Methods for treating implantable device infections

Abstract

To provide a method for treating implantable device infections. [Solution] The present invention relates to the field of phage therapy, and more particularly to providing phage-based compositions and methods for treating or preventing infections associated with implantable devices. The compositions may be administered directly to the infection site of the device, as a single dose and / or by other means as needed, potentially avoiding the need to replace the implantable device. The method may include (a) obtaining a biological sample from the subject; (b) culturing bacteria present in the biological sample; and (c) inoculating the cultured bacteria with a phage to lyse them.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to the field of phage therapy, and more particularly to providing phage-based compositions and methods for treating or preventing acute or chronic bacterial infections associated with implantable devices using phage-based compositions. [Background technology]

[0002] Consideration of related technologies The following description provides context and introduces specific articles and methods. Nothing included herein should be construed as an "acknowledgment" of prior art. The applicant expressly reserves the right, where appropriate, to expressly indicate that the articles and methods referenced herein do not constitute prior art under applicable legal provisions.

[0003] Partial or total knee and hip replacement, most commonly performed, is a life-improving procedure that millions of patients undergo each year. In most cases, the procedure is highly effective, relieving pain and restoring function and independence to patients. In a relatively small number of cases, complications can occur, such as malfunction of the artificial joint device (e.g., caused by wear, device breakage, or misalignment) or the development of periarthritis of the joint (PJI), also known as periarthritis of the joint (PJI), an infection involving the artificial joint device and surrounding tissues. Studies have shown that the incidence of PJI in the United States is approximately 2% for knee replacements and up to approximately 1.5% for hip replacements (Tande AJ & R Patel, Clin. Microbiol. Rev. 27(2):302-345, 2014). However, although this number is relatively small, PJI is a very significant risk for patients, resulting in a considerable pathological condition with pain and swelling, and in some cases, potentially leading to amputation and / or death. The diagnosis, treatment, and management of PJI also represent a significant cost burden on healthcare systems, projected to exceed US$1.5 billion in the United States alone in 2020 (Kurtz SM et al., J. Arthroplasty 27:61-65.e61, 2012).

[0004] Generally, PJI is broadly classified into three types; each is distinguished by the timing of the onset of infection after surgery. Therefore, PJI includes (i) infections that occur early, within three months of surgery ("early-onset" PJI); (ii) infections that occur later, three months after surgery but before 12 or 24 months ("delayed-onset" PJI); and (iii) infections that occur 12 to 24 months or later ("late-onset" PJI). PJI infections can also be classified as "acute" infections that occur within one month of surgery, or "chronic" infections that occur more than one month after surgery. Regardless of the classification system used, early PJI infections are primarily caused by relatively virulent microorganisms such as Staphylococcus aureus and / or aerobic Gram-negative bacteria such as Pseudomonas aeruginosa, while delayed-type PJI is commonly caused by less virulent bacteria such as coagulase-negative S. aureus (CoNS), Staphylococcus epidermidis, and / or Enterococci spp. Both early and delayed types are thought to be acquired during surgery, but late-type PJI infections can frequently occur as secondary infections from another bacterial infection site (e.g., resulting from hematogenous transmission of bacteria via the blood from another infection site). S. aureus is considered a common causative microorganism of late-onset PJI resulting from hematogenous transmission (Tande & Patel, cited above).

[0005] To successfully treat PJI, surgical intervention and / or medical treatment (e.g., treatment with antimicrobial agents such as antibiotics) are usually required in the majority of cases. Some procedures, known as DAIR procedures, involve debridement (i.e., surgical removal of infected / damaged tissue), antibiotic treatment, and implant retention (artificial joint device), while others require replacement of the artificial joint device in a one- or two-stage arthroplasty (Tande & Patel, cited above) combined with antibiotic treatment. Two-stage arthroplasty is generally considered "the most reliable strategy for eradicating infection and preserving joint function," with reported success rates as high as 87–100% for hip replacement and 72–95% for knee replacement (Tande & Patel, cited above). However, these two-stage procedures can be complex and expensive, requiring at least two surgeries, sometimes spaced 2–3 months or longer, and prolonged antibiotic treatment. This means that some patients are unsuitable for or unwilling to undergo two-stage (or even one-stage) arthroplasty, in which case there are few options other than attempting to treat PJI with antibiotics. However, this is not recommended, and generally, patients are treated with oral antibiotics for an extended or indefinite period, with careful therapeutic monitoring continuing (Tande & Patel, cited above).

[0006] An additional complicating problem for the effective treatment of PJI with antibacterial agents is that the infection can be a "polymicrobial infection," which means that there are two or more causative microorganisms present in the infection. This is particularly the case with early-onset PJI, where it is estimated that up to 35% of such infections are polymicrobial infections that typically involve infections by aerobic gram-negative bacteria such as S. aureus, Enterococci spp., and P. aeruginosa (Berbari EF et al., Clin. Infect. Dis. 27:1247-1254, 1998; Peel TN et al., Antimicrob. Agents Chemother. 56:2386-2391, 2012). Therefore, careful selection of antibacterial agents is necessary for treatment to be successful. In recent years, the effectiveness and options for the treatment of PJI have improved significantly, but there is a great need to identify and develop new or improved treatment and / or management strategies to effectively treat and / or prevent these infections in patients undergoing joint replacement surgery as well as other infections associated with implanted devices.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Means for Solving the Problem

[0008] Summary of the Invention This summary is provided to introduce, in a simplified form, a selection of concepts that are further described in the detailed description below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will become apparent from the following detailed description, which includes the aspects illustrated in any accompanying drawings and defined in the appended claims.

[0009] Specifically, the disclosed invention is a method for treating an infectious disease caused by one or more bacteria associated with a device implanted within a subject, the method comprising (a) Identify at least one identified phage capable of infecting bacteria, wherein the identified phages are APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-P JI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI -21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29 , APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, A PT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT- The library is to be selected from a collection of at least one phage selected from any other lytic phages that have a genome sequence having at least 70% sequence identity to the genome sequence of PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50, or any of the aforementioned phages, and that can induce bacterial lysis; and (b) Administering a therapeutically effective amount of a composition containing identified phages to a subject, wherein the composition is effective in treating and / or reducing an infection. Describe the method, including the following:

[0010] In other embodiments, the disclosed invention is a method for identifying a subject that is suffering from, at risk of suffering from, or is suitable to receive treatment for, a method which is (a) A step of obtaining a biological sample from the subject; (b) A step of culturing bacteria present in the biological sample; (c) APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07 in the cultured bacteria. , APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, A PT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, AP The steps include: inoculating an identified phage selected from a library containing T-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50, as well as at least one phage selected from one or more other lytic phages that have a genome sequence having at least 70% sequence identity to the genome sequence of any of the aforementioned phages and are capable of causing bacterial lysis; and (d) A step of determining whether cultured bacteria are lysed by identified phages, wherein if any of the cultured bacteria are lysed by the phage, the subject is determined to be (1) eligible for bacteriophage treatment for bacterial infection, (2) suffering from a bacterial infection; and / or (3) at risk of suffering from a bacterial infection. Describe the method, including the following:

[0011] Further embodiments include compositions used in any of the methods: APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJ I-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-P JI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT- PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT -PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, AP The phage comprises at least one identified phage selected from the group consisting of T-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50, or any other lytic phages having a genome sequence with at least 70% sequence identity to the genome sequence of any of the aforementioned phages, and capable of causing bacterial lysis.

