CA3174729A1 - Method for treating implantable device infections - Google Patents

Abstract

The invention relates to the field of phage therapy and is particularly directed at providing a phage-based composition and method for treating or preventing infections associated with implantable devices. The composition may be directly administered to the infected location of the device, optionally as a single dose, and/or by other modes of administrations, and may potentially avoid any requirement for replacing the implantable device.

Description

2 METHOD FOR TREATING IMPLANTABLE DEVICE INFECTIONS
BACKGROUND OF THE INVENTION
Field of the Invention [001] The invention relates to the field of phage therapy and is particularly directed at providing a phage-based composition and method for treating or preventing acute or chronic bacterial infections associated with an implantable device using phage-based compositions.
Discussion of the Related Art [002] In the following discussion, certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an "admission" of prior art. The Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

[003] Partial or total joint replacement surgery, most commonly of knees and hips, is a common, life-enhancing procedure undertaken by millions of patients each year. In most cases, the procedure is highly successful, bringing pain relief and restoration of function and independence to patients. In a relatively small number of cases, complications can be experienced such as prosthetic device failure (e.g.
caused by wear, device fracture or malpositioning), or the appearance of prosthetic joint infection (PJI), also known as periprosthetic joint infection, which is an infection involving the joint prosthetic device and adjacent tissues. Studies have determined that the incidence of PJI
in the United States is in the order of about 2% for knee replacements and up to about 1.5 % for hip replacements (Tande AJ & R Patel, Clin. Microbiol. Rev. 27(2):302-345, 2014).
However, while this number is relatively low, PJI represents a very serious risk to the patient bringing with it considerable morbidity involving pain and swelling, and in some cases, can necessitate amputation and/or lead to patient death. The diagnosis, treatment and management of PJI is also a very significant cost burden on the health care system, and has been predicted to be greater than USD1.5 billion in the United States alone in 2020; Kurtz SM et al., J. Arthroplasty 27:61-65.e61, 2012).

[004] Generally, doctors have categorized PJI into three major kinds of PJI; each distinguished by the period of time, after surgery, that the infection appears. Thus, PJI
includes infections that: (i) occur early, within 3 months of surgery ("early onset" PJI);
(ii) are delayed, appearing after 3 months but before 12 or 24 months of the surgery ("delayed onset" PJI); and (iii) infections that occur later than 12 to 24 months following the surgery ("late onset" PJI). Others have categorized PJI infections as "acute"
(infections occurring within 1 month after surgery or "chronic" which occur after 1 month from surgery. Regardless of which classification system is used, early PJI
infections are mostly caused by relatively virulent microorganisms such as Staphylococcus aureus and/or aerobic gram-negative bacteria such as Pseudomonas aeruginosa, whereas the delayed PJI types are commonly due to less virulent bacteria such as coagulase-negative S. aureus (CoNS), Staphylococcus epidermidis and/or Enterococci spp. Both the early and delayed kinds are believed to be acquired during the surgery, whereas late PJI
infections may frequently eventuate as a secondary infection from another site or sites of bacterial infection (e.g. arising from hematogenous spread of bacteria from other infectious foci through the blood). S. aureus appears to be the common causative microorganism for late onset PJI arising from hematogenous spread (Tande &
Patel, supra).

[005] The successful treatment of PJI typically requires surgical intervention and/or medical therapy (e.g. with antimicrobial agent(s) such as antibiotics) in the majority of cases. Some treatments involve a debridement (i.e. surgical removal of infected/damaged tissue), antibiotic treatment, and implant (prosthetic device) retention, and are referred to as a DAIR procedure, while in other cases, the treatment requires the replacement of the prosthetic device in a one- or two-stage arthroplasty exchange (Tande & Patel, supra) in combination with treatment with an antimicrobial agent(s). Two-stage arthroplasty exchange procedures, are typically regarded to be the "most definitive strategy in terms of infection eradication and preservation of joint function" with success rates reported as high as 87-100% for hip replacements and 72-95% for knee replacements (Tande & Patel, supra). However, these two-stage procedures can be complex and costly, requiring at least two surgeries, sometimes separated by a period of 2-3 months or more, and extensive periods of treatment with an antimicrobial agent(s). This means that some patients are not suitable for a two-stage arthroplasty exchange (or even a one-stage procedure) or are unwilling to undertake further surgery, in which case there is little option other than to attempt to treat the PJI
with an antimicrobial agent(s). However, this is not recommended and commonly leads to the patient being placed on prolonged or indefinite treatment with oral antimicrobial agent(s) and careful ongoing therapeutic monitoring (Tande Sr Patel, supra).

[006] An added complication to the effective treatment of PJI with antimicrobial agent(s) is that the infection can be "polymicrobial", meaning that there is more than one causative microorganism present in the infection. This is particularly the case with early onset PJI, where it has been estimated that up to 35% of such infections are polymicrobial commonly involving infection by S. aureus, Enterococci spp. and aerobic gram-negative bacteria such as P. aeruginosa (Berbari EF et al., Clin. Infect. Dis. 27:1247-1254, 1998;
Peel TN et al., Antimicrob. Agents Chemotherap. 56:2386-2391, 2012).
Consequently, a careful selection of antimicrobial agent(s) is needed if the treatment is to be successful.

[007] While the efficacy and options for treatments of PJI has significantly improved in recent years, there is a great ongoing need to identify and develop new or improved treatments and/or management strategies for effectively treating and/or preventing these infections in joint replacement patients, as well as other infections associated with implantable devices.
SUMMARY OF THE INVENTION

[008] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description.
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 be apparent from the following written Detailed Description including those aspects illustrated in any accompanying drawings and defined in the appended claims.

