci containing 8 μg / mL nitrofurantoin (NIT8) (EA NIT8) and 32 μg / mL erythromycin. Colony-forming units (CFUs) were counted after incubation at 37°C for 48 hours and used to calculate log10 CFU / g feces.

[0130] The effectiveness of phage treatment in reducing VRE gastrointestinal colonization was determined by daily quantification of VRE in weighed fecal samples from mice treated with a phage cocktail, compared to a PBS-treated control group. Administration of the phage cocktail reduced the overall load of VRE gastrointestinal colonization by approximately 0.5 log after phage administration compared to the PBS control group, with the maximum reduction in fecal VRE load achieved on day 6. VRE colonization tended to be lower in the phage cocktail-treated group compared to the PBS control at all time points examined.

[0131] Example 7: Efficacy in an in vitro human intestine simulator model The efficacy of the bacteriophages described was demonstrated using the in vitro human gut simulator model described herein. Specifically, a cocktail of five bacteriophages, VREML237-2, VREML110-1, VREML105, VREML202-1, and VREML85-2, was shown to be effective against VRE in the SHIME® model (human gut microbiota simulator or SHIME®), as outlined below. See Moye et al, J. Food Prot. 82(8):1336-1349 (2019).

[0132] Stool samples from healthy, VRE-negative human donors from three subjects were used in a SHIME® model, which simulates the ascending, transverse, and descending colon using inoculum preparation, retention time, pH, temperature settings, and reactor supply composition. The SHIME® setup was adapted to include 12 proximal colon compartments to test the effects of inter-individual variability among the three donors. The SHIME® system, using stool samples from healthy donors from three VRE-negative healthy individuals, was stabilized for two weeks in a healthy state or a state of intestinal symbiotic imbalance (i.e., treatment with 33.9 ppm clindamycin over 7 days (-8 to -2 days)). The ascending colon reactor was treated twice daily at 8-hour intervals for 5 days, starting on day -1, with PBS or a phage cocktail (6 × 10⁶). 10 The reactor was treated with PFU. Four hours after the third and fifth doses of PBS or phage cocktail, VRE (1 × 10⁻¹⁰) was added to the reactor at 0 hours (day 0) and 24 hours (day 1). 9 A CFU was loaded.

[0133] The efficacy of phage cocktails was compared to PBS treatment in both healthy and intestinal flora symbiotic imbalances (i.e., under the antibiotic treatments described below). Efficacy was calculated by quantifying VRE load (CFU) over time in the proximal colon (1 hour after each PBS or phage cocktail treatment, and at each VRE loading). The phage cocktails were effective in reducing VRE load to levels more than 50% lower compared to controls (PBS or clindamycin). In all three healthy stool samples, the phage cocktails promoted E. faecium clearance and reduced E. faecium levels by more than 83-99% within 21 hours after loading compared to placebo control. In three of three stool samples with enteromicrobial symbiosis imbalance (healthy donor stool treated with clindamycin), the phage cocktail promoted E. faecium clearance and reduced E. faecium abundance by over 51–99% within 21 hours of loading compared to antibiotic-treated samples. The impact on overall endogenous enterococcal abundance was limited, indicating specific antipathogenic targeting of E. faecium VRE by phages in the phage cocktail. In all samples, phage treatment did not affect short-chain fatty acid levels and / or improved levels of beneficial bite markers compared to enteromicrobial symbiosis imbalance (antibiotic-treated) samples. In addition, the phage cocktail did not alter the endogenous microbial composition of healthy donors, and in two of the three donors, the cocktail stimulated microbial recovery of primary substrate degradation products after antibiotic treatment.

[0134] Example 8: Phase 1 / 2A Clinical Trial Phase 1: This double-blind, placebo-controlled, randomized Phase 1 trial evaluated the safety of a single dose of the VRE phage cocktail described herein (i.e., a composition containing two or more bacteriophages from the VRE bacteriophage strains described herein, such as two or more or all of VREML237-2, VREML110-1, VREML105, VREML202-1, and VREML85-2) administered orally two or three times daily over 7 days in approximately 10-20 healthy adults, who were followed up for up to 3 months compared to the placebo control group. For example, approximately 1 × 10⁶ 10 PFU phages may be administered orally two or three times daily, at least one hour before meals. Clinical safety evaluations include observation of clinical symptoms and signs, analysis of blood parameters, and measurement of fecal calprotectin content. The primary objective is to determine the safety of a series of oral administrations of the bacteriophage cocktail. Exploratory objectives include determining the effects of the phage cocktail on the human fecal microbiome and evaluating phage efflux using state-of-the-art next-generation sequencing and bioinformatics technologies.

[0135] Phase 2a: This double-blind, placebo-controlled, randomized Phase 2a trial evaluated the safety and efficacy of the VRE phage cocktail described herein (i.e., a composition containing two or more bacteriophages from the VRE bacteriophage strains described herein, such as two or more or all of VREML237-2, VREML110-1, VREML105, VREML202-1, and VREML85-2) administered orally two or three times daily for two weeks. Approximately 30–50 adult subjects with VRE colonization were followed up as outpatients for up to 6 months compared to a placebo control group. For example, approximately 1 × 10⁻⁶ 10PFU phages may be administered orally two or three times daily, at least one hour before meals. Clinical safety evaluations are as described above for Phase 1. Efficacy will be determined by quantitative assessment of VRE fecal excretion. The primary objective is safety and efficacy compared to placebo, assessed by the effect of phage administration on VRE levels in the GI tube. Exploratory objectives include evaluating phage excretion and determining the effects of the phage cocktail on the human gut microbiome.