[0012] Other embodiments are described further below. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for treating an infection caused by one or more bacteria associated with a device implanted within a subject, wherein the method is: (a) Identifying at least one identified phage capable of infecting the bacteria, wherein the identified phage is APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT -PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJ I-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-2 9, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, A PT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT- The library is to be selected from a library containing at least one phage selected from any other lytic phages that has a genome sequence having at least 70% sequence identity with the genome sequence of PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50, or any of the aforementioned phages, and that can cause lysis of the bacteria; and (b) Administering to the subject a therapeutically effective amount of a composition containing the identified phage, wherein the composition is effective in treating and / or reducing the infection. Methods that include... (Item 2) A method for identifying a person who is suffering from or at risk of suffering from a bacterial infection, wherein the method is: (a) A step of obtaining a biological sample from the subject; (b) A step of culturing the bacteria present in the biological sample; (c) The cultured bacteria are given APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-P JI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-1 8, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, AP T-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45 The steps include: inoculating an identified phage selected from a library containing at least one phage selected from one or more other lytic phages that have a genome sequence having at least 70% sequence identity to the genome sequence of APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50 and any of the aforementioned phages, and that can cause lysis of the bacteria; and (d) A step of determining whether the cultured bacteria are lysed by the identified phage, wherein if any of the cultured bacteria are lysed by the phage, the subject is determined to be (1) eligible for bacteriophage treatment for the bacterial infection, (2) suffering from a bacterial infection; and / or (3) at risk of suffering from a bacterial infection. Methods that include... (Item 3) The composition is APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, PT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-1 5, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJ I-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT- The method according to item 1 or 2, comprising at least one identified phage selected from the group consisting of PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50, or any other lytic phages having a genome sequence having at least 70% sequence identity with the genome sequence of any of the aforementioned phages, and capable of causing lysis of the bacteria. (Item 4) The composition may be APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT -PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, AP T-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, A PT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30 , APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-3 6, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI- 42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI The method according to any of the above items, comprising at least two identified phages selected from the group consisting of any other lytic phages having a genome sequence having at least 70% sequence identity to the genome sequence of -48, APT-PJI-49 and / or APT-PJI-50, or any of the aforementioned phages, and capable of causing lysis of the bacteria. (Item 5) The aforementioned at least two identified phages, (a) The identified phage causes lysis in the same bacterial strain; (b) The identified phage causes lysis in different bacterial strains; (c) At least one of the identified phages is APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, Selected from the group consisting of APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, and APT-PJI-46, as well as any other lytic phages having a genome sequence with at least 70% sequence identity to the genome sequence of any of the aforementioned phages; (d) At least one of the identified phages is selected from the group consisting of APT-PJI-13, APT-PJI-14, and any other lytic phages having a genome sequence that has at least 70% sequence identity with the genome sequence of any of the aforementioned phages; (e) At least one of the identified phages is selected from the group consisting of APT-PJI-15, APT-PJI-16, APT-PJI-17, and APT-PJI-18, as well as any other lytic phages having a genome sequence that has at least 70% sequence identity with the genome sequence of any of the aforementioned phages; (f) At least one of the identified phages is selected from the group consisting of APT-PJI-20 and APT-PJI-21 and any other lytic phages having a genome sequence that has at least 70% sequence identity with the genome sequence of any of the aforementioned phages. (g) at least one of the identified phages is selected from the group consisting of APT-PT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45 or APT-PJI-46 and any other lytic phage having a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the aforementioned phages; (h) at least one of the identified phages is selected from the group consisting of APT-PJI-47, APT-PJI-48, APT-PJI-49 or APT-PJI-50 and any other lytic phage having a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the aforementioned phages; (i) at least one of the identified phages is selected from any of the combinations of (a) to (h); The method according to item 4, characterized in that. (Item 6) The composition provides each phage at a dose within the range of 10 9 , 8 , 11 , 7 , 11 , 6 , 7 , 12 , 8 , 10 , 6 , 11 , , 9 ~10 13 pfu, preferably the dose of each phage is 10 6 ~10 12 pfu; 10 7 ~10 11 pfu; 10 8 ~10 11 pfu; 10 9 ~10 11 pfu; 10 9 ~10 10 pfu, or within the range of 10 or approximately 10 6 pfu, 10 7 pfu, 10 8 pfu, 109 pfu, 10 10 pfu, 10 11 pfu, 10 12 pfu or 10 13 The dosage is pfu. The method described in any one of the items above. (Item 7) The method according to any of the above items, wherein the bacteria are selected from at least one of the following: Enterococcus spp. including S. aureus, S. epidermidis, E. fecalis and Enterococcus fecium; other coagulase-negative Staphylococcus species including S. simulans, S. caprae and S. lugdunensis; Lancefield groups A, B, C and G; Streptococcus spp. including S. agalactiae and S. pneumoniae; aerobic gram-negative bacteria including Escherichia coli; other Enterobacteriaceae including Enterobacter clocae; Clostridium spp.; Actinomyces spp.; Peptostreptococcus spp.; Cutibacterium acnes, Klebsiella pneumoniae, P. aeruginosa and Bacteroides fragilis. (Item 8) The aforementioned bacteria include Enterococcus spp., including S. aureus, S. epidermidis, E. fecalis, and Enterococcus fecium; other coagulase-negative Staphylococcus species, including S. simulans, S. caprae, and S. lugdunensis; Lancefield groups A, B, C, and G; Streptococcus spp., including S. agalactiae and S. pneumoniae; aerobic Gram-negative bacteria, including Escherichia coli; other Enterobacteriaceae, including Enterobacter clocae; Clostridium spp.; Actinomyces spp.; Peptostreptococcus spp.; Cutibacterium acnes; Klebsiella pneumoniae; P. aeruginosa, and Bacteroides. The method described in the preceding item, selected from two or more different bacterial strains selected from fragilis. (Item 9) The method according to any one of the above items, wherein the bacterium is selected from S. aureus, S. epidermidis, E. fecalis, S. caprae and / or S. lugdunensis. (Item 10) The method according to the preceding item, wherein the composition comprises at least two phages having different lytic specificities that can infect at least two of S. aureus, S. epidermidis, E. fecalis, S. caprae and / or S. lugdunensis. (Item 11) The method according to the preceding item, wherein the composition comprises at least three phages having different lytic specificities, and each phage is capable of infecting at least one of S. aureus, S. epidermidis, E. fecalis, S. caprae, and / or S. lugdunensis. (Item 12) The method according to any one of the above items, wherein the other lytic phage has a genomic sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to any of the genomic sequences of the aforementioned phages. (Item 13) The composition is (a) Identified phages that are compatible with bacterial strains known to exist in a particular geographical location; (b) APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT- PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, A The method described in any one of the above items, comprising a phage selected from PT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50. (Item 14) The aforementioned device (a) permanently planted in the subject; (b) temporarily planted in the subject; (c) Removable; and / or (d) Artificial joints, left ventricular assist devices (LVADs), stents, metal rods, indwelling catheters, spinal devices and / or spinal instruments, and / or bone devices and / or bone instruments Selected from, The method described in any one of the items above. (Item 15) The method according to any one of the above items, wherein the infection is selected from prosthesis joint infection (PJI), chronic bacterial infection, acute bacterial infection, refractory infection, biofilm-related infection, and implantable device-related infection. (Item 16) The composition is (a) By IV injection; (b) By direct injection into the site of infection; (c) By intra-articular injection; (d) By IM injection; (e) Preventively; (f) Before surgery; (g) Instead of surgery; (h) During surgery; (i) in one go (i.e., as a "single shot"), and / or (j) As a treatment process lasting two weeks or longer, The method described in any one of the items above, to be administered. (Item 17) Dissolution, (a) Growth inhibition; (b) optical density; (c) metabolic output; (d) Photometric (e.g., fluorescence, absorption, and transmission assays); and / or (e) Plaque formation The method described in any one of the above items, measured by the change in [the specified value]. (Item 18) The method described above, wherein the photometric change is measured using an additive that causes and / or enhances the detection of the photometric signal, preferably the additive being a tetrazolium dye. (Item 19) The aforementioned biological sample (a) Synovial fluid; (b) Area surrounding the implanted device; (c) site of infection; (d) Samples during surgery; (e) A swab of the device; (f) Biofilm; (g) fistula; and / or (h) Aspirate from the site of infection A method obtained from any one of the items described above. (Item 20) The aforementioned bacterial infection, (a) It is resistant to multiple drugs; (b) Clinically refractory to antibiotic treatment; (c) Clinically refractory to antibiotic treatment due to biofilm formation; and / or (d) The subject is clinically refractory to treatment due to an inability to tolerate the antibacterial agent as a result of an adverse reaction, The method described in any one of the items above. (Item 21) The method described in any one of the above items, wherein the subject is suffering from an equipment-related infection. (Item 22) The method described in any of the above items, wherein the subject is suffering from prosthetic joint infection (PJI). (Item 23) The method according to any one of the preceding items, wherein the library includes phages that have been pre-screened to exclude phages having undesirable and / or toxic characteristics. (Item 24) The method according to the preceding item, wherein the excluded phages having undesirable and / or toxic characteristics are selected from toxin genes or other bacterial pathogenic factors, phages having lysogenic properties and / or possessing lysogenic genes, phages transmitting bacterial pathogenic factor genes or antibiotic resistance genes, phages possessing any antibiotic resistance gene or capable of conferring antibiotic resistance to bacterial strains, and phages that induce an inappropriate immune response and / or elicit a strong allergic response in mammalian systems. (Item 25) The method described in item 23 or 24, wherein the aforementioned undesirable and / or toxic characteristics are eliminated by genome editing. [Modes for carrying out the invention]

[0013] Detailed explanation The following definitions are provided for the specific terms used in the following written explanation.

[0014] definition As used herein and in the claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Also, as will be understood by those skilled in the art, the term “phage” can be used to refer to a single phage or two or more phages.

[0015] The present invention may "comprise" (open-ended) or "essentially consist of" the components of the present invention. As used herein, "comprising" means the enumerated elements, or their equivalents in structure or function, as well as any other elements or components not enumerated. The terms "having" and "including" should also be interpreted as open-ended unless otherwise suggested by the context.

[0016] The terms “about” or “approximately” mean an acceptable range of a particular value as determined by those skilled in the art, which will depend in part on how the value is measured or determined, for example, on the limitations of the measuring system. For example, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or methods, the term may mean within one order of magnitude, for example, within five times or twice the value. Unless otherwise specified, the term “about” means an acceptable range of error for a particular value, for example, ±1 to 20%, preferably ±1 to 10%, and more preferably ±1 to 5%. In further embodiments, “about” should be understood to mean + / - 5%.

[0017] Where a range of values ​​is provided, it is understood that each intermediate value between the upper and lower limits of that range, and any other stated or intermediate values ​​within that stated range, are included in the present invention. The upper and lower limits of these smaller ranges may independently be included within smaller ranges and are included in the present invention, subject to any specifically excluded limits within the stated range. If a stated range includes one or both limits, the range excluding both of those included limits is also included in the present invention.

[0018] All ranges listed herein include endpoints that enumerate the range "between" two values.

[0019] Terms such as “about,” “generally,” “substantially,” and “approximately” should be interpreted as modifying a term or value so as not to be read in the prior art, rather than being absolute. Such terms are defined by the context and the terms they modify so that they are understood by those skilled in the art. This includes, at a minimum, the degree of expected experimental error, technical error, and instrumental error with respect to a given technique used to measure a value.

[0020] As used herein, the term "and / or" when used in a list of two or more items means that any one of the listed characteristics may be present, or any combination of two or more listed characteristics may be present. For example, if a composition is described as containing features A, B and / or C, the composition may contain feature A only, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0021] The term "bacteriophage" or "phage," as understood by those skilled in the art, refers to a noncellular infectant that replicates only in appropriate host cells, namely bacterial host cells.

[0022] The term "phage therapy" refers to any treatment that addresses a bacterial infection, or a disease or condition caused by bacteria (e.g., PJI), or a disease or condition that exhibits the onset or progression of symptoms or disease / symptoms associated with the presence of bacteria. Phage therapy may involve administering to a patient who requires treatment with one or more therapeutic phage compositions that can be used to infect bacteria, kill bacteria, or inhibit bacterial growth, the compositions containing one or more viable phages as antimicrobial agents (e.g., a composition containing one phage type or two or more phage types in a phage "cocktail"). The one or more phage types may be obtained from a stock of phages, which may be stored as inventory. If phage therapy involves the administration of two or more therapeutic phage compositions, those compositions may have different host ranges (e.g., one may have a broad host range, one may have a narrow host range, and / or one or more compositions may act synergistically with each other). Furthermore, as those skilled in the art will readily understand, therapeutic phage compositions used in phage therapy typically also include a set of inert components selected from a variety of conventional pharmaceutically acceptable excipients, carriers, buffers, and / or diluents.