[009] Specifically, the disclosed invention describes a method of treating an infection caused by one or more bacterium associated with a device implanted within a subject, wherein said method comprises: (a) identifying at least one identified phage capable of infecting the bacterium, wherein said identified phage is selected from a library comprising at least one phage selected from APT-PJI-01, APT-PM-02, APT-PJI-03, APT-PM-04, APT-PM-05, APT-PJI-06, APT-PM-07, APT-PJI-08, APT-PM-09, APT-PM-io, APT-PJI-12, APT-PM-13, APT-PM-14, APT-PM-15, APT-PM-16, APT-PJI-17, APT-PM-18, APT-PJI-20, APT-PM-21, APT-PJI-22, APT-PJI-23, APT-PM-24, APT-PJI-25, APT-PM-26, APT-PM-27, APT-PM-28, APT-PM-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PM-34, APT-PJI-35, APT-PJI-36, APT-PM-37, APT-PM-38, APT-PM-39, APT-PM-4o, APT-PM-41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PM-45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PJI-50, or any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage and which is capable of causing lysis of the bacterium; and (b) administering to said subject a therapeutically effective amount of a composition comprising said identified phage, wherein said composition is effective in treating and/or reducing said infection.
[ow] In other embodiments, the disclosed invention describes a method of identifying a subject suffering, at risk of suffering, or eligible for receiving treatment for a bacterial infection, comprising the steps of: (a) obtaining a biological sample from the subject; (b) culturing a bacterium present in the biological sample; (c) inoculating the cultured bacterium with an identified phage selected from a library comprising at least one phage selected from one or more of APT-PJI-oi, APT-PM-02, APT-PM-03, APT-PJI-04, APT-PM-05, APT-PM-06, APT-PM-07, APT-PJI-o8, APT-PM-09, APT-PM-io, APT-PJI-ii, APT-PJI-12, APT-PJI-13, APT-PM-14, APT-PM-15, APT-PJI-16, APT-PJI-17, APT-PM-18, APT-PJI-2o, APT-PM-21, APT-PJI-22, APT-PJI-23, APT-PM-24, APT-PJI-25, APT-PM-26, APT-PM-27, APT-PM-28, APT-PM-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PM-34, APT-PJI-35, APT-PJI-36, APT-PM-37, APT-PM-38, APT-PM-39, APT-PM-4o, APT-PM-41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PJI-45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PJI-5o, and any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage and which is capable of causing lysis of the bacterium; and (d) determining whether the cultured bacterium are lysed by the identified phage, wherein when any of the cultured bacterium are lysed by the phage, the subject is determined (1) to be eligible for treatment by bacteriophage for said bacterial infection (2) to be suffering from a bacterial infection; and/or (3) at risk of suffering from a bacterial infection.
[on] In further described embodiments, the composition used in either method comprises at least one identified phage selected from the group consisting of APT-PJI-oi, APT-PM-02, APT-PJI-03, APT-PM-04, APT-PM-05, APT-PJI-o6, APT-PM-07, APT-PJI-o8, APT-PM-09, APT-PM-io, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PM-14, APT-PJI-15, APT-PM-16, APT-PM-17, APT-PM-18, APT-PM-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PM-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PM-31, APT-PJI-32, APT-PJI-33, APT-PM-34, APT-PJI-35, APT-PM-36, APT-PM-37, APT-PJI-38, APT-PM-39, APT-PJI-40, APT-PM-41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PM-45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PJI-49, and/or APT-PM-50, or any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage and which is capable of causing lysis of the bacterium.
[01.2] Other embodiments are further described below.
DETAILED DESCRIPTION
[013] The following definitions are provided for specific terms which are used in the following written description.
Definitions [014] As used in the specification and claims, the singular form "a", "an"
and "the"
include plural references unless the context clearly dictates otherwise. Also, as understood by one of ordinary skill in the art, the term "phage" can be used to refer to a single phage or more than one phage.
[015] The present invention can "comprise" (open ended) or "consist essentially of' the components of the present invention. As used herein, "comprising"
means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms "having" and "including" are also to be construed as open ended unless the context suggests otherwise.
[016] The term "about" or "approximately" means within an acceptable range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean a range of up to 20%, preferably up to io%, more preferably up to 5%, and more preferably still up to 1% of a given value.
Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 fold, and more preferably within 2 fold, of a value. Unless otherwise stated, the term "about" means within an acceptable error range for the particular value, such as 1-20%, preferably i-io% and more preferably 1-5%. In even further embodiments, "about" should be understood to mean-q-5%.
[017] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
[o18] All ranges recited herein include the endpoints, including those that recite a range "between" two values.
[01.9] Terms such as "about," "generally," "substantially,"
"approximately" and the like are to be construed as modifying a term or value such that it is not an absolute, but does not read on the prior art. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by one of ordinary skill in the art.
This includes, at very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value.
[020] Where used herein, the term "and/or" when used in a list of two or more items means that any one of the listed characteristics can be present, or any combination of two or more of the listed characteristics can be present. For example, if a composition is described as containing characteristics A, B, and/or C, the composition can contain A
feature alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.
[021] The term "bacteriophage" or "phage", as understood by one of ordinary skill in the art, refers to a non-cellular infective agent that reproduces only in a suitable host cell, that is, a bacterial host cell.
[022] The term "phage therapy" refers to any therapy to treat a bacterial infection or a disease or condition that is caused by bacteria (e.g. PJI) or which shows symptoms or disease/condition development or progress associated with the presence of bacteria.
Phage therapy may involve the administration to a patient requiring treatment of one or more therapeutic phage composition that can be used to infect, kill or inhibit growth of a bacterium, which comprises one or more viable phage as an antibacterial agent (e.g. a composition comprising one phage type or two or more phage type in a phage "cocktail").
The one or more phage types may be obtained from stocks of the phage, which may be held in storage in an inventory. AAThere a phage therapy involves the administration of more than one therapeutic phage composition, then the compositions may have a different host range (e.g. one may have a broad host range and one may have a narrow host range, and/or one or more of the compositions may act synergistically with one another). Further, as will be readily understood by one of ordinary skill in the art, the therapeutic phage composition(s) used in a phage therapy will also typically comprise a range of inactive ingredients selected from a variety of conventional pharmaceutically acceptable excipients, carriers, buffers, and/or diluents.
[023] The term "pharmaceutically acceptable" is used to refer to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. Examples of pharmaceutically acceptable excipients, carriers, buffers, and/or diluents are familiar to one of ordinary skill in the art and can be found, e.g. in Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton, Pa. For example, pharmaceutically acceptable excipients include, but are not limited to, wetting or emulsifying agents, pH buffering substances, binders, stabilizers, preservatives, bulking agents, adsorbents, disinfectants, detergents, sugar alcohols, gelling or viscosity enhancing additives, flavoring agents, and colors.
Pharmaceutically acceptable carriers include macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, 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 saline.
[024] The present invention is directed at providing a phage-based composition and method for treating an infection associated with an implantable device, such as for example, a prosthetic joint infection ("PJI"). The composition and method may offer considerable simplicity inasmuch as the treatment may comprise administering the composition directly to the infected area (e.g., joint), optionally as a single dose, such that, for example, the phage therapy may potentially avoid any requirement for replacing the implantable device (e.g., the prosthetic device of the infected joint).
[025] Thus, in a first aspect, the invention provides a pharmaceutical composition comprising at least two different bacteriophage that are capable of lysis of an infection associated with an implantable device (e.g., a prosthetic joint infection (PJI) in a patient), wherein the pharmaceutical composition is formulated for administration to the patient either directly to the location of the infection, by IV, and/or orally.
[026] In one embodiment, the at least two different bacteriophage may be phage types selected for their capability of causing lysis (i.e. killing) of a single bacterial species or two or more bacterial species (i.e. in the case of a polymicrobial infection) that may be present in the implantable device infection.
[027] The selection of the phage types for therapeutic indications may be based upon the results of testing to determine the type(s) of bacteria present in the implantable device infection (i.e. the causative microorganisms); for example, by synovial fluid aspiration and subsequent culture of the fluid on solid or in liquid media, bacteria present in the culture can be readily determined by standard techniques well known to one of ordinary skill in the art including 16S rRNA gene sequence analysis (e.g. as described in Whelan FJ et al., Ann. Am. Thorac. Soc. 11:513-521, 2014). The biological sample may also be used in assays to assess the bacteria for their susceptibility to individual phage, which can be invaluable in making the selection of the phage types for inclusion in the composition.
[028] The selection of the phage types may also be based upon a prediction or expectation of which causative microorganisms are present in the implantable device infection. Similarly, the selection of phage types may also be based upon a prediction or expectation of which causative microorganisms are present at a particular geographical location.
[029] For example, the phage selected for inclusion in the composition may include types that are capable of lysis of bacteria commonly causative of delayed onset a device infection, such as for example, a delayed onset PJI, namely S. aureus (e.g.
coagulase-negative S. aureus) and Enterococcus spp. (e.g. E. fecalis and Enterococcus fecium). Such a composition might also be effective for treating late onset PJI, which is commonly caused by S. aureus. Alternatively, the phage selected can be used to treat an acute infection or a chronic infection. Other bacteria that may be targeted by inclusion of appropriately selected phage type(s) include: other coagulase-negative Staphylococcus species that have been reported to cause PJI, such as S. simulans (Razonable RR et al., Mayo CHn. 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); various Streptococcus spp. that have been associated with PJI, including 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., South.
Med. J. 93:1217-1220, 2000), S. agalactiae and S. pneumoniae (Raad J et al., Semin.
Arthritis Rheum. 34:559-569, 2004), aerobic gram-negative bacteria such as E.
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., Cutibacterium acnes (formerly Propionibacterium acnes), Klebsiella pneumoniae, P. aeruginosa and Bacteroides fragilis (Tande & Patel, supra).
[030] Moreover, it is also contemplated that the identified phage to be included in a composition could be a matched phage composition based on the infections commonly experienced in a certain geographic location. Examples of such -geomatched"
phage are described in USSN 62/956,729, filed on January 3, 2020, and which is hereby incorporated by reference in its entirely.
[031] Thus, one aspect of the invention is a method of treating an infection caused by one or more bacterium associated with a device implanted within a subject, wherein said method comprises:
(a) identifying at least one identified phage capable of infecting the bacterium, wherein said identified phage is selected from a library comprising at least one phage selected from APT-PJI-oi, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-o5, APT-PJI-06, APT-PM-07, APT-PJI-08, APT-PM-09, APT-PJI-io, APT-PM-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-Pa-15, APT-PJI-16, APT-PM-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PM-24, APT-PJI-25, APT-PJI-26, APT-PM-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PM-33, APT-PM-34, APT-PM-35, APT-PM-36, APT-PM-37, APT-PM-38, APT-PJI-39, APT-PJI-40, APT-PA-41, APT-PM-42, APT-PA-43, APT-PM-44, APT-PJI-45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PM-50 as described in Table 1 or any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage and which is capable of causing lysis of the bacterium; and (b) administering to said subject a therapeutically effective amount of a composition comprising said identified phage, wherein said composition is effective in treating and/or reducing said infection.
[032] Table 1 provides relevant information for the public phage (e.g, APT-PJI-oi, APT-PJI-02, APT-PJI-o3, APT-PJI-04, APT-PJI-o5, APT-PJI-o6, APT-PJI-o7, APT-PJI-o8, APT-PJI-09, APT-PJI-io, APT-PJI-n, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-APT-PJI-16, APT-PJI-17, and APT-PJI-18) as well as the proprietary, not publicly available phage (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-3o, 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-PM-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PM-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49, and/or APT-PJI-50).
[033] In preferred embodiments, the compositions and methods are performed with any combination of the proprietary phage. Specifically, at least one, at least two, at least three, at least four and/or at least five phage selected from the group consisting of 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-PM-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 are used to treat a patient suffering from a PJI infection as described herein.