Claims

1. An isolated bacteriophage that infects and lyses one or more strains of vancomycin-resistant enterococcus (VRE), wherein the bacteriophage is bacteriophage strain VREML237-2 (SEQ ID NO: 1), deposited with ATCC under accession number PTA-126934, or a variant thereof, and the variant strain is bacteriophage strain VREML237-2 (SEQ ID NO: 1) or a variant thereof, having at least 90% of the mean nucleotide identity (gANI) across the entire genome relative to bacteriophage strain VREML237-2.

2. The isolated bacteriophage according to claim 1, wherein the bacteriophage is a variant strain of the deposited bacteriophage strain having at least 95% of the gANI of the deposited bacteriophage strain.

3. The isolated bacteriophage according to claim 1, wherein the bacteriophage is a variant strain of the deposited bacteriophage strain having an RFLP DNA profile equivalent to that of the deposited bacteriophage strain.

4. An isolated offspring bacteriophage of a deposited bacteriophage strain according to Claim 1, which infects and lyses one or more strains of VRE, wherein the isolated offspring bacteriophage has 90% or more of the gANI relative to the deposited bacteriophage strain.

5. An isolated offspring bacteriophage of a deposited bacteriophage strain according to Claim 1, which infects and lyses one or more strains of VRE, wherein the isolated offspring bacteriophage has 95% or more of the gANI relative to the deposited bacteriophage strain.

6. An isolated progeny bacteriophage of a deposited bacteriophage strain according to Claim 1, which infects and lyses one or more strains of VRE, wherein the isolated progeny bacteriophage has 98% or more or 99.9% or more of the gANI relative to the deposited bacteriophage strain.

7. (i) Bacteriophages of one or more strains as described in any one of claims 1 to 6; and (ii) Pharmaceutically acceptable carriers A composition containing the following.

8. (i) Bacteriophage strain VREML110-1 or a variant strain thereof deposited with ATCC under accession number PTA-126932, wherein the variant strain has at least 90% of the gANI of bacteriophage strain VREML110-1; (ii) Bacteriophage strain VREML105 or a variant strain thereof deposited with ATCC under accession number PTA-126931, wherein the variant strain has at least 90% of the gANI of bacteriophage strain VREML105; (iii) bacteriophage strain VREML202-1 or a variant strain thereof deposited with ATCC under accession number PTA-126933, wherein the variant strain has at least 90% of the gANI of bacteriophage strain VREML202-1; and (iv) Bacteriophage strain VREML85-2 or a variant strain thereof deposited with ATCC under accession number PTA-126930, wherein the variant has at least 90% of the gANI of bacteriophage strain VREML85-2. A composition further comprising one or more bacteriophage strains comprising or comprising the above, wherein the variant strains (i) to (iv) infect and lyse one or more strains of VRE, according to claim 7.

9. The composition according to claim 7, comprising or comprising (i) bacteriophage strain VREML237-2; (ii) bacteriophage strain VREML110-1; (iii) bacteriophage strain VREML105; (iv) bacteriophage strain VREML202-1; and (v) bacteriophage strain VREML85-2.

10. The composition according to claim 7, wherein the composition is provided in an oral dosage form.

11. The composition according to claim 7, wherein the composition is provided in a dosage form selected from a rectal dosage form, a vaginal dosage form, a pulmonary dosage form, a topical dosage form, and an injectable dosage form.

12. The composition according to claim 7, further comprising probiotics.

13. The composition according to claim 12, wherein the probiotic comprises probiotic bacteria.

14. The aforementioned probiotic bacteria include L. acidophilus, L. rhamnosus, L. gasseri, L. reuteri, L. bulgaricus, L. plantarum, L. johnsonii, L. paracasei, L. casei, L. salivarius, L. lactis, B. bifidum, B. longum, B. breve, B. The composition according to claim 13, which is one or more selected from the group consisting of Bacillus infantis, Bacillus lactis, Bacillus adolescentis, Streptococcus thermophilus, Bacillus cereus, Bacillus subtilis, and any combination thereof.

15. The composition according to claim 12, wherein the probiotics include probiotic yeast.

16. The aforementioned probiotic yeasts include Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces cerevisiae var. boulardii, Issachenkia occidentalis, Lachancea thermotolerance, Metschnikowia ziziphylla, and Torulaspora delbruckii. The composition according to claim 15, selected from the group consisting of delbrüeckii, and any combination thereof.

17. The composition according to claim 7, for use in reducing, preventing, or treating the risk of VRE colonization or infection in a target subject, or in modulating the microbiome of a human subject.

18. The composition according to claim 17, wherein the subject has a risk of colonization or infection by VRE, or is colonized or infected by VRE.

19. The composition according to claim 17, wherein the subject is suffering from VRE intestinal colonization, and the composition is effective in reducing or eliminating VRE intestinal colonization.

20. The composition according to claim 17, wherein the treatment further comprises administering probiotics to the subject.

21. The composition according to claim 17, wherein the composition is administered orally.

22. The composition according to claim 17, wherein the composition is administered rectally.

23. The composition according to claim 17, wherein the composition is administered topically.

24. The composition according to claim 17, wherein the composition is administered via vaginal delivery.

25. A composition comprising a lytic enzyme produced by a bacteriophage according to any one of claims 1 to 6.

26. A vaccine comprising a VRE bacterial lysate obtained by lysing a VRE strain with a bacteriophage or its lytic enzyme according to any one of claims 1 to 6.

27. A composition for use in targeted vaccination against VRE infection, comprising a VRE bacterial lysate obtained by lysing a VRE strain with a bacteriophage or its lytic enzyme according to any one of claims 1 to 6.

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