[0023] The term "pharmaceutically acceptable" is used to refer to a non-toxic substance that is compatible with a biological system, such as cells, cell cultures, tissues, or organisms. Examples of pharmaceutically acceptable excipients, carriers, buffers, and / or diluents are well known to those skilled in the art, e.g., Remington's Pharmaceutical Sciences (latest edition), Mack This can be found in Publishing Company, Easton, Pa. For example, pharmaceutically acceptable excipients include, but are not limited to, wetting or emulsifying agents, pH buffers, binders, stabilizers, preservatives, bulking agents, adsorbents, disinfectants, detergents, sugar alcohols, gelling or thickening agents, flavoring agents, and coloring agents. Pharmaceutically acceptable carriers include polymers, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, trehalose, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Pharmaceutically acceptable diluents include, but are not limited to, water and physiological saline.

[0024] The present invention relates to providing phage-based compositions and methods for treating infections associated with implantable devices, such as prosthesis infections ("PJI"). Since the treatment may involve directly administering the composition to the infected area (e.g., a joint) as a single dose as needed, the compositions and methods may offer considerable simplicity, and for example, phage therapy may potentially avoid the need for replacement of implantable devices (e.g., prosthesis devices in infected joints).

[0025] Accordingly, in a first embodiment, the present invention provides a pharmaceutical composition comprising at least two different bacteriophages capable of dissolving infections associated with implantable devices (e.g., prosthesis joint infections (PJIs) in patients), the pharmaceutical composition being formulated to be administered to a patient directly and / or orally at the site of infection by IV.

[0026] In one embodiment, at least two different bacteriophages may be phage types selected for their ability to lyse (i.e., kill) a single bacterial species or two or more bacterial species (i.e., in the case of a polymicrobial infection) that may be present in an implantable device infection.

[0027] The selection of phage types for therapeutic indications may be based on the results of tests to determine the type of bacteria (i.e., causative microorganism) present in implantable device infections; for example, synovial fluid aspiration and subsequent culturing of that synovial fluid on solid or liquid media can easily determine the bacteria present in the culture using standard techniques well known to those skilled in the art, including 16S rRNA gene sequencing analysis (e.g., Whelan FJ et al., Ann. Am. Thorac. Soc. 11:513-521, 2014). Biological samples may also be used in assays to evaluate the susceptibility of bacteria to individual phages, which can be very useful in selecting phage types to include in the composition.

[0028] The selection of phage types may also be based on predictions or expectations about which causative microorganisms are present in implantable device infections. Similarly, the selection of phage types may also be based on predictions or expectations about which causative microorganisms are present in specific geographical locations.

[0029] For example, the phages selected for inclusion in this composition may include types capable of lysing bacteria that commonly cause delayed-onset device infections, such as late-onset PJIs, namely S. aureus (e.g., coagulase-negative S. aureus) and Enterococcus spp. (e.g., E. fecalis and Enterococcus fecium). Such compositions may also be effective in treating late-onset PJIs commonly caused by S. aureus. Alternatively, the selected phages can be used to treat acute or chronic infections.Other bacteria that could be targeted by including appropriately selected phage types include other coagulase-negative Staphylococcus species reported to cause PJI, e.g., S. simulans (Razonable RR et al., Mayo Clin. Proc. 76:1067-1070, 2001), S. caprae (Allignet J et al., Microbiology 145 (Part 8):2033-2042, 1999), and S. lugdunensis (Sampathkumar P et al., Mayo Clin. Proc. 75:511-512, 2000); Lancefield groups A, B, C, and G (Meehan AM et al., Clin. Infect. Dis. 36:845-849, 2003; Zeller V et al., Presse Med. 38:1577-1584, 2009; and Kleshinski J et al.) Various Streptococcus spp. associated with PJI, including S. agalactiae and S. pneumoniae (Raad J et al., Semin. Arthritis Rheum. 34:559-569, 2004), aerobic Gram-negative bacteria, such as Escherichia coli (Jaen N et al., Rev. Esp. Quimioter. 25:194-198, 2012), other Enterobacteriaceae (Hsieh PH et al., Clin. Infect. Dis. 49:1036-1043, 2009), such as Enterobacter clocae; and others, such as Clostridium spp., Actinomyces spp., Peptostreptococcus spp., and Cutibacterium acnes (formerly Propionibacterium). Examples include acnes, Klebsiella pneumoniae, P. aeruginosa, and Bacteroides fragilis (Tande & Patel, mentioned above).

[0030] Furthermore, it is intended that the identified phages included in the composition may be a matched phage composition based on infectious diseases that commonly occur in specific geographical locations. An example of such a “geomatched” phage is described in USSN 62 / 956,729, filed January 3, 2020 (the entire document is incorporated herein by reference).

[0031] Therefore, one aspect of the present invention is a method for treating an infection caused by one or more bacteria associated with a device implanted in a subject, the method being: (a) Identify at least one identified phage capable of infecting bacteria, wherein the identified phages are APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11 as listed in Table 1. , APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, AP T-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJ I-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-3 7, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, AP The library is to be selected from a collection of at least one phage selected from any other lytic phages that have a genome sequence having at least 70% sequence identity to the genome sequence of T-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50, or any of the aforementioned phages, and that can induce bacterial lysis; and (b) Administering a therapeutically effective amount of a composition containing identified phages to a subject, wherein the composition is effective in treating and / or reducing an infection. This method includes [something].

[0032] Table 1 shows the publicly released phages (e.g., APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, A PT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17 and APT-PJI-18) as well as proprietary phages that are not publicly available (e.g., APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT- PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, AP T-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, A Information related to PT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50) is provided.

[0033] In a preferred embodiment, the composition and method are carried out using any combination of proprietary phages. Specifically, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-3 9. Treat patients suffering from PJI infections as described herein using at least one, at least two, at least three, at least four, and / or at least five phages selected from the group consisting of APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49, and / or APT-PJI-50. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0034] In another aspect, the disclosed invention is a method for identifying subjects who are suffering from, at risk of suffering from, and / or who are suitable for phage treatment for a bacterial infection, the method is (a) A step of obtaining a biological sample from the subject; (b) A step of culturing bacteria present in the biological sample; (c) APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07 in the cultured bacteria. , APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, A PT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, AP The steps include: inoculating an identified phage selected from a library containing T-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50, as well as at least one phage selected from one or more other lytic phages that have a genome sequence having at least 70% sequence identity to the genome sequence of any of the aforementioned phages and are capable of causing bacterial lysis; and (d) A step of determining whether cultured bacteria are lysed by identified phages, wherein if any of the cultured bacteria are lysed by the phage, the subject is determined to be (1) eligible for bacteriophage treatment for bacterial infection, (2) suffering from a bacterial infection; and / or (3) at risk of suffering from a bacterial infection. This method includes [something].

[0035] In a preferred embodiment, compositions are APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-1 4, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-P JI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, AP T-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-3 8, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-P The phage comprises at least one identified phage selected from the group consisting of any other lytic phages having a genomic sequence that has at least 70% sequence identity with the genomic sequence of JI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50 or any of the aforementioned phages, and which is capable of causing bacterial lysis.

[0036] In a more preferred embodiment, compositions are APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-P JI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, AP T-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30 , APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI -38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT- The phage comprises at least two identified phages selected from the group consisting of any other lytic phages having a genome sequence that has at least 70% sequence identity with the genome sequence of PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and / or APT-PJI-50 or any of the aforementioned phages, and which are capable of causing bacterial lysis.

[0037] In a further embodiment, at least two identified phages (a) induce lysis in the same bacterial strain; and (b) induce lysis in different bacterial strains.

[0038] In other further embodiments, one of the two identified phages described above is (a) APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI- 26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI- 36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45 and APT-PJI -46 and any other lytic phages having a genome sequence with at least 70% sequence identity to any of the phages mentioned in (a) above are selected from the group consisting of (b) APT-PJI-13, APT-PJI-14 and any other lytic phages having a genome sequence with at least 70% sequence identity to any of the phages mentioned in (b) above are selected from the group consisting of (c) APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18 and any other lytic phages having a genome sequence with at least 70% sequence identity to any of the phages mentioned in (c) above are selected from the group consisting of (d) APT-PJI-20 or APT-PJI-21 and any other lytic phages having a genome sequence with at least 70% sequence identity to any of the phages mentioned in (d) above are selected from the group consisting of (d) APT-PJI-20 or APT-PJI-21 and any other lytic phages having a genome sequence with at least 70% sequence identity to any of the phages mentioned in (d) above are selected from the group consisting of (b) APT-PJI-13, APT-PJI-14 at least 70% sequence identity to any of the phages mentioned in (d) above are selected from the group consisting of (b) APT-PJI-13, APT-PJI-14 and any other lytic phages having at least 70% sequence identity to any of the phages mentioned in (d) above are selected from the group consisting of (b) APT-PJI-13, AAlternatively, (e) selected from the group consisting of APT-PJI-47, APT-PJI-48, APT-PJI-49, or APT-PJI-50 and any other lytic phages having a genome sequence that has at least 70% sequence identity with the genome sequence of any of the aforementioned phages in (e).

[0039] In a further embodiment, the two identified phages are selected from each of (a) and (b); selected from each of (a) and (c); selected from each of (a) and (d); selected from each of (a) and (e); and / or selected from each of (b) and (c), selected from each of (b) and (d); selected from each of (b) and (e); and / or selected from each of (c) and (d); selected from each of (c) and (e); and / or selected from each of (d) and (e). In a further embodiment, the composition comprises at least three identified phages, at least one of which is selected from each of (a), (b), (c), (d) and / or (e) derived from the phages described in the preceding paragraph.

[0040] In another further embodiment, one of the two identified phages described above is (a) APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT -PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45 and APT-PJI-46 and (a (b) selected from a group consisting of any other lytic phages having a genome sequence that has at least 70% sequence identity with the genome sequence of any of the aforementioned phages in (b); (b) selected from a group consisting of any other lytic phages having a genome sequence that has at least 70% sequence identity with the genome sequence of APT-PJI-20 or APT-PJI-21 and any of the aforementioned phages in (b); or (c) selected from a group consisting of any other lytic phages having a genome sequence that has at least 70% sequence identity with the genome sequence of any of the aforementioned phages in (c).