,-, ), E' APT-14-PCT Final '.' Table 1 Phage Bacterial GenBank Phage ID
SEQ ID
Bacteria Name host Comments Accession code NO:i (HER#)* HER#*
NO.
Podoviridae, species APT-PJI-oi P68 (49) 1049 44AHJD; also lyses NC 004679 2 HERnoi Podoviridae, species 44AHJD NC_oo4678 APT-PJI-02 1101 44AHJD;
also lyses 1 (ioi) HER1:49 NC _007053 APT-PJI-03 3A (225) 1225 Siphoviridae, species 3A 6 APT-PJI-04 77(226) 1226 Siphoviridae, species 77 NC 005356 3 species 11 hoviridae, APT-PJI-o5 71 (238) 1238 SipNC _007059 7 (P11-M15) IV
APT-PJI-06 187 (239) 1239 Siphoviridae, species 187 NC_007047 4 S. auretts 2638A
APT-PJI-o7 1283 Siphoviridae NC _007051 5 (283) APT-PJI-o8 CSi (466) 1466 Siphoviridae APT-PJI-o9 DW2 (467) 1466 Siphoviridae NC 024391 10 APT-PJI-io K (474) 1474 Myoviridae NC_005880 9 APT-PJI-ii 812 (475) 1475 Myoviridae NC_029080 12 XI
m -a Myoviridae, Remus Staphylococcus species NC0 (528) m Remus E
m Z
APT-PJI-o 2 2 16 ¨I
_______________________________________________________________________________ _____________________________________ Cl, I
M
M
¨I

`,7 APT-14-PCT Final '.' Tablet Phage Bacterial GenBank Phage ID
SEQ ID
Bacteria Name host Comments Accession code NO:t (HER#)* HER#*
NO.

23 i-(A) 28 xj M

C.) m m z ¨i 31 0) I
m m ,-, ), E' APT-14-PCT Final '.' Table 1 Phage Bacterial GenBank Phage ID SEQ ID
Bacteria Name host Comments Accession NO:i code (HER#)* HER#*
NO.
APT-PJI-o38 APT-Pa-039 APT-Pa-043 APT-Pa-044 4.

APT-PJI-o46 APT-PJI-13 392 (292) 1292 Siphoviridae S. epidermidis APT-PJI-14 6ec (555) 1555 Siphoviridae APT-PJI-15 VD13 ( Siphoviridae, species 44) 1044 VD13 NC 024212.1 8 x m APT-PJI-16 182 (8o) io8o Podoviridae species 182 E. fecalis 0m E
APT-PJI-17 1323 Siphoviridae m (323) z APT-PJI-18 1 (339) 1339 Myoviridae, species 1 (1) _______________________________________________________________________________ ___________________________________ I
M
M
-I

-,1 APT-14-PCT Final .

õ
. Table 1 Phage Bacterial GenBank Phage ID
SEQ ID
Bacteria Name host Comments Accession code NO:i (HER#)* HER#*
NO.

S. caprae S. lug dunensis APT-PJI-047 41 * d'Herelle (HER) numbers as indicated at www.phage.ulaval.ca i-u-.
x m -a m E
m z Cl, mx m [034]
In another aspect, the invention disclosed is a method of identifying a subject suffering, at risk of suffering, and/or eligible for phage treatment for a bacterial infection, comprising the steps of:
(a) obtaining a biological sample from the subject;
(b) culturing a bacterium present in the biological sample;
(c) inoculating the cultured bacterium with an identified phage selected from a library comprising at least one phage selected from one or more of APT-PM-oi, APT-PM-02, APT-PM-03, APT-PM-04, APT-PM-o5, APT-PM-o6, APT-PM-07, APT-PJI-o8, APT-PM-09, APT-PM-io, APT-PJI-n, APT-PM-12, APT-PM-13, APT-PM-14, APT-PM-15, APT-PM-16, APT-PM-17, APT-PM-18, APT-PJI-20, APT-PM-21, APT-PM-22, APT-PM-23, APT-PM-24, APT-PM-25, APT-PM-26, APT-PM-27, APT-PM-28, APT-PJI-29, APT-PJI-3o, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PM-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PM-39, APT-PM-40, APT-PJI-41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PM-45, APT-PM-46, APT-PJI-47, APT-PM-48, APT-PM-49, and/or APT-PJI-5o, and any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage and which is capable of causing lysis of the bacterium; and (d) determining whether the cultured bacterium are lysed by the identified phage, wherein when any of the cultured bacterium are lysed by the phage, the subject is determined (1) to be eligible for treatment by bacteriophage for said bacterial infection (2) to be suffering from a bacterial infection; and/or (3) at risk of suffering from a bacterial infection.
[035]
In preferred embodiments, the composition comprises at least one identified phage selected from the group consisting of APT-PJI-oi, APT-Pa-02, APT-PM-03, APT-PM-04, APT-PM-05, APT-PM-06, APT-PM-07, APT-PM-o8, APT-PM-09, APT-PM-io, APT-PM-11, APT-PM-12, APT-PM-13, APT-PM-14, APT-PM-15, APT-PM-16, APT-PM-17, APT-PM-18, APT-PM-20, APT-PM-21, APT-PJI-22, APT-PM-23, APT-PJI-24, APT-PM-25, APT-PM-26, APT-PM-27, APT-PM-28, APT-PM-29, APT-PM-30, APT-PM-31, APT-p,11-32, APT-PM-33, APT-PM-34, APT-PM-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-Pa-42, APT-PM-43, APT-PJI-44, APT-PM-45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PJI-50, or any other lytic phage that has a genomic sequence with at least 70%
sequence identity to the genomic sequence of any of the foregoing phage and which is capable of causing lysis of the bacterium.
[036] In even further preferred embodiments, the composition comprises at least two identified phage selected from the group consisting of APT-PM-oi, APT-PJI-02, APT-PJI-o3, APT-PJI-o4, APT-PJI-o5, APT-PJI-o6, APT-PJI-o7, APT-PJI-o8, APT-PJI-o9, APT-PM-10, APT-PM-11, APT-PJI-12, APT-PM-13, APT-PM-14, APT-PJI-15, APT-PJI-16, APT-PM-17, APT-PM-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-M-32, APT-PM-33, APT-PM-34, APT-PJI-35, APT-PJI-36, APT-PM-37, APT-PJI-38, APT-PM-39, APT-PM-40, APT-PM-41, APT-PM-42, APT-PM-43, APT-RII-44, APT-PM-45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PJI-50, and any other lytic phage that has a genomic sequence with at least 70%
sequence identity to the genomic sequence of any of the foregoing phage and which is capable of causing lysis of the bacterium.
[037] In further aspects, the at least two identified phage: (a) cause lysis in the same strain of bacterium; (b) cause lysis in different strains of bacterium.
[038] In other further aspects, one of the two identified phage: (a) is selected from the group consisting of APT-PJI-01, APT-PJI-02, APT-PM-03, APT-PM-04, APT-PM-05, APT-PJI-o6, APT-PM-07, APT-PJI-08, APT-PM-09, APT-PM-io, APT-PM-n, APT-PJI-12, APT-PJI-22, APT-PJI-23, APT-PM-24, APT-PJI-25, APT-PJI-26, APT-PM-27, APT-PJI-28, APT-PM-29, APT-PJI-30, APT-PM-31, APT-PM-32, APT-PJI-33, APT-PM-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PM-4o, APT-PM-41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PM-45, and APT-PM-46, and any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage in (a); (b) is selected from the group consisting of APT-PJI-13, APT-PM-14 and any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage in (b); (c) is selected from the group consisting of selected from APT-PJI-15, APT-PM-16, APT-PM-17, APT-PM-18 and any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage in (c); (d) is selected from the group consisting of APT-PJI-20 or APT-PJI-21 and any other lytic phage that has a genomic sequence with at least 70%
sequence identity to the genomic sequence of any of the foregoing phage in (d); or (e) is selected from the group consisting of APT-PJI-47, APT-PJI-48, APT-PJI-49, or APT-PJI-5o, and any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage in (e).
[039] In further aspects, the two identified phage 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) from the phage described in the previous paragraph. In further aspects, the composition comprises at least three identified phage, where at least one of the identified phage is selected from each of (a), (b), (c), (d) and/or (e) from the phage described in the previous paragraph.
[040] In other further aspects, one of the two identified phage: (a) is selected from the group consisting of (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 any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage in (a); (b) is selected from the group consisting of APT-PJI-20 or APT-PJI-21 and any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage in (b); or (c) 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 that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage in (c).
[041] In further aspects, the two identified phage are: selected from each of (a) and (b); selected from each of (a) and (c); and/or selected from each of (b) and (c) from the phage described in the previous paragraph. In further aspects, the composition comprises at least three identified phage, where at least one of the identified phage is selected from each of (a), (b), and (c) from the phage described in the previous paragraph.
[042] In further aspects, the composition provides a dose of each phage in the range of 105 to 1013 pfu, and preferably, at a range of lob to 1012 pfu; 107 to loll pfu; 108 to 1011 pfu; 109 to 1011 pfu; 109 to 1010 pfu; or at a dose of approximately 106 pfu, 107 pfu, 108 pfu, 1o9 pfu, 1010 pfu, io pfu, 1012 pfu, or 1013 pfu. In most preferred embodiments, the dose of each phage in the composition is approximately 109 pfu.
[043] Thus, the composition may be formulated so as to provide a suitable dose of each phage type included in the composition. By way of example only, a suitable dose of the phage(s) may be in the range of 105 to 1013 pfu, and more preferably 109 to 1012 pfu.
Most preferably, the composition is formulated to provide a dose of each phage type present of about 109 pfu, 1010 pfu, or 1011 pfu.
[044] In preferred embodiments, the bacterium is selected from at least one of S.
aureus, S. epidermidis, Enterococcus spp., including E. fecalis and Enterococcus fecium, other coagulase-negative Staphylococcus species, including S. simulans, S.
caprae and S.
lugdunensis, Streptococcus spp., including Lancefield groups A, B, C and G, S.
agalactiae and S. pneumoniae, aerobic gram-negative bacteria including E. coil, other Enterobacteriaceae including Enterobacter clocae, Clostridium spp., Actinomyces spp., Peptostreptococcus spp., Cutibacterium acnes, Klebsiella pneumoniae, P.
aeruginosa and Bacteroides fragilis.
[045] In other preferred embodiments, the bacterium is selected from two or more different strains of bacterium selected from S. aureus, S. epidermidis, Enterococcus spp., including E. fecalis and Enterococcus fecium, other coagulase-negative Staphylococcus species, including S. simulans, S. caprae and S. lugdunensis, Streptococcus spp., including Lancefield groups A, B, C and G, S. agalactiae and S. pneumoniae, aerobic gram-negative bacteria including E. coil, other Enterobacteriaceae including Enterobacter clocae, Clostridium spp., Actinomyces spp., Pep tostreptococcus spp., Cuti bacterium acnes, Klebsiella pneumoniae, P. aeruginosa and Bacteroides fragilis.
[046] In even further preferred embodiments, the bacterium is selected from S.
aureus, S. epidermidis and/or E. fecalis. To treat such an infection, the composition could comprise at least two phage having different lytic specificities capable of infecting at least two of S. aureus, S. epidermidis and/or E. fecalis. In other aspects, the composition comprises, for example, at least three phage having different lytic specificities, each phage capable of infecting at least one of S. aureus, S.
epidermidis and E. fecalis.
[047] Preferably, the lytic phage has a genomic sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the genomic sequence of any of the foregoing phage.
[048] Sequence identity percentages referred to herein are to be understood as having been calculated by comparing two polynucleotide sequences using the BLAST
algorithm from the National Center for Biotechnology Information database (NCBI;
Bethesda, MD, United States of America).
[049] The terms "percent (%) sequence similarity", "percent (%) sequence identity", and the like, generally refer to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of polypeptides that may or may not share a common evolutionary origin (see Reeck et al., supra).
Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, PASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin), etc.
[050] To determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions/total number of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are, or are about, of the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted.
[051] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990, 87: 2 2 64, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al, J. Mol.
Biol. 1990;
215: 403. BLAST nucleotide searches can be performed with the NBLAST program, score = loo, wordlength = 12, to obtain nucleotide sequences homologous to sequences of the invention. BLAST protein searches can be performed with the XBLAST program, score =
50, wordlength = 3, to obtain amino acid sequences homologous to protein sequences of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST
can be utilized as described in Altschul et al, Nucleic Acids Res. 1997, 25:3389.
Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationship between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov/BLAST/ on the WorldWideWeb.
[052] Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of 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 utilizing the ALIGN program for comparing amino acid sequences, a PAM12o weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
[053] In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol.
Biol.
1970, 48:444-453), which has been incorporated into the GAP program in the GCG