[0041] In a further embodiment, the two identified phages are selected from each of (a) and (b); selected from each of (a) and (c); and / or selected from each of (b) and (c). In a further embodiment, the composition comprises at least three identified phages, at least one of which is selected from each of (a), (b), and (c) of the phages described in the preceding paragraph.

[0042] In a further embodiment, the composition, 10 5 ~10 13 Within the range of pfu, preferably 106 ~10 12 pfu;10 7 ~10 11 pfu;10 8 ~10 11 pfu;10 9 ~10 11 pfu;10 9 ~10 10 A dose within the range of pfu or approximately 10 6 pfu, 10 7 pfu, 10 8 pfu, 10 9 pfu, 10 10 pfu, 10 11 pfu, 10 12 pfu or 10 13 The present invention provides each phage in a dose of pfu. In the most preferred embodiment, the dose of each phage in the composition is approximately 10 9 It is pfu.

[0043] Therefore, the composition can be formulated so that each phage type is contained in an appropriate dose. As just one example, an appropriate dose of phage is 10 5 ~10 13 pfu, fua10 9 ~10 12 It is within the range of pfu. Most preferably, the composition is about 10 9 pfu, 10 10 PFU or 10 11 The formulation is designed so that each phage type has a corresponding dose of pfu.

[0044] In preferred embodiments, the bacteria are selected from at least one of the following: Enterococcus spp., including S. aureus, S. epidermidis, E. fecalis and Enterococcus fecium; other coagulase-negative Staphylococcus species, including S. simulans, S. caprae and S. lugdunensis; Lancefield groups A, B, C and G; Streptococcus spp., including S. agalactiae and S. pneumoniae; aerobic gram-negative bacteria, including Escherichia coli; other Enterobacteriaceae, including Enterobacter clocae; Clostridium spp.; Actinomyces spp.; Peptostreptococcus spp.; Cutibacterium acnes, Klebsiella pneumoniae, P. aeruginosa and Bacteroides fragilis.

[0045] In other preferred embodiments, the bacteria are selected from two or more different bacterial strains selected from Enterococcus spp., including S. aureus, S. epidermidis, E. fecalis and Enterococcus fecium; other coagulase-negative Staphylococcus species, including S. simulans, S. caprae and S. lugdunensis; Lancefield groups A, B, C and G; Streptococcus spp., including S. agalactiae and S. pneumoniae; aerobic gram-negative bacteria, including Escherichia coli; other Enterobacteriaceae, including Enterobacter clocae; Clostridium spp.; Actinomyces spp.; Peptostreptococcus spp.; Cutibacterium acnes, Klebsiella pneumoniae, P. aeruginosa and Bacteroides fragilis.

[0046] In a more preferred embodiment, the bacteria are selected from S. aureus, S. epidermidis, and / or E. fecalis. To treat such an infection, the composition may include at least two phages having different lytic specificities that can infect at least two of S. aureus, S. epidermidis, and / or E. fecalis. In another embodiment, the composition may include, for example, at least three phages having different lytic specificities, each phage capable of infecting at least one of S. aureus, S. epidermidis, and E. fecalis.

[0047] Preferably, the lytic phage has a genomic sequence that has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the aforementioned phage genomic sequences.

[0048] The sequence identity percentages referred to herein are from the National Center It is understood that this was calculated by comparing two polynucleotide sequences using the BLAST algorithm from the Biotechnology Information database (NCBI; Bethesda, MD, United States of America).

[0049] The terms "sequence similarity percentage (%)" and "sequence identity percentage (%)" generally refer to the degree of identity or agreement between different nucleotide sequences of nucleic acid molecules or different amino acid sequences of polypeptides, whether or not they share a common evolutionary origin (see Reeck et al., cited above). Sequence identity can be determined using one of several publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, or GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin).

[0050] To determine the percentage of identity between two amino acid sequences or two nucleic acid molecules, those sequences are aligned for optimal comparison. The percentage of identity between two sequences is a function of the number of identical positions they share (i.e., percentage of identity = number of identical positions / total number of positions (e.g., overlapping positions) × 100). In one embodiment, the two sequences are of the same length or approximately the same length. The percentage of identity between two sequences can be determined using techniques similar to those described below, with or without gap tolerance. When calculating percentage sequence identity, perfect matches are typically counted.

[0051] The determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. A non-restrictive example of a mathematical algorithm used to compare two sequences is provided in Karlin and Altschul, Proc.Natl.Acad.Sci.USA 1993, 90:5873-5877, as modified by Karlin and This is the algorithm described in Altschul, Proc. Natl. Acad. Sci. USA 1990, 87:2264. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 1990; 215:403. By performing a BLAST nucleotide search using the NBLAST program, score=100, and word length=12, nucleotide sequences homologous to the sequence of the present invention can be obtained. By performing a BLAST protein search using the XBLAST program, score=50, and word length=3, amino acid sequences homologous to the protein sequence of the present invention can be obtained. To obtain a gapped alignment for comparison purposes, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 1997, 25:3389. Alternatively, iterative searches can be performed to detect intermolecular distance relationships using PSI-Blast. See Altschul et al. (1997), cited above. When using the BLAST, Gapped BLAST, and PSI-Blast programs, you can use the default parameters for each program (e.g., XBLAST and NBLAST). See ncbi.nlm.nih.gov / BLAST / on the World Wide Web.

[0052] Another non-restrictive example of a mathematical algorithm used to compare sequences is the algorithm described by Myers and Miller, CABIOS 1988;4:1 1-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0053] In a preferred embodiment, the percentage of identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (J.Mol.Biol.1970,48:444-453) incorporated into the GAP program in the GCG software package (Accelrys, Burlington, MA; available at accelrys.com on the World Wide Web), using a Blossum62 matrix or PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using an NWSgapdna.CMP matrix, gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and one that can be used when practitioners are unsure what parameters to apply to determine whether a molecule is the limit of sequence identity or sequence homology in the present invention) is to use the Blossum62 score matrix with a gap-open penalty of 12, a gap-extension penalty of 4, and a frame-shift gap penalty of 5.

[0054] Another non-restrictive example of a method for determining identity percentage is found in Current Protocols In Molecular Biology (FMAusubel et al., eds., 1987), Supplement 30, section 7.7.18, Table. This involves using a software program, such as the software program described in 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST with default parameters. In particular, preferred programs are BLASTN and BLASTP with the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sort = by high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: http: / / www.ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0055] Statistical analysis of the properties described herein may be performed by standard tests, such as t-tests, ANOVA, or chi-squared tests. Typically, statistical significance is measured at the level of p=0.05 (5%), more preferably p=0.01, p=0.001, p=0.0001, and p=0.000001.

[0056] In a preferred embodiment, the method described herein relates to a device, which is (a) permanently implanted in a subject; (b) temporarily implanted in a subject; (c) removable; and / or (d) selected from an artificial joint, a left ventricular assist device (LVAD), a stent, a metal rod, an indwelling catheter, a spinal device, and / or a bone device.

[0057] In a further embodiment, the infection is selected from prosthetic joint infections (PJI), chronic bacterial infections, acute bacterial infections, refractory infections, biofilm-related infections, and / or implantable device-related infections.

[0058] In further embodiments, the methods described herein relate to compositions, for example, It is administered (a) by IV injection; (b) by direct injection into the site of infection; (c) prophylactically; (d) before surgery; (e) instead of surgery; (f) during surgery; (g) in a single dose (i.e., as a “single shot”), and / or (h) as part of a treatment course lasting two weeks or longer.

[0059] In further embodiments, bacterial lysis by phages can be measured using assays known in the art, including but not limited to (a) growth inhibition; (b) optical density; (c) metabolic output; (d) photometrics (e.g., fluorescence, absorption, and transmission assays); and / or (e) plaque formation.

[0060] In a preferred embodiment, the photometric assay used to measure dissolution uses additives that induce and / or enhance the detection of the photometric signal. Examples of such additives include, but are not limited to, tetrazolium dyes.

[0061] In other preferred embodiments, the biological sample may include (a) synovial fluid; (b) the area surrounding the implanted device; (c) the site of infection; (d) a sample taken during surgery; (e) a swab of the device; (f) biofilm; (g) a fistula; and / or (h) an aspirate from the site of infection. It is obtained from.

[0062] In a further aspect, bacterial infections are clinically refractory to antibiotic treatment due to (a) multidrug resistance; (b) clinically refractory to antibiotic treatment due to biofilm formation; and / or (d) clinically refractory due to the subject's inability to tolerate antibiotics due to adverse reactions.

[0063] As understood herein, terms such as “multidrug resistance,” “multidrug resistance,” and “MDR” may be used interchangeably herein and are known to those skilled in the art, namely, multidrug-resistant bacteria are organisms that exhibit resistance to multiple different antimicrobial agents, e.g., antibiotics; more specifically, to multiple different classes of antibiotics. Bacterial infections treated are understood herein to include bacteria in biofilms and / or bacteria in planktonic growth modes.

[0064] Typical MDR bacteria that can be treated include, but are not limited to, "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.), which are often hospital-acquired infections and can cause severe local and systemic infections. Specifically, these include, for example, methicillin-resistant Staphylococcus aureus (MRSA); vancomycin-resistant Enterococcus faecium (VRE); carbapenem-resistant Klebsiella pneumonia (NDM-1); MDR-Pseudomonas aeruginosa; and MDR-Acinetobacter baumannii.

[0065] Among the ESKAPE pathogens, A. baumannii is a Gram-negative, encapsulated opportunistic pathogen that spreads readily in hospital intensive care units. For example, A. baumannii infections are typically found in the respiratory tract, urinary tract, and wounds. Many A. baumannii clinical isolates are also MDRs, which severely limits the available treatment options. Untreated infections in trauma often result in prolonged healing, the need for extensive surgical debridement, and, in some cases, further or complete amputation of the limb. Blast-related injuries in military groups, in particular, carry a high risk of infectious complications, as they involve significant tissue destruction and simultaneous massive blood loss. Given that A. baumannii and other MDR ESKAPE pathogens can colonize and survive in many environmental settings, those skilled in the art will understand the urgent need for new therapeutic agents against these pathogens.

[0066] In a further embodiment, the subject is suffering from an equipment-related infection.

[0067] In a preferred embodiment, the infection is prosthesis joint infection (PJI). For example, the compositions of the present invention may be useful in treating PJI of any type. That is, by appropriately selecting two or more phages, the compositions may be beneficial in treating early-onset, delayed-onset, and late-onset PJI, as well as PJI resulting from multiple bacterial infections. Similarly, in other preferred embodiments, the compositions described herein can be used to treat acute and / or chronic infections, preferably PJI infections. The compositions of the present invention may be particularly useful in treating PJI associated with hip, knee, shoulder, and elbow prosthesis replacement.

[0068] In some embodiments, the library used in the methods described herein includes phages that have been pre-screened to exclude phages having undesirable and / or toxic characteristics. Examples of such undesirable and / or toxic characteristics are selected from toxin genes or other bacterial pathogenic factors, phages having lysogenic properties and / or possessing lysogenic genes, phages transmitting bacterial pathogenic factor genes or antibiotic resistance genes, phages possessing any antibiotic resistance gene or capable of conferring antibiotic resistance to bacterial strains, and phages that induce an inappropriate immune response and / or elicit a strong allergic response in mammalian systems. An example of constructing such a pre-screened phage library is described, for example, in US10,357,522 (the entire disclosure of which is incorporated herein by reference).

[0069] The consequences of using bacteriophages to treat bacterial infections are known to include antibiotic resistance, toxin formation, and unwanted innate immune responses in mammals (Quiros et al., Antimicrob Agents Chemother 58:606-609, 2014; do Vale et al., Front. Microbiol. 7:42, 2016). In some respects, phages containing unwanted and / or toxic characteristics can be excluded from the library in the first place. It is well understood that genetically modifying phages to exclude and / or reduce unwanted and / or toxic phenotypes is also within the scope of the art. Harmful phenotypes can be traced back to genes located on the phage's single-stranded DNA. To remove these genes and prevent phages from horizontally transferring these characteristics to bacteria associated with chronic infections within implantable devices, genetic material can be edited using systems such as CRISPR (clustered and regularly arranged short palindromic sequence repeats). Phages with DNA or RNA genomes can be manipulated using CRISPR systems. Gene editing using CRISPR / Cas9

[0070] The CRISPR and CRISPR-related protein 9 (Cas9), collectively known as the CRISPR / Cas9 system, can be used for gene editing. Gene editing includes, but is not limited to, gene insertion, gene substitution, gene deletion, frameshift, single nucleotide changes, nonsense mutations, and missense mutations. Gene editing may also include editing of regulatory sequences to regulate gene expression. Based on these techniques, genes can be mutated, repaired, or regulated in various cell lines and germline sequences. A review of the CRISPR / Cas9 system can be found in Hsu et al., “Development This information may be found in "and Applications of CRISPR-Cas9 for Genome Engineering" Cell (2014) vol.157:1262. Guidelines for using CRISPR / Cas9 systems may be found in Addgene (addgene.org / CRISPR / guide).

[0071] The CRISPR / Cas9 system can be used to "knock out" and "knock in" specific genes in various cell types and organisms, as well as to selectively activate or repress specific genes, purify specific regions of DNA, image DNA in living cells using fluorescence microscopy, and for many other applications.

[0072] According to embodiments of the present invention, the CRISPR / Cas9 system can be used to edit genes within phages to eliminate and / or reduce unwanted and / or toxic phenotypes. By reducing or eliminating toxic phenotypes, unwanted effects of bacteriophage therapy for treating bacterial infections, including antibiotic resistance, toxin formation, and unwanted innate immune responses in mammals are reduced or eliminated, and thus these phages are expected to be useful in treating infections.

[0073] According to another embodiment of the present invention, phages edited by the CRISPR / Cas9 system may be administered to a patient. In yet further embodiments of the present invention, phages edited by the CRISPR / Cas9 system may be used in combination with one or more further active agents described herein.

[0074] Phage genome editing, which was traditionally a time-consuming and highly complex process, can now be performed efficiently using the CRISPR / Cas9 system. In some cases, editing with the CRISPR / Cas9 system can occur within 24 hours of delivery of the Cas9 gene, guide RNA, and, where applicable, the nucleotide substitution sequence. Components of a CRISPR / Cas9 system

[0075] Generally, the CRISPR / Cas9 system comprises at least two components: (i) the Cas9 protein, which is a nuclease capable of cleaving both strands of a DNA double helix; and (ii) at least one RNA sequence (e.g., a guide RNA (gRNA) sequence, e.g., a single-stranded guide RNA (sgRNA) sequence) designed to target Cas9 to a specific location or locus of a target gene (genomic target). The gRNA contains a targeting sequence homologous to the genomic target, as well as a scaffold domain that binds to Cas9, in order to recruit Cas9 to the genomic target. Non-homologous end joining

[0076] In some embodiments, the CRISPR / Cas9 system induces double-strand DNA breaks at genomic targets. These breaks are thought to stimulate cellular repair mechanisms that utilize non-homologous end joining (NHEJ). Non-homologous end joining, which can lead to gene disruption through random insertions or deletions, is considered a major mechanism for repairing double-strand breaks (DSBs). NHEJ-mediated DSB repair can disrupt the open reading frame (ORF) of genomic targets, resulting in non-functional proteins.

[0077] In the NHEJ method, Cas9 and gRNA are introduced into host cells, for example, as a pre-assembled complex or inserted into one or more expression vectors. Homology Directed Repair

[0078] In other embodiments, the CRISPR / Cas9 system is used to repair, replace, or insert one or more nucleotides in a genomic target. In this method, a substitution nucleotide sequence is introduced into a cell, where the substitution nucleotide sequence contains desired edit and homology regions upstream and downstream of the genomic target. Homologous recombination repair (HDR) requires a gRNA, e.g., an in vitro transcribed sgRNA, and Cas9, e.g., recombinant Cas9 from Streptococcus pyogenes, complexed with the substitution nucleotide sequence.

[0079] Homologous recombination repair (HDR) can be used to perform gene editing ranging from single nucleotide base changes to large nucleotide sequence insertions. To utilize HDR for gene editing, a substitution nucleotide sequence is delivered into the cell along with gRNA and Cas9. The substitution nucleotide sequence contains the desired genome edit, as well as further homologous sequences (referred to as the left homology arm and right homology arm) immediately upstream and downstream of the genomic target sequence. Depending on the specific application, the substitution nucleotide sequence may be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. Generally, the substitution nucleotide sequence does not contain protospacer-adjacent motif (PAM) sequences to avoid being targeted by Cas9 cleavage. SHRNA

[0080] As discussed earlier, gRNAs contain a targeting sequence and a scaffolding domain. When expressed, Cas9 and gRNA form a riboprotein complex via the interaction between the gRNA and the scaffolding domain. Upon gRNA binding, Cas9 undergoes a conformational change to a DNA-binding form, while simultaneously allowing the targeting sequence to freely interact with the genomic target DNA. In the case of Cas9 cleaving genomic target DNA, the targeting sequence should exhibit high homology to the genomic target sequence. Generally, the target sequence contains 20 nucleotides or approximately 20 nucleotides and may be synthetic RNA.

[0081] Generally, a "target sequence" or "targeted sequence" refers to a gRNA sequence (for example, a portion that does not associate with the scaffold domain). A "genome target" or "genome target sequence" refers to a genomic sequence or locus targeted for editing.

[0082] The gRNA uses a target sequence to concentrate the nuclease activity of Cas9 on a genomic target. Once the target sequence binds to or hybridizes with the genomic target, Cas9 cleaves the genomic DNA at or near that site. By altering the target sequence, the genomic target of Cas9 can be modified.

[0083] In some embodiments, gRNAs may be synthesized using commercially available kits, such as the Guide-it sgRNA In Vitro Transcription Kit. By using in vitro transcription, gRNAs that can be purified using techniques known in the art may be produced. Genome targeting

[0084] To avoid off-target effects, genome target sequences should be unique compared to the rest of the genome of the organism or cell in question.

[0085] Furthermore, the genome target sequence should be located immediately upstream of a protospacer-adjacent motif (PAM) sequence in the genome. The PAM sequence is necessary for Cas9 binding, and the exact PAM sequence depends on the species of Cas9 used. Cas9 from Streptococcus pyogenes is widely used in genome engineering. Generally, Cas9 binds to a PAM sequence, and a DNA break occurs approximately three base pairs upstream of the PAM sequence.

[0086] An example of a PAM sequence is 5'-NGG-3'. Genome target sequences can be located on either strand of genomic DNA.

[0087] Several online tools (e.g., http: / / crispr.mit.edu / or https: / / chopchop.rc.fas.harvard.edu / ) are available for selecting PAM sequences and also provide a list of potential genomic target sequences within the desired genomic locus or genomic location (e.g., protein-coding genomic locations such as CXCR4, PD-1, etc.). These tools also predict off-target effects to enable the selection of genomic target sequences that minimize cleavage of genomic DNA at other locations. Vectors and host cells

[0088] Constructions of various types of vectors, including vectors for CRISPR / Cas9 systems, may be found in U.S. Patent Application No. 2014 / 0273226 (materially incorporated herein by reference).

[0089] In general, polynucleotides, such as polynucleotides encoding Cas9, polynucleotides encoding gRNA sequences, and polynucleotides encoding substitution nucleotide sequences, can be incorporated into any desired DNA or RNA-based vector without limitation. For example, polynucleotides can be cloned into expression vectors, subcloning vectors, shuttle vectors, vectors designed for use in in vitro transcription reactions, cosmids, phagemids, and vectors derived from mammalian viruses, including retroviruses (e.g., lentiviruses), adenoviruses, and adeno-associated viruses (AAVs), as well as episomal EBNA-based vectors derived from Epstein-Barr virus.

[0090] In some embodiments, the vector may be in a circular or linear form. The linear form may be used in a subcloning step, for example, in the step of cloning a gRNA target sequence into a host vector.

[0091] Those skilled in the art will understand that a wide variety of expression vectors for expressing gene products are within the scope of embodiments of the present invention. Expression vectors can be optimally designed to express proteins, such as Cas9, variant Cas9, etc., in host cells. For example, a vector containing a nucleotide sequence encoding a protein (e.g., a Cas9 open reading frame (ORF)) and appropriate regulatory elements can be delivered to host cells by any suitable method, such as transfection or transduction. Any type and amount of regulatory elements involved in the control of transcription or translation may be incorporated into the expression vector and may be positioned upstream or downstream of the ORF. Once in the host cell, the host cell's own mechanisms, such as endogenous RNA polymerase, may be used to synthesize mRNA, which is then translated to produce a protein.