software package (Accelrys, Burlington, MA; available at accelrys.corn on the WorldWideWeb), using either a Blossum 62 matrix or a PAM25o matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package using a NWSgapdna.CMP matrix, a gap weight of 40, 50, 6o, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A
particularly preferred set of parameters (and the one that can be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is a sequence identity or homology limitation of the invention) is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
[054] Another non-limiting example of how percent identity can be determined is by using software programs such as those described in Current Protocols In Molecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1.
Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN
and BLASTP, using the following default parameters: Genetic code=standard;
filter=none;
strand=both; cutoff=6o; expect=io; Matrix=BLOSUM62; Descriptions =50 sequences;
sort by=HIGH SCORE;
Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.
[055] Statistical analysis of the properties described herein may be carried out by standard tests, for example, t-tests, ANOVA, or Chi squared tests. Typically, statistical significance will be measured to a level of p=o.05 (5%), more preferably p=o.oi, p=o.00l, p=o.000l, p=o.00000i [056] In preferred aspects, the methods described herein relate to devices, wherein said device is: (a) permanently implanted in the subject; (b) temporarily implanted in the subject; (c) removable; and/or (d) is selected from a prosthetic joint, a left-ventricular assist device (LVAD), a stent, a metal rod, an in-dwelling catheter, spinal hardware, and/or bone hardware.
[057] In further aspects, the infection is selected from: a prosthetic joint infection (PJI), a chronic bacterial infection, an acute bacterial infection, a refractory infection, an infection associated with a biofilm, and/or an infection associated with an implantable device.
[058] In further aspects, the methods described herein relate to compositions, wherein said composition is administered, for example, (a) by IV injection;
(b) by direct injection to the site of infection; (c) prophylactically; (d) prior to surgery; (e) in lieu of surgery; (g) during surgery; (h) on a single occasion (i.e., as a "single shot"); and/or (i) as a therapeutic course over 2 weeks or more.
[059] In preferred aspects, lysis of a bacterium by a phage can be measured using assays known in the art, such as but not limited to (a) growth inhibition; (b) optical density; (c) metabolic output; (d) photometry (e.g., fluorescence, absorption, and transmission assays); and/or (e) plaque formation.
[o6o] In preferred aspects, the photometric assay used to measure lysis utilizes an additive that causes and/or enhances the photometric signal detection. An example of such an additive, include, but is not limited to tetrazolium dye.
[061] In other preferred aspects, the biological sample is obtained from:
(a) synovial fluid; (b) an area surrounding an implantable device; (c) a site of infection;
(d) an intra-operative sample; (e) a swab of the device; (f) a biofilm; (g) a fistula;
and/or (h) an aspiration of a site of infection.
[062] In further aspects, the bacterial infection is (a) multi-drug resistant; (b) clinically refractory to antimicrobial treatment; (c) clinically refractory to antimicrobial treatment due to biofilm production; and/or (d) clinically refractory due to the subject's inability to tolerate antimicrobials due to adverse reactions.
[063] As understood herein, the terms, "multidrug resistant", "multi drug resistant", "multi drug resistance", "MDR" and like terms may be used interchangeably herein, and are familiar to one of skill in the art, i.e., a multidrug resistant bacteria is an organism that demonstrates resistance to multiple different antibacterial drugs, e.g., antibiotics; and more specifically, resistance to multiple different classes of antibiotics.
It is understood herein that bacterial infections to be treated comprise bacteria in biofilm and/or planktonic growth modes.
[064] Examples of typically MDR bacteria that maybe treated include, but are not limited to the "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp), which are often nosocomial in nature and can cause severe local and systemic infections. Specifically, these include, e.g., methicillin-resistant Staphylococcus aureus (MRSA); vancomycin-resistant Enterococcus faecium (VRE); carbapenem-resistant Klebsiella pneumonia (NDM-1); MDR-Pseudomonas aeruginosa; and MDR-Acinetobacter baumannii.
[065] Among the ESKAPE pathogens, A. baumannii is a Gram-negative, encapsulated, opportunistic pathogen that is easily spread 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 MDR, which severely restricts the available treatment options, with untreatable infections in traumatic wounds often resulting in prolonged healing times, the need for extensive surgical debridement, and in some cases the further or complete amputation of limbs. Notably, blast-related injuries in military populations are associated with significant tissue destruction with concomitant extensive blood loss and therefore these injuries are at high risk for infectious complications. One of skill in the art will appreciate that given the ability for A. baumannii and other MDR ESKAPE pathogens to colonize and survive in a host of environmental settings, there is an urgent need for new therapeutics against these pathogens.
[o66] In further aspects, the subject is suffering from a hardware related infection.
[067] In preferred embodiments, the infection is a prosthetic joint infection (PJI).
For example, the composition of the invention may be useful for treating PJI, regardless of type. That is, by appropriately selecting the two or more phage, the composition may be beneficial in treating early, delayed and late onset types of PJI, as well as PJI that are the result of a polymicrobial infection. Similarly, in other preferred embodiments, the compositions described herein can be used to treat an acute and/or chronic infection, and preferably a PJI infection. The composition of the invention may be useful for treating PJI associated particularly with prosthetic hip, knee, shoulder and elbow replacements.
[o68] In some aspects, the library used in the methods described herein comprises phage pre-screened to exclude phage comprising undesirable and/or toxic characteristics.
Examples of such undesirable and/or toxic characteristics are selected from toxin genes or other bacterial virulence factors, phages which possess lysogenic properties and/or carry lysogeny genes, phages which transduce bacterial virulence factor genes or antibiotic resistance genes, phages which carry any antibiotic-resistance genes or can confer antibiotic resistance to bacterial strains, and phages which elicit an inappropriate immune response and/or provoke a strong allergenic response in a mammalian system.
Examples of producing such pre-screened libraries of phage are described in, for example, US 10,357,522 (the disclosure of which is hereby incorporated by reference in its entirety).
[069] It is known that antibiotic resistance, toxin formation, and unwanted mammalian innate immune responses are possible outcomes from the use of bacteriophages to treat bacterial infections (Quiros et. al., Antimicrob Agents Chemother 58: 606-609, 2014; do Vale et. al., Front. Microbiol. 7:42, 2016).
In some respects, phages comprising undesirable and/or toxic characteristics can be eliminated initially from the library. It is also well understood within the skill in the art to genetically alter the phage to eliminate and/or reduce undesirable and/or toxic phenotype.
The detrimental 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, systems such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) can he used to edit the genetic material. Phages with DNA or RNA
genom es may be engineered using CRISPR systems.
Gene Editing Using CRISPR/Cas9 [070] CRISPR and CRISPR-associated protein 9 (Cas9), referred to as the CRISPR/Cas9 system, may be used to perform gene editing. Gene editing includes, but is not limited to, gene insertions, gene replacements, gene deletions, frame shifts, single nucleotide changes, nonsense mutations, missense mutations, etc. Gene editing may also include editing of regulatory sequences to modulate gene expression. Based on these techniques, genes may be mutated, repaired, or even modulated in a variety of cell lines and germline sequences. A review of the CRISPR/Cas9 system may be found in Hsu, et al., "Development and Applications of CRISPR-Cas9 for Genome Engineering" Cell (2014) vol. 157:1262. A guide to using the CRISPR/Cas9 system may be found at Addgene (add.gene.org/CRiSPR/gu.ide).

[071] The CRISPR/Cas9 system can be employed to "knock-out" and "knock-in"
specific genes in various cell types and organisms as well as selectively activate or repress specific genes, purify specific regions of DNA, image DNA in live cells using fluorescence microscopy, as well as many other uses.
[072] According to embodiments of the present invention, the CRISPR/Cas9 system can be used to edit the gene in phages to eliminate and/or reduce undesirable and/or toxic phenotype. By reducing or eliminating toxic phenotype, the undesirable effects of bacteriophage therapy to treat bacterial infections including antibiotic resistance, toxin formation, and unwanted mammalian innate immune responses be reduced or eliminated, and it is expected that the phages are useful in treatment of infection.
[073] According to other embodiments of the present invention, the phages that have been edited by the CRISPR/Cas9 system may be administered to the patient. In still further embodiments of the invention, the phages that have been edited by the CRISPR/Cas9 system may be combined with one or more additional agents as described herein.
[074] Genome editing of phages, traditionally a challenging and time-consuming process, may be efficiently performed using the CRISPR/Cas9 system. In some cases, editing by the CRISPR/Cas9 system may occur within 24 hours of delivery of the Cas9 gene, the guide RNA, and as applicable, the nucleotide replacement sequence.
Components of the CRISPR/Cas9 system [075] In general, the CRISPR/Cas9 system comprises at least two components:
(i) a Cas9 protein, which is a nuclease capable of cutting both stands of a DNA
double helix;
and (ii) at least one RNA sequence (e.g., a guide RNA (gRNA) sequence, such as a single-guide RNA (sgRNA) sequence) that is designed to target Cas9 to a specific location or locus of a gene of interest (a genomic target). The gRNA comprises a targeting sequence homologous to the genomic target, as well as a scaffolding domain that binds to Cas9, in order to recruit Cas9 to the genomic target.

Non-Homologous End Joining [076] In some embodiments, the CRISPR/Cas9 system induces double stranded DNA breaks in the genomic target, which are thought to stimulate cell repair mechanisms using non-homologous end joining (NHEJ). Non-homologous end joining, which can lead to gene disruption by random insertions or deletions, is thought to be a primary mechanism for repairing double strand breaks (DSBs). NHEJ-mediated DSB repair can result in disruption of the open reading frame (ORF) of the genomic target, leading to a non-functional protein.
[077] For NHEJ methods, Cas9 as well as a gRNA is introduced into the host cell, e.g., either as a preassembled complex or inserted into one or more expression vectors.' Homology Directed Repair [078] In other embodiments, the CRISPR/Cas9 system is utilized to repair, replace or insert one or more nucleotides at the genomic target. In this method, a replacement nucleotide sequence is introduced into a cell, wherein the replacement nucleotide sequence contains the desired edits as well as regions of homology upstream and downstream of the genomic target. To perform homology directed repair (HDR), Cas9, e.g., a recombinant Cas9 from Streptococcus pyogenes, complexed with a gRNA, e.g., an in vitro transcribed sgRNA, and a replacement nucleotide sequence is needed.
[079] Homology Directed Repair (HDR) may be used to generate gene edits ranging from a single nucleotide base change to large nucleotide sequence insertions.
To utilize HDR for gene editing, a replacement nucleotide sequence is delivered into the cell along with the gRNA and Cas9. The replacement nucleotide sequence contains the desired genomic edit(s) as well as additional homologous sequences immediately upstream and downstream of the genomic target sequence (referred to as a left homology arm and a right homology arm). The replacement nucleotide sequence can be a single stranded oligonucleotide, a double-stranded oligonucleotide, a double-stranded DNA
plasmid depending on the specific application, and so forth. Generally, the replacement nucleotide sequence will not contain a Protospacer Adjacent Motif (PAM) sequence so as not to become a target for Cas9 cleavage.