[0092] In other embodiments, the expression vector is optimally designed to express a functional RNA molecule, such as a guide RNA for tethering Cas9 to a genomic target sequence.

[0093] In some embodiments, Cas9 mRNA and gRNA are expressed from the same expression vector. In other embodiments, Cas9 mRNA and gRNA are expressed from different expression vectors. The Cas9 gene inserted into the expression vector may be a native bacterial Cas9 gene, for example, derived from Streptococcus pyogenes, Staphylococcus aureus, etc. In some embodiments, the expression vector may be a mammalian expression vector, such as a human expression vector, and may contain one or more promoter elements, such as a bacteriophage promoter element, such as a T7 promoter.

[0094] In a further embodiment, a gene encoding Cas9 may be operably ligated to one or more genes encoding nuclear localization signals, thereby targeting the expressed Cas9 / gRNA to the nucleus of a host cell.

[0095] In other embodiments, the Cas9-encoding gene is optimized to reflect the preferred codon usage frequency for the organism undergoing gene editing. For example, when gene editing is performed in a phage, the Cas9-encoding gene in the expression vector construct is optimized to reflect the preferred codon utilization.

[0096] In some embodiments, phages may be isolated, and pre-assembled gRNA / Cas9 complexes may be electroporated into host cells along with freely selected nucleotide substitution sequences. The gRNA sequences may be designed to have homology to the exon-coding regions of one or more genes.

[0097] In another embodiment, the CRISPR / Cas9 system enables targeting of multiple gene loci (genomic targets) by cloning multiple gRNAs into a single vector.

[0098] This embodiment also provides compositions and methods for in vivo gene replacement, gene mutation, and gene repair using the CRISPR / Cas9 system described herein. This system is useful for manipulating cell genomes in a highly specific manner. In some embodiments, manipulated cell lines produced by the CRISPR / Cas9 system are useful for therapeutic transplantation to treat human diseases. Human diseases resulting from bacterial infections can be treated with the compositions and methods of the present invention, or with edited cell lines produced by the compositions and methods of the present invention.

[0099] Embodiments of the present invention provide a method for specific genomic modification of a host cell or phage, the method comprising (i) providing an expression vector construct comprising a first polynucleotide encoding a Cas9 protein or a variant thereof and a second polynucleotide encoding a gRNA, wherein the gRNA comprises a target sequence homologous to a genomic locus of interest, (ii) providing a host cell containing the genomic locus of interest, (iii) delivering the expression vector construct into the host cell, and (iv) expressing the first and second polynucleotides in the host cell. In some embodiments, the method may further include visualizing, identifying, or selecting host cells having gene editing at a genomic target.

[0100] Another embodiment of the present invention provides a method for specific genome modification of a host cell, the method comprising (i) providing a first expression vector comprising a first polynucleotide encoding a Cas9 protein and having a transcriptional regulatory region or a variant thereof, and a second expression vector comprising a second polynucleotide encoding a gRNA, wherein the gRNA comprises a target sequence homologous to a genomic target; (ii) providing a host cell containing the genomic target; (iii) delivering the two expression vector constructs into the host cell (e.g., via transfection); and (iv) expressing the first and second polynucleotides in the host cell. In some embodiments, the method may include visualizing, identifying, or selecting a host cell having gene editing at the genomic target. Expression can be regulated, e.g., repressed or activated, by targeting a regulatory region, such as a promoter region, that controls the expression of the target gene.

[0101] Further embodiments of the present invention provide a method for specific genome modification of host cells, the method comprising (i) providing a Cas9 protein complexed with a gRNA, wherein the gRNA contains a target sequence homologous to a genomic target; (ii) electroporating the Cas9 protein / gRNA complex into a host cell containing the genomic target; and (iii) selecting host cells based on the expression level of the protein encoded by the genomic target (genomic locus). In some embodiments, the method may further include staining the host cells for the presence of a cell surface protein encoded by the genomic target, and selecting host cells expressing low levels of the cell surface protein using flow cytometry.

[0102] In yet another embodiment of the present invention, two or more genomic targets (target loci) are simultaneously targeted for gene editing. Therefore, the expression vector may contain multiple nucleotide sequences, each encoding a different gRNA having a different targeting sequence, and each targeting sequence corresponding to a genomic target. Alternatively, multiple expression vectors may be used, each containing one or more targeting sequences, each targeting sequence corresponding to a genomic target.

[0103] Those skilled in the art will understand that embodiments of the present invention are not limited to the polynucleotide or polypeptide sequences referred to herein, and also encompass variant polypeptide sequences and variant polynucleotide sequences. Variant polypeptide sequences include polypeptides having conservative amino acid substitutions in their amino acid sequence. Variant polynucleotide sequences include polynucleotides that have been modified to have a change in their nucleotide sequence, resulting in a polypeptide that, when expressed, has essentially the same function or activity as the polypeptide expressed by the unmodified nucleotide sequence.

[0104] In some embodiments, a CRISPR-CAS9 system is used to edit phages. In some other embodiments, a type IE CRISPR-Cas system is used to edit phages. In yet another embodiment, a type III CRISPR-CAS10 system is used to edit phages.

[0105] For example, protocols described in recent publications may be modified to delete toxic and / or undesirable phenotypes in phages of the families Myoviridae, Siphoviridae, and Podoviridae (Bari et al., Synth Biol. 2017;6(12):2316-2325; Box et al., Journal of Bacteriology Jan 2016,198(3)578-590; Tao et al., ACS Synth Biol. 2017;6(10):1952-1961; and Park et al., Sci Rep. 2017;7:42458). Bari et al. describe a type III-A CRISPR-Cas10 system for editing phages targeting S. aureus strains, with or without the use of a natural CRISPR-Cas system. This system provides a mechanism for selecting phage-derived sequences, such as sequences with point mutations at multiple loci, and for restoring recombination of phages that have acquired the desired mutations. When using RNA sequences as maps, Cas enzymes cleave targeted genes that have harmful and / or toxic characteristics. Another publication (Yosef et al., Proceedings of the National Academy of Sciences Jun 2015, 112(23)7267-7272) describes lysogenic phage-based CRISPR-Cas delivery to the genomes of antibiotic-resistant bacteria.

[0106] In some embodiments, phages or phagemids are used to deliver DNA to bacterial cells.

[0107] In some embodiments, CRISPR systems can be used to eliminate specific bacterial strains by removing them from the microbiome, or for specific therapeutic purposes.

[0108] In a more preferred embodiment, the method relies on a CRISPR system, such as the CRISPR / Cas9 system, to edit unwanted and / or toxic genes, preferably bacterial pathogenicity genes, such as endogenous bacterial genes, single nucleotide polymorphisms (SNPs), epichromosome genes, antibiotic resistance genes, genes encoding one or more pathogenic factors, genes encoding one or more toxins, genes highly conserved between species, genera or phylum, and / or genes encoding enzymes involved in biochemical pathways, along with products that can modulate host physiological functions.

[0109] In preferred embodiments, the CRISPR system targets toxin and antitoxin loci involved in important biological functions, including plasmid maintenance, phage defense, persistence, and virulence, such as those described in Akarsu et al. (2019) PLoS Comput Biol 15(4):e1006946, Xie et al., Nucleic Acids Res. 2018;46(D1):D749-D753, Harms et al., Mol Cell. 2018;70(5):768-784, and do Vale et al., 2016.

[0110] Examples of bacterial pathogens include Escherichia coli, Shigella dysenteriae, Yersinia pestis, Francisella tularensis, Bacillus anthracis, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholerae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and Salmonella enterica Typhi. In a more preferred embodiment, the bacteria may be selected from S. aureus, S. epidermidis, and / or E. fecalis.

[0111] Examples of toxins include pertussis toxin and adenylyl cyclase toxin (ACT) secreted by Bordetella pertussis, anthrax toxin from Bacillus anthracis and leukotoxin from Staphylococcus aureus, AIP56 from Photobacterium damselaepiscicida (Phdp), mycolactones, polyketide molecules produced by Mycobacterium ulcerans, other bacterial secretory products not officially called toxins, such as S. aureus superantigen-like protein (SSL) and phenol-soluble modulin (PSM), Clostridium C3 toxin, Shiga toxin, cholera toxin, hemolysin, leucocidine, ciliary and afimbriatal adhesins, proteases, lipases, endonucleases, endotoxins and exotoxins, microcin and cholinin. In some embodiments, the toxin is encoded by a superantigen enterotoxin gene, such as the Sek gene of S. aureus. In some embodiments, the toxin is an exotoxin such as toxic shock syndrome toxin-1 (TSST-1); an enterotoxin such as SEA, SEB, SECn, SED, SEE, SEG, SEH, and SEI; and an exfoliating toxin such as ETA and ETB.

[0112] Examples of genes that confer virulence to Escherichia coli (e.g., O157:H7) include, but are not limited to, stx1 and stx2 (encoding Shiga toxin-like toxins) and espA (involved in inducing enteric cell effusion (LEE) A / E lesions). Other examples of genes that confer virulence to Escherichia coli include fimA (a large pili subunit), csgD (a Curli regulator), and csgA. An example of a gene that confers virulence to Yersinia pestis is yscF (a plasmid-borne (pCD1)T3SS external needle subunit). An example of a gene that confers virulence to Francisella tularensis is fslA. An example of a gene that confers virulence to Bacillus anthracis is pag (anthrax toxin, a cell-binding protective antigen). Examples of genes that confer the virulence trait to Vibrio cholerae include, but are not limited to, ctxA and ctxB (cholera toxin), tcpA (toxin coregulating ciliary), and toxT (master virulence regulator). Examples of genes that confer the virulence trait to Pseudomonas aeruginosa include, but are not limited to, genes encoding the production of siderophoapioverdin (e.g., sigma factor pvdS, biosynthetic genes pvdL, pvdl, pvdJ, pvdH, pvdA, pvdF, pvdQ, pvdN, pvdM, pvdO, pvdP, transporter genes pvdL, pvdR, pvdT, and opmQ), genes encoding the production of siderophoapiochelin (e.g., pchD, pchC, pchB, pchA, pchE, pchF, and pchG), and genes encoding toxins (e.g., exoU, exoS, and exoT). Examples of genes that confer the virulence trait to Klebsiella pneumoniae include, but are not limited to, fimA (stickiness, type I pili-large subunit) and cps (capsular polysaccharide). Examples of genes that confer the virulence trait to Acinetobacter baumannii include, but are not limited to, ptk (capsular polymerization) and epsA (assembly).Examples of genes that confer the virulence trait to Salmonella enterica Typhi include, but are not limited to, hilA (invasion, SPI-1 regulator), ssrB (SPI-2 regulator), and bile resistance-related genes, including the efflux pump genes acrA, acrB, and tolC.