gRNA
[080] As discussed previously, gRNA comprises a targeting sequence and a scaffolding domain. Once expressed, Cas9 and the gRNA form a riboprotein complex through interactions between the gRNA scaffolding domain. Upon gRNA binding, Cas9 undergoes a conformational change into a form that binds DNA, while allowing the targeting sequence to remain free to interact with genomic target DNA. For Cas9 to cleave genomic target DNA, the targeting sequence should exhibit high homology to the genomic target sequence. In general, the target sequence comprises 20 nucleotides, or about 20 nucleotides, and may be a synthetic RNA.
[081] In general, "target sequence" or "targeting sequence" refers to a sequence (e.g., the portion that is not associated with the scaffolding domain) of the gRNA.
"Genomic target" or "Genomic target sequence" refers to a sequence or locus of the genome targeted for editing.
[082] The gRNA focuses the nuclease activity of Cas9 to the genomic target using the target sequence. Once the target sequence binds or hybridizes to the genomic target, Cas9 cleaves the genomic DNA at or near that site. By changing the target sequence, the genomic target of Cas9 can be modified.
[083] In some embodiments, gRNA may be synthesized using commercially available kits, e.g., a Guide-it sgRNA In Vitro Transcription Kit. In vitro transcription may be used to produce gRNAs that may be purified using techniques known in the art.
Genomic target [084] The genomic target sequence should be unique as compared to the remainder of the genome of the organism or cell in order to avoid off target effects.
[085] Additionally, the genomic target sequence should be present immediately upstream of a genomic Protospacer Adjacent Motif (PAM) sequence. The PAM
sequence is needed for Cas9 binding, with the exact PAM sequence being dependent upon the species of Cas9 used. Cas9 from Streptococcus pyo genes is widely used in genomic engineering. In general, Cas9 binds to the PAM sequence, and DNA cleavage occurs approximately 3 base pairs upstream of the PAM sequence.
[086] An example of a PAM sequence is 5'-NGG-3". Genomic target sequences may reside on either strand of genomic DNA.
[087] 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 a genomic locus or location of interest (e.g., within a genomic location encoding for a protein such as CXCR4, PD-1, etc.). These tools also predict off target effects in order to allow selection of a genomic target sequence that minimizes cleavage of genomic DNA at other locations.
Vectors and Host Cells [088] Construction of various types of vectors, including vectors for the CRISPR/Cas9 system, maybe found in U.S. Patent Application No. 2014/0273226, which is incorporated herein by reference.
[089] In general, polynucleotides, e.g., polynucleotides encoding Cas9, polynucleotides encoding a gRNA sequence, polynucleotides encoding a replacement nucleotide sequence, etc., can be incorporated into any desired DNA or RNA
based vector, without limitation. For example, a polynucleotide may be cloned into an expression vector, a subcloning vector, a shuttle vector, a vector designed for use with in vitro transcription reactions, cosmids, phagemids, and vectors derived from mammalian viruses, including retroviruses (for example, lentiviruses), adenoviruses, adenoassociated viruses (AAV), and episomal EBNA-based vectors of Epstein-Barr virus origin.
[090] In some embodiments, vectors may be in circular form or in linearized form.
The linearized form may be used in subcloning steps, e.g., cloning the gRNA
target sequence into a host vector.
[091] One of skill in the art will understand that a wide variety of expression vectors for expression of gene products are within the scope of present invention embodiments.
An expression vector can be optimally designed to express a protein, e.g., Cas9, a variant Cas9, etc., in a host cell. For example, a vector comprising the nucleotide sequence encoding a protein (e.g., a Cas9 open reading frame (ORF)) and suitable regulatory elements can be delivered into the host cell by any suitable method of transfection, transduction, etc. Any type and any quantity of regulatory elements, involved in regulation of transcription or translation, may be incorporated into the expression vector and may be located upstream or downstream of the ORF. Once in the host cell, the host cell's own machinery, e.g., endogenous RNA polymerases, etc., may be employed to synthesize mRNA, which is translated to produce the protein.
[092] In other embodiments, expression vectors are optimally designed to express a functional RNA molecule, e.g., a guide RNA for tethering Cas9 to a genomic target sequence.
[093] In some embodiments, the Cas9 mRNA and the gRNA are expressed from the same expression vector. In other embodiments, the Cas9 mRNA and the gRNA are expressed from different expression vectors. The Cas9 gene inserted into the expression vector may be a native bacterial Cas9 gene from, e.g., Streptococcus pyo genes, Staphylococcus aureus, etc. In some embodiments, the expression vectors may be mammalian expression vectors, e.g., such as human expression vectors, and may contain one or more promoter elements, e.g., a bacteriophage promoter element such as a T7 promoter.
[094] In further embodiments, the gene encoding for Cas9 may be operably linked with one or more genes encoding nuclear localization signals, resulting in targeting of the expressed Cas9/gRNA to the host cell nucleus.
[095] In still other embodiments, the gene encoding for Cas9 is optimized to reflect preferred codon usage for the organism in which gene editing is being performed. For example, if gene editing is being performed in phages, then the gene encoding for Cas9 in the expression vector construct, is optimized to reflect preferred codon utilization.
[096] In some embodiments, phages may be isolated, and a preassembled gRNA/Cas9 complex along with an optional nucleotide replacement sequence may be electroporated into host cells. The gRNA sequences may be designed to have homology with exonic coding regions of one or more genes.

[097] In other embodiments, CRISPR/Cas9 systems allow targeting of multiple genetic loci (genomic targets) by cloning multiple gRNAs into a single vector.
[098] Present embodiments also provide 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 engineering cell genomes in a highly specific manner. In some embodiments, engineered cell lines produced by the CRISPR/Cas9 system are useful for therapeutic transplantation to treat human diseases. Human diseases arising from bacterial infections can be treated with compositions and methods of the invention, or with edited cell lines produced by the compositions and methods of the invention.
[099] According to embodiments of the invention, methods are provided for specific genomic modification of host cells or phages, including: (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 comprising the genomic locus of interest, (iii) delivering the expression vector construct into the host cell, and (iv) expressing the first and second polynucleotides within the host cell. In some embodiments, the method may further comprise visualizing, identifying, or selecting for host cells having gene edits at the genomic target.
[loo] According to other embodiments of the invention, methods are provided for specific genomic modifications of host cells, including: (i) providing a first expression vector comprising a first polynucleotide encoding a Cas9 protein and having a transcriptional regulatory domain, 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 comprising 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 within the host cell. In some embodiments, the method may comprise visualizing, identifying or selecting for host cells having gene edits at the genomic target. By targeting a regulatory region such as a promoter region, controlling expression of a gene of interest, expression may be modulated, e.g., repressed or activated.

[101] According to still other embodiments of the invention, methods are provided for specific genomic modification of host cells, including: (i) providing a Cas9 protein complexed with a gRNA, wherein the gRNA comprises a target sequence homologous to a genomic target, (ii) electroporating the Cas9 protein/gRNA complex into a host cell comprising the genomic target, (iii) selecting host cells based upon expression levels of the protein encoded by the genomic target (genomic locus). In some embodiments, the method may further comprise staining the host cell 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.
[102] In still other embodiments of the invention, more than one genomic target (loci of interest) is targeted for gene editing at the same time. Accordingly, the expression vector may comprise multiple nucleotide sequences, each encoding a different gRNA with a different targeting sequence, each targeting sequence corresponding to a genomic target. Alternatively, multiple expression vectors may be used, each expression vector comprising one or more_targeting sequences, each targeting sequence corresponding to a genomic target.
[103] One of skill in the art will appreciate that present invention embodiments are not limited to the polynucleotide or polypeptide sequences referred to herein, and will also encompass variant polypeptide and polynucleotide sequences. Variant polypeptide sequences include polypeptides having conservative amino acid substitutions in their amino acid sequences. Variant polynucleotide sequences include polynucleotides that are modified to have a change in a nucleotide sequence, and upon expression, result in a polypeptide having essentially the same function or activity as the polypeptide expressed by the unmodified nucleotide sequence.
[104] In some embodiments, CRISPR-CAS9 system is used for editing phages.
In some other embodiments, a type I-E CRISPR-Cas system is used for editing phages. In yet other embodiments, a type III CRISPR-CASio system is used for editing phages.
[105] For example, the protocols described in recent publications may be modified to delete a toxic and/or undesirable phenotype in phages of Myoviridae, Siphoviridae, and Podoviridae families (Ban et. al., Synth Biol. 2017;6(12):2316-2325; Box et. al, Journal of Bacteriology Jan 2016, 198 (3) 578-590, Tao et. At, ACS Synth Biol.

2017;6(10):1952-1961; and Park et. al., Sci Rep. 2017;7:42458). Bari et. al., describes a Type III-A CRISPR-Casio system for editing phages targeting S. aureus strain with or without a natural CRISPR-Cas system. The system provides a mechanism to select phage-derived sequences such as those harboring point mutations at multiple genetic loci and recover phage recombinants that have acquired desired mutations. Using RNA
sequences as a map, the Cas enzyme cleaves the targeted gene carrying the deleterious and/or toxic characteristic. Another publication (Yosef et. al., Proceedings of the National Academy of Sciences Jun 2015, 112 (23) 7267-7272) describes temperate phage based CRISPR-Cas delivery into the genome of antibiotic-resistant bacteria.
[106] In some embodiments, a phage or phagemid is used to deliver DNA to the bacterial cell.
[107] In some embodiments, a CRISPR system can be used to eliminate specific bacterial strains by removing them from the microbiome or for specific therapy.
[108] In further preferred embodiments, the method relies on the CRISPR
system such as the CRISPR/Cas9 system, to edit undesirable and/or toxic genes, and preferably bacterial pathogenic gene such as bacterial endogenous gene, a single nucleotide polymorphism(SNP), an epichromosomal gene, an antibiotic resistance gene, a gene encoding one or more virulence factors, a gene encoding one or more toxins, highly conserved genes amongst a species, genera or phyla, and/or genes that encode enzymes involved in biochemical pathways with products that can modulate host physiology.
[109] In a preferred embodiment, the CRISPR system targets a toxin and antitoxin loci involved in key biological functions including plasmid maintenance, defense against phages, 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(M.):D749-D753., Harms et. al., Mol Cell. 2018;70(5):768-784. and do Vale et. al., 2016.
[no] 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 even further preferred embodiments, the bacterium is selected from S. aureus, S.
epidermidis and/or E. fecalis.
[111] Examples of toxins include pertussis toxin and adenylatecyclase toxin(ACT) secreted by Bordetella pertussis, anthrax toxin from Bacillus anthracis and Staphylococcus aureus leukotoxins, AIP56 from Photobacterium damselaepiscicida (Phdp), mycolactone, a polyketide molecule produced by Mycobacteriumulcerans, other bacterial secreted products not formally termed as toxins, such as S. aureus super antigens-like proteins (SSLs) and phenol-soluble modulins (PSMs), Clostridial C3 toxins, Shiga toxin, cholera toxin, hemolysins, leucocidin, fimbrial and afimbrial adhesins, proteases, lipases, endonucleases, endotoxins and exotoxins cytotoxic factors, microcins and colicins. In some embodiments, the toxin is encoded by superantigen enterotmdn gene such as the S. aureus Sek gene. In some embodiments, the toxin is a exotoxin such as toxic shock syndrome toxin-i (TSST-1); enterotoxins such as SEA, SEB, SECn, SED, SEE, SEG, SEH, and SET, and exfoliative toxins, such as ETA and ETB.
[112] Examples of genes that confer virulence traits to Escherichia coli (e.g., 0157:H7) include, without limitation, stx1 and 5tx2 (encode Shiga-like toxins) and espA
(responsible for induction of enterocyte effacement (LEE) A/E lesions). Other examples of genes that confer virulence traits to Escherichia coli include fimA
(fimbriae major subunit), csgD (curli regulator) and csgA. An example of a gene that confers virulence traits to Yersinia pestis is yscF (plasmid-borne (pCD1) T3SS external needle subunit). An example of a gene that confers virulence traits to Francisella tularensis is fs1A. An example of a gene that confers virulence traits to Bacillus anthracis is pag (Anthrax toxin, cell-binding protective antigen). Examples of genes that confer virulence traits to Vibrio cholerae include, without limitation, ctxA and ctxB (cholera toxin), tcpA
(toxin co-regulated pilus), and toxT (master virulence regulator).Examples of genes that confer virulence traits to Pseudomonas aeruginosa include, without limitation, genes that encode for the production of siderophore pyoverdine (e.g., sigma factor pvdS, biosynthetic genes pvdL, pvdl, pvdJ, pvdH, pvdA, pvdF, pvdQ, pydN, pvdM, pvd0, pvdP, transporter genes pvdL, pydR, pvdT and opmQ), genes that encode for the production of siderophore pyochelin (e.g., pchD, pchC, pchB, pchA, pchE, pchF and pchG, and genes that encode for toxins (e.g., exoU, exoS and exoT). Examples of genes that confer virulence traits to Klebsiella pneumoniae include, without limitation, fimA
(adherence, type I fimbriae major subunit), and cps (capsular polysaccharide). Examples of genes that confer virulence traits to Acinetobacter baumannii include, without limitation, ptk (capsule polymerization) and epsA (assembly). Examples of genes that confer virulence traits to Salmonella enterica Typhi include, without limitation, hilA
(invasion, SPI-1 regulator), ssrB (SPI-2 regulator), and those associated with bile tolerance, including efflux pump genes acrA, acrB and to1C.
[113] Examples of antibiotic resistance genes or various methods for identifying the same are known in the art. Genomic methods of identifying resistance phenotype of strain are described in the art such as whole-genome sequencing for antimicrobial susceptibility testing (WGS-AST) described in Su et. al., Journal of Clinical Microbiology Feb 2019, 57 (3) e01405-18 and antibiotic resistance determinants (ARDs) identified from intestinal microbiota in Ruppe et al., Nat Microbial. 2019;4W:112-123. In some embodiments, the resistance gene confers resistance to a narrow-spectrum beta-lactam antibiotic of the penicillin class of antibiotics. In other embodiments, the resistance gene confers 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, quinupristin/dalfopristin. In some embodiments, the resistance genes is selected from fosfomycin resistance gene fosB, tetracycline resistance gene tetM, kanamycin nucleotidyltransferase aadD, bifunctional aminoglycoside modifying enzyme genes aacA-aphD, chlorampheni col acetyltransferase cat, mupirocin-resistance gene ileS2, vancomycin resistance genes vanX, vanR, vanH, vraE, vraD, methicillin resistance factor femA, fmtA, mecl, streptomycin adenylyltransferase spci, spc2, anti, ant2, pectinomycin adenyltransferase spd, ant9, aadA2, and any other resistance gene.
[114] In a preferred embodiment, the CRISPR system targets one or more of antibiotic resistance genes selected from an extended-spectrum beta-lactamase resistance factor (ESBL factor), CTX-M-15, beta lactamase, New Delhi metallo-lactamase (NDM)-1,2,5,6 and tetracycline A (tetA).

[115] Examples of genes that confer resistance to aminoglycoside include, without limitation, aph, aac and aad variants and other genes that encode aminoglycoside-modifying enzymes. Examples of genes with SNPs that confer aminoglycoside resistance include, without limitation, rpsL, rrnA and rrnB. Examples of genes that confer beta-lactam resistance include, without limitation, genes that encode beta-lactamase (bla) (e.g., TEM, SHV, crx-m, OXA, AmpC, IMP, VIM, KPC, NDM-1, family beta-lactamases) and mecA. Examples of genes with SNPs that confer daptomycin resistance include, without limitation, mprF, yycFG, rpoB and rpoC. Examples of genes that confer macrolide-lincosamide-streptogramin B resistance include, without limitation, ermA, ermB and ermC. Examples of genes that confer quinolone resistance include, without limitation, qnrA, qnrS, qnrB, qnrC and qnrD. Examples of genes with SNPs that confer quinolone resistance include, without limitation, gyrA and parC. Examples of genes with SNPs that confer trimethoprim/sulfonamide resistance include, without limitation, the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes.
Examples of genes that confer vancomycin resistance include, without limitation, vanA
(e.g., vanRS
and vanHAX), vanB and vanC operons.
[116] In some embodiments, the CRISPR system may be used to target SNPs, which cause overexpression of genes that encode multi-drug efflux pumps, such as acrAB, mexAB, mexXY, mexCD, mefA, msrA and tetL.
[117] Examples of highly conserved genes that can typify microbial species, genera or phyla for the purposes of remodeling complex microbial communities (referred to herein as "remodeling" genes) include, without limitation, ribosomal components rrnA, rpsL, rpsJ, rp10, rpsM, rp1C, rpsH, rp1P, and rpsK, transcription initiation factor infB, and tRNA synthetase pheS.
[118] Examples of genes encoding enzymes involved in biochemical pathways with products that can modulate host physiology (referred to herein as "modulatory"
genes include, without limitation, (1) genes encoding enzymes involved in deoxycholate production linked to hepatocellular carcinoma, (2) genes encoding enzymes involved in polysaccharide A production by Bacteroides fragilis, leading to development of regulatory T cells (TREG), IL-io response and increased TH1 cell numbers, (3) genes encoding enzymes involved in butyrate production leading to secretion of inducible antimicrobial peptides, (4) genes encoding enzymes involved in short-chain fatty acid production leading to increased energy harvest, obesity, inflammatory modulation and gastrointestinal wound healing, (5) genes encoding enzymes involved in transformation of choline into methylamines, which can disrupt glucose homeostasis, leading to non-alcoholic fatty liver disease and cardiovascular disease, (6) genes encoding enzymes involved in the generation of neuromodulatory compounds such as y-aminobutyric acid, noradrenaline, 5-HT, dopamine and acetylcholine, and (7) genes encoding enzymes involved in the formation of lactic acid and propionic acid linked to anxiety.
[119] Besides CRISPR/Cas9 systems, other genome editing tools can be used to modify and/or eliminate toxic and/or undesirable characteristics. Examples of such phage genome editing tools include, but are not limited to other CRISPR based systems, engineered TALEN (Transcription Activator-Like Effector Nuclease) variants, engineered zinc finger nuclease (ZFN) variants and other systems such as those described in Chen et. al., (Front. Microbiol., 03 May 2019) including homologous recombination-based technologies, bacteriophage recombineering of electroporated DNA (BRED), rebooting phages using assembled phage genomic DNA.
[120] As understood herein, terms such as "effective amount" and "therapeutically effective amount" of a pharmaceutical composition of the instant invention, refer to an amount of a composition suitable to elicit a therapeutically beneficial response in the subject, e.g., by eradicating a bacterial pathogen in the subject and/or altering the virulence or antibiotic susceptibility of surviving phage-resistant bacterial pathogens and/or by providing an added benefit when the composition is simultaneously administered with either effective and/or ineffective antibiotics. Such response may include e.g., preventing, ameliorating, treating, inhibiting, and/or reducing one of more pathological conditions associated with a bacterial infection. One of skill in the art will appreciate that it is desirable that the initial dose of a composition described herein be sufficient to control the bacteria population before it reaches a lethal threshold. Animal models suggest that 109 to loll pfu/ml phage particles per dose would likely be the maximum dosage tenable based on protein load presented acutely to the liver in an adult (which would be scaled down in a pediatric population). It is suspected that this is enough of an acute bolus to reduce the bacterial burden sufficiently to potentiate an immune response. Notably, phage "viremia" may be measured in the blood after administration.
Animal models suggest that viremia is quite transient given the host immune response and sequestration in the reticuloendothelial system (liver and spleen).
[121] Suitable effective amounts of the compositions of the instant invention can be readily determined by one of skill in the art and can depend upon the age, weight, species (if non-human) and medical condition of the subject to be treated. In addition, one of skill in the art will appreciate that the type of infection (e.g., systemic or localized), and the accessibility of the infection to treatment may also impact the dosage amount that is deemed effective. One of skill in the art will appreciate that initial information may be gleaned in laboratory experiments and an effective amount of a composition described herein for humans subsequently determined through dosing trials and routine experimentation.
[122] It is even further contemplated that the compositions of the instant invention may be administered to a subject by a variety of routes according to conventional methods, including but not limited to systemic, parenteral (e.g., by intracisternal injection and infusion techniques), intradermal, transmembranal, transdermal (including topical), intramuscular, intraperitoneal, intravenous, intra-arterial, intralesional, subcutaneous, oral, and intranasal (e.g., inhalation) routes of administration. Administration can also be by continuous infusion or bolus injection.
[123] In addition, the compositions of the instant invention can be administered in a variety of dosage forms. These include, e.g., liquid preparations and suspensions, including preparations for parenteral, subcutaneous, intradermal, intramuscular, intraperitoneal or intravenous administration (e.g., injectable administration), such as sterile isotonic aqueous solutions, suspensions, emulsions or viscous compositions that may be buffered to a selected pH. In a particular embodiment, it is contemplated herein that the compositions of the instant invention are administered to a subject as an injectable, including but not limited to injectable compositions for delivery by intramuscular, intravenous, subcutaneous, or transdermal injection. Such compositions may be formulated using a variety of pharmaceutical excipients, carriers or diluents familiar to one of skill in the art.