[0113] Examples of antibiotic resistance genes or various methods for identifying them are known in the art. Genomic methods for identifying strain resistance phenotypes, such as the week-chain antimicrobial susceptibility testing (WGS-AST) described by Su et al., Journal of Clinical Microbiology Feb 2019, 57(3)e01405-18, and whole-genome sequencing for antibiotic resistance determinants (ARDs) identified from the gut microbiota by Ruppe et al., Nat Microbiol. 2019;4(1):112-123, are described in the art. In some embodiments, resistance genes confer resistance to narrow-spectrum beta-lactam antibiotics of the penicillin class. In other embodiments, resistance genes confer resistance to methicillin (e.g., methicillin or oxacillin), or flucloxacillin, or dicloxacillin, or some or all of these antibiotics. In selected embodiments, the CRISPR system is suitable for selectively targeting methicillin-resistant S. aureus (MRSA) and / or vancomycin-resistant S. aureus (VRSA). In certain embodiments, the resistance gene may confer resistance to linezolid, daptomycin, and quinupristin / dalfopristin. In some embodiments, the resistance gene is selected from the fosfomycin resistance gene fosB, the tetracycline resistance gene tetM, the kanamycin nucleotidyltransferase aadD, the bifunctional aminoglycoside modifying enzyme gene aacA-aphD, the chloramphenicol acetyltransferase cat, the mupirosine resistance gene ileS2, the vancomycin resistance genes vanX, vanR, vanH, vraE, vraD, the methicillin resistance factors femA, fmtA, mecl, the streptomycin adenyltransferases spc1, spc2, antl, ant2, the spectinomycin adenyltransferases spd, ant9, aadA2, and any other resistance gene.

[0114] In a preferred embodiment, the CRISPR system targets one or more antibiotic resistance genes selected from broad-spectrum beta-lactamase resistance factors (ESBL factors), CTX-M-15, beta-lactamase, New Delhi metallo-β-lactamase (NDM)-1,2,5,6, and tetracycline A (tetA).

[0115] Examples of genes that confer resistance to aminoglycosides include, but are not limited to, the aph, aac, and aad variants, as well as other genes encoding aminoglycoside modifying enzymes. Examples of genes containing SNPs that confer aminoglycoside resistance include, but are not limited to, rpsL, rrnA, and rrnB. Examples of genes that confer beta-lactam resistance include, but are not limited to, genes encoding beta-lactamases (bla) (e.g., TEM, SHV, CTX-M, OXA, AmpC, IMP, VIM, KPC, NDM-1, the beta-lactamase family) and mecA. Examples of genes containing SNPs that confer daptomycin resistance include, but are not limited to, mprF, yycFG, rpoB, and rpoC. Examples of genes that confer macrolide-lincosamide-streptogramin B resistance include, but are not limited to, ermA, ermB, and ermC. Examples of genes that confer quinolone resistance include, but are not limited to, qnrA, qnrS, qnrB, qnrC, and qnrD. Examples of genes containing SNPs that confer quinolone resistance include, but are not limited to, gyrA and parC. Examples of genes containing SNPs that confer trimethoprim / sulfonamide resistance include, but are not limited to, the dihydrofolate reductase (DHFR) gene and the dihydropteroinate synthase (DHPS) gene. Examples of genes that confer vancomycin resistance include, but are not limited to, vanA (e.g., vanRS and vanHAX), vanB, and vanC operons.

[0116] In some embodiments, the CRISPR system may be used to target SNPs that cause overexpression of genes encoding multidrug efflux pumps, such as acrAB, mexAB, mexXY, mexCD, mefA, msrA, and tetL.

[0117] Examples of highly conserved genes (referred to herein as “remodeling” genes) that can typically represent a microbial species, genus, or phylum for the purpose of reconstructing complex microbial communities include, but are not limited to, the ribosome components rrnA, rpsL, rpsJ, rplO, rpsM, rplC, rpsH, rplP, and rpsK, the transcription initiation factor infB, and the tRNA synthetase pheS.

[0118] Examples of genes that encode enzymes involved in biochemical pathways along with products that can regulate host physiological functions (referred to herein as “regulatory” genes) include (1) genes that encode enzymes involved in deoxycholate production related to hepatocellular carcinoma, and (2) Bacteroides that lead to the development of regulatory T cells (TREGs), IL-10 response, and an increase in TH1 cell numbers. (3) genes encoding enzymes involved in the production of polysaccharide A by fragilis; (4) genes encoding enzymes involved in butyrate production leading to the secretion of inducible antimicrobial peptides; (5) genes encoding enzymes involved in short-chain fatty acid production leading to increased energy recovery, obesity, regulation of inflammation, and healing of gastrointestinal wounds; (6) genes encoding enzymes involved in the conversion of choline to methylamine, which can disrupt glucose homeostasis and lead to non-alcoholic fatty liver disease and cardiovascular disease; (7) genes encoding enzymes involved in the production of neuromodulatory compounds such as γ-aminobutyric acid, norepinephrine, 5-HT, dopamine, and acetylcholine; and (8) genes encoding enzymes involved in the formation of lactic acid and propionic acid, which are related to anxiety.

[0119] In addition to the CRISPR / Cas9 system, other genome editing tools can be used to modify and / or eliminate toxic and / or undesirable features. Examples of such phage genome editing tools include, but are not limited to, other CRISPR-based systems, engineered TALEN (transmission activator-like effector nuclease) variants, engineered zinc finger nuclease (ZFN) variants, and homologous recombination-based technologies, including bacteriophage reconciliation (BRED) of electroporated DNA, and phage rebooting using assembled phage genomic DNA, as described by Chen et al. (Front. Microbiol., 03 May 2019).

[0120] As understood herein, terms such as “effective dose” and “therapeutic dose” of the pharmaceutical composition of the present invention refer to an amount of the composition suitable to induce a therapeutically beneficial response in a subject, for example, by eliminating bacterial pathogens in the subject and / or altering the virulence or antibiotic susceptibility of surviving phage-resistant bacterial pathogens, and / or by providing additional benefits when the composition is administered concurrently with an effective and / or ineffective antibiotic. Such a response may include, for example, preventing, reversing, treating, inhibiting and / or reducing one or more pathological symptoms associated with a bacterial infection. Those skilled in the art will understand that it is desirable that the initial dose of the composition described herein be sufficient to control the bacterial population before reaching a lethal threshold. Animal models have been found to be based on a protein load acutely presented to the liver of an adult, with 10 per dose. 9 ~10 11The pfu / ml phage particle dose is likely to be the maximum tolerable dose (which would be scaled down in a pediatric population). This is presumably an acute bolus sufficient to reduce the bacterial load enough to enhance the immune response. In particular, phage "viremia" can be measured in the blood after administration. Given the host immune response and isolation in the reticuloendothelial system (liver and spleen), animal models suggest that viremia is fairly transient.

[0121] The appropriate effective dose of the composition of the present invention can be easily determined by those skilled in the art and may depend on the age, weight, species (in the case of non-humans) and condition of the subject being treated. Furthermore, those skilled in the art will understand that the type of infection (e.g., systemic or localized) and the availability of treatment for the infection may also influence the dose considered effective. Those skilled in the art will understand that initial information may be gathered in laboratory experiments, and thereafter, the effective dose of the composition described herein for humans will be determined by administration trials and conventional experimental methods.

[0122] It is still intended that the compositions of the present invention may be administered to subjects by various routes of administration according to conventional methods, including but not limited to systemic, parenteral (e.g., by intracisional injection and intracisional infusion techniques), intradermal, transmembrane, transdermal (including topical), intramuscular, intraperitoneal, intravenous, intraarterial, intrafocal, subcutaneous, oral, and intranasal (e.g., inhalation) routes of administration. Administration may also be by continuous infusion or bolus injection.

[0123] Furthermore, the compositions of the present invention can be administered in a variety of dosage forms. These include, for example, liquid preparations and suspensions, including preparations for parenteral administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration or intravenous administration (e.g., injectable administration), such as sterile isotonic aqueous solutions, suspensions, emulsions or viscous compositions that can be buffered to a selected pH. In certain embodiments, the compositions of the present invention are intended herein to be administered to a target as injectable compositions, including but not limited to injectable compositions for delivery by intramuscular injection, intravenous injection, subcutaneous injection or transdermal injection. Such compositions can be formulated using a variety of pharmaceutical excipients, carriers or diluents well known to those skilled in the art.

[0124] In another specific embodiment, the compositions of the present invention, and / or pharmaceutical formulations administered together therewith, such as antibiotics, may be administered orally. Oral formulations for administration according to the method of the present invention may include various dosage forms, such as liquids, powders, suspensions, tablets, pills, capsules, caplets, sustained-release formulations, or formulations having a sustained-release preparation or liquid filling, such as a gelatin-coated liquid (where the gelatin dissolves in the stomach and is delivered to the intestines). Such formulations may include various pharmaceutically acceptable excipients described herein, including but not limited to mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate.

[0125] In certain embodiments, the composition for oral administration may be a liquid formulation or may exist as part of a dissolvable paper placed on the tongue, as intended herein. Such formulations may contain pharmaceutically acceptable thickeners that can produce a highly viscous composition that facilitates mucosal delivery of the active substance, for example, by resulting in prolonged contact with the gastric lining. Such viscous compositions may be prepared by those skilled in the art using conventional methods and pharmaceutically excipients and reagents, such as methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and carbomer.

[0126] Other dosage forms suitable for nasal or respiratory (mucosal) administration, such as squeeze spray dispensers, pump dispensers, or aerosol dispensers, are intended herein. Dosage forms suitable for rectal or transvaginal delivery are also intended herein. Where appropriate, compositions for use with the methods of the present invention may be lyophilized and delivered to the subject with or without rehydration using conventional methods.

[0127] Accordingly, as can be understood, the methods of the present invention involve administering the compositions of the present invention to a subject in a variety of regimens, i.e., in an amount and manner sufficient to provide a clinically meaningful benefit to the subject, and for such a sufficient period of time. Suitable administration regimens for use in the present invention can be determined by those skilled in the art according to conventional methods. For example, it is intended herein that effective doses may be administered to a subject as a single dose, a series of repeated doses administered over several days, or one or more further “boosting” doses after a single dose. As used herein, “dose” or “administered dose” means a physically discontinuous unit suitable for administration to a subject, and each administered dose contains a predetermined amount of the pharmacopoeia active ingredient calculated to produce a desired response.

[0128] The administrative regimen, such as the amount administered, the number of treatments, and the effective dose per unit dose, is subject to practitioner's judgment and is specific to each subject. Factors to consider in this regard include the subject's physical and clinical condition, the route of administration, the intended treatment objective, and the potency, stability, and toxicity of the particular composition. As will be understood by those skilled in the art, a “boosting dose” may include the same dose as the initial dose or a different dose. In fact, when a series of doses are administered to produce a desired response in a subject, those skilled in the art will understand that the “effective dose” may include two or more doses.

[0129] In preferred embodiments, the compositions described herein may be administered both topically and systemically, for example, by IV, intra-articular, IM, and / or direct injection into the site of infection. For example, in the case of PJI, the compositions described herein may be injected directly into the infected joint. Thus, the compositions may be formulated as injectable fluids, semi-solids, or depot-type formulations, as will be readily apparent to those skilled in the art. However, PJI may also be treated by IV injection, and / or direct injection into the site of infection, as well as in combination with administration by IV, intra-articular, and / or IM.

[0130] If the infected joint is also treated by debridement and / or replacement of the prosthesis device in a one- or two-stage arthroplasty, the composition may be administered directly to the joint during surgery. For example, the composition may be administered in a single dose (i.e., a "single shot") or in multiple doses as needed (this "single shot" includes both direct injection into the site of infection or IV). However, it is considered that the composition may potentially be administered as a single shot, and furthermore, the need to replace the prosthesis device in the infected joint may be avoided.

[0131] In some embodiments, the composition may further comprise additional active agents or preparations, such as one or more antibiotics (e.g., rifampin and / or fluoroquinolones, e.g., ciprofloxacin), one or more bactericides, and / or one or more other therapeutic molecules, such as small molecules or biologics having bactericidal activity. These additional agents may be administered as part of the composition or concurrently with but separately from the administration of the phage composition.

[0132] In a further embodiment, the present invention provides a method for treating a patient having prosthetic joint infection (PJI) or a patient at risk of developing PJI, the method comprising administering the pharmaceutical composition described in the first embodiment.

[0133] In some embodiments, patients have PJI caused by bacteria selected from S. aureus, S. epidermidis, E. fecalis, S. caprae and / or S. lugdunensis.

[0134] In other embodiments, the patient has PJI determined to be caused by bacteria selected from S. aureus, S. epidermidis, E. fecalis, S. caprae and / or S. lugdunensis. This determination may be made, for example, by culturing synovial fluid obtained by aspiration from the affected joint and determining the identity of the bacteria in the culture, for example, by 16S rRNA gene sequence analysis.

[0135] In other embodiments, patients are at risk of developing PJI caused by bacteria selected from S. aureus, S. epidermidis, E. fecalis, S. caprae, and / or S. lugdunensis.

[0136] The method of the present invention may be useful for the treatment or prevention of PJI of any type. That is, by appropriately selecting two or more phages to be included in the composition, the method may be beneficial in the treatment of early, delayed, and late-onset PJI, as well as PJI resulting from multiple bacterial infections.

[0137] This method may be performed to provide at least two different phages in an effective dose, i.e., an amount sufficient to cause lysis (i.e., death) of bacteria present in the treated infection, e.g., PJI (or potentially developing PJI), and / or to prevent the development of a bacterial infection, so that a beneficial or desired clinical outcome is achieved. The effective dose may be administered in one or more doses. Typically, the effective dose is sufficient to treat the disease or symptoms, or, if not, sufficient to reduce, restore, stabilize, reverse, slow, delay, or prevent the progression or development of the PJI.

[0138] The infections treated include, but are not limited to, bacterial infections already present in the patient, as well as the prevention of future infections that may or may not occur in patients with implanted devices. For example, bacterial infections of specific bacterial strains known to occur frequently in a particular geographical location (e.g., geographically adapted compositions) may be treated with the compositions of this disclosure in a prophylactic manner with the aim of preventing future bacterial infections.

[0139] In further detail, the method may include direct administration of the composition to an infected joint or an artificial joint at risk of developing PJI. In the latter case, the method may be performed during surgery (i.e., the composition may be administered at the time the joint replacement surgery is being performed). When the method is used to treat an infected joint, the method may avoid the need to replace the artificial joint device. In other cases, the method may be performed instead of surgery. In other cases where the infected joint is also treated by debridement and / or replacement of the artificial joint device in a one- or two-stage joint replacement surgery, the method may conveniently be performed during surgery (i.e., the composition may be administered to the joint during surgery). The composition may be administered in a single dose (i.e., a "single shot") or in multiple doses as needed.

[0140] In some embodiments, the methods of the present invention may be carried out as combination therapy. For example, a composition comprising at least two different phages may be administered to a patient as combination therapy, comprising the administration of further active agents or preparations, such as one or more antibiotics (e.g., rifampin and / or fluoroquinolones, e.g., ciprofloxacin), one or more bactericides, and / or one or more other therapeutic molecules, such as small molecules or biologics having bactericidal activity.

[0141] When administered as a combination therapy with further active agents or preparations, the composition may contain at least two different phages together with their further active agents or preparations (i.e., as a single composition), or, if not, separate compositions may be used. When administered as separate compositions, the therapeutic phage composition and the further active agents or preparations may be administered simultaneously or sequentially in any order (e.g., within seconds or minutes (e.g., 5 to 60 minutes) or within hours (e.g., 2 to 48 hours)).

[0142] While the present invention has been described herein with reference to embodiments, it should be understood that these embodiments and examples provided herein are merely illustrative of the principles and applications of the present invention. Therefore, it should be understood that numerous modifications can be made to the exemplary embodiments and examples, and that other configurations can be devised without departing from the spirit and scope of the invention as defined by the appended claims. All patent applications, patents, documents and references cited herein are incorporated herein by reference in their entirety. [Examples]

[0143] The present invention will now be further illustrated with reference to the following examples. These are merely illustrative examples and can be modified in detail while remaining within the scope of the present invention. Example 1: Selection of phages to be included in the PJI composition

[0144] Design a composition for treating PJIs that may be caused by one or more of the following: S. aureus (MRSA and / or MSSA), S. epidermidis, E. fecalis, S. caprae, and / or S. lugdunensis. Candidate phages to be included in the composition will be identified from phages publicly available from the Felix d'Herelle Reference Center for Bacterial Viruses of the Universite Laval (Quebec City, Quebec, Canada; www.phage.ulaval.ca) and proprietary phages listed in Table 1 (above). Formulation of injectable compositions

[0145] about 10 11 In one particular example, an injectable fluid composition is prepared by combining each of the following phages of pfu, APT-PJI-01, APT-PJI-13, and APT-PJI-15, in a pharmaceutically acceptable diluent (e.g., isotonic saline). The composition may be provided as a pre-filled syringe. Example 2: Selection of phages for inclusion in PJI compositions

[0146] The composition of Example 1 can be readily modified by adding one or more phages targeted to lyse other pathogenic bacteria of implantable device infections (e.g., PJI, etc.) (e.g., S. lugdunensis, E. fecium, S. agalactiae, P. aeruginosa, K. pneumoniae, Escherichia coli, and E. clocae) to the composition, or by replacing one or more phages in the composition. Some examples of candidate phages to be included in such a composition are Felix. d'Herelle Reference Center for Bacterial Viruses of the Universite Laval (Quebec These phages were identified from publicly available sources (City, Quebec, Canada; www.phage.ulaval.ca) and are listed in Table 2 below. [Table 2] Example 3: A case study on a desk

[0147] Two months after undergoing ceramic-on-polyethylene hip replacement surgery, a 55-year-old male patient reported significant pain in the joint to his orthopedic surgeon. He was advised to undergo a CT scan of the affected joint, which revealed joint swelling and suggested an infection of the tissue surrounding the prosthesis. Blood tests also provided further evidence for PJI. Subsequently, a synovial fluid sample was collected from the hip joint by athrocentesis, and the bacteria present in the synovial fluid were cultured in liquid medium. The extracted bacterial DNA was then subjected to sequencing analysis of the V1-V3 region of the 16S rRNA gene, and the bacterial species present were identified using a 16S rRNA sequence database. It was determined that the patient's PJI was caused by S. aureus and E. fecalis.

[0148] The patient was given approximately 10 units of pharmaceutical saline solution. 9 A single dose therapeutic phage composition containing pfu, each of the APT-PJI-01 (targeting S. aureus) and APT-PJI-15 (targeting E. fecalis) phages, is administered by direct injection into the affected joint. Subsequently, the patient is treated with intravenous (IV) phages (APT-PJI-01 and / or APT-PJ-15) for 10 days. After treatment, the patient is closely monitored for clinical response. After 4 weeks, the patient reports little to no pain remaining. Aspiration may be performed from the joint to determine if any infection remains. If necessary, a further CT scan may be performed to determine that the joint is no longer infected.

[0149] The present invention is not limited to the embodiments described herein, and the embodiments may be modified in configuration and detail without departing from the spirit of the invention. Any entire teaching of any patent, patent application or other publication referenced herein is incorporated herein by reference as if it were entirely contained herein.

Claims

[Claim 1] The invention described in the specification.

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