[124] In another particular embodiment, the compositions of the instant invention, and/or pharmaceutical formulations administered in conjunction therewith, e.g., antibiotics, may be administered orally. Oral formulations for administration according to the methods of the present invention may include a variety of dosage forms, e.g., solutions, powders, suspensions, tablets, pills, capsules, caplets, sustained release formulations, or preparations which are time-released or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the gut. Such formulations may include a variety of pharmaceutically acceptable excipients described herein, including but not limited to mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.
[125] In a particular embodiment, it is contemplated herein that a composition for oral administration maybe a liquid formulation or as part of a dissolvable paper placed on the tongue. Such formulations may comprise a pharmaceutically acceptable thickening agent which can create a composition with enhanced viscosity which facilitates mucosal delivery of the active agent, e.g., by providing extended contact with the lining of the stomach. Such viscous compositions may be made by one of skill in the art employing conventional methods and employing pharmaceutical excipients and reagents, e.g., methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, and carbomer.
[126] Other dosage forms suitable for nasal or respiratory (mucosal) administration, e.g., in the form of a squeeze spray dispenser, pump dispenser or aerosol dispenser, are contemplated herein. Dosage forms suitable for rectal or vaginal delivery are also contemplated herein. Where appropriate, compositions for use with the methods of the instant invention may also be lyophilized and may be delivered to a subject with or without rehydration using conventional methods.
[127] Thus, and as would be understood, the methods of the instant invention comprise administering the compositions of the invention to a subject according to various regimens, i.e., in an amount and in a manner and for a time sufficient to provide a clinically meaningful benefit to the subject. Suitable administration regimens for use with the instant invention may be determined by one of skill in the art according to conventional methods. For example, it is contemplated herein that an effective amount may be administered to a subject as a single dose, a series of multiple doses administered over a period of days, or a single dose followed by one or more additional "boosting" doses thereafter. The term "dose" or "dosage" as used herein refers to physically discrete units suitable for administration to a subject, each dosage containing a predetermined quantity of the active pharmaceutical ingredient calculated to produce a desired response.
[128] The administrative regimen, e.g., the quantity to be administered, the number of treatments, and effective amount per unit dose, etc. will depend on the judgment of the practitioner and are peculiar to each subject. Factors to be considered in this regard include physical and clinical state of the subject, route of administration, intended goal of treatment, as well as the potency, stability, and toxicity of the particular composition. As understood by one of skill in the art, a "boosting dose" may comprise the same dosage amount as the initial dosage, or a different dosage amount.
Indeed, when a series of doses are administered in order to produce a desired response in the subject, one of skill in the art will appreciate that in that case, an "effective amount"
may encompass more than one administered dosage amount.
[129] In a preferred embodiment, the compositions described herein can be administered both topically and systemically, e.g. via IV, intra-articular, IM, and/or an injection directly to the site of infection. For example, in the case of a PJI, the composition described herein can be directly injected into the infected joint.
Accordingly, the composition may be formulated as an injectable fluid, semi-solid or depot-type formulation as will be readily apparent to one of ordinary skill in the art.
However, PJI
could also be treated by IV injection, and/or even as a combination of both direct injection to the site of infection along with administration by IV, intra-articular, and/or IM.
[130] In cases where the infected joint is also being treated by debridement and/or replacement of the prosthetic device in a one- or two-stage arthroplasty exchange, the composition may be directly administered to the joint during the surgery. For example, the composition may be administered on a single occasion (i.e. a "single shot") or on multiple occasions as may be required (this "single shot" encompassing both a direct injection to the site of infection or by IV). It is however considered that the composition may potentially be administered as a single shot and, moreover, may avoid any requirement for replacing the prosthetic device of the infected joint.

[131] In some embodiments, the composition may further comprise a further active agent or preparation such as, for example one or more antibiotics (e.g.
rifampin and/or a fluoroquinolone such as ciprofloxacin), one or more bactericides, and/or one or more other therapeutic molecules such as a small molecule or biologic that has bactericidal activity. These further agents could be administered as part of the composition, or separately yet concurrently with the administration of the phage compositions.
[132] In a further aspect, the invention provides a method of treating a patient with a prosthetic joint infection (PJI) or at risk of developing a PJI, comprising administering a pharmaceutical composition according to the first aspect.
[133] In some embodiments, the patient has a PJI caused by bacteria selected from S. aureus, S. epidermidis, E. fecalis, S. caprae, and/or S. lugdunensis.
[134] In other embodiments, the patient has a PJI that has been determined as being caused by bacteria selected from S. aureus, S. epidermidis, E. fecalis, S. caprae, and/or S. lugdunensis. Said determination may be conducted by, for example, culturing synovial fluid obtained from the affected joint by aspiration and determining the identity of bacteria in the culture by, for example, 16S rRNA gene sequence analysis.
[135] In other embodiments, the patient is at risk of developing a PJI
caused by bacteria selected from S. aureus, S. epidermidis, E. fecalis, S. caprae, and/or S.
lugdunensis.
[136] The method of the invention may be useful for treating or preventing PJI, regardless of type. That is, by appropriately selecting the two or more phage for inclusion in the composition, the method may be beneficial in treating early, delayed and late onset types of PJI, as well as PJI that are the result of a polymicrobial infection.
[137] The method may be performed so as to provide an effective amount of the at least two different phage; that is, an amount that is sufficient to cause lysis (i.e. kill) of the bacteria present at an infection being treated, such as a PJI being treated (or in a PJI
that may develop) such that a beneficial or desired clinical result is achieved and/or to prevent the occurrence of a bacterial infection. An effective amount can be administered in one or more administrations. Typically, an effective amount is sufficient for treating a disease or condition or otherwise to palliate, ameliorate, stabilize, reverse, slow, delay or prevent the progression or development of the PJI.
[138] The infection to be treated includes, but is not only limited to, a bacterial infection already existing in a patient, but also the prevention of a future infection that may or may not occur in a patient with an implantable device. For example, bacterial infections of particular strain(s) of bacterium known to frequently occur in a specific geographical location (e.g., a geomatched composition) can be treated with the disclosed compositions in a prophylactic manner in an attempt to prevent a future bacterial infection.
[139] In further details, the method may involve directly administering the composition to an infected joint or a prosthetic joint at risk of the development of PJI. In the latter case, the method may be performed at the time of surgery (i.e. the composition may be administered at the time that the joint replacement is being performed). In cases where the method is being used to treat an infected joint, the method may avoid any requirement for replacing the prosthetic device. That is, the method may be performed in lieu of surgery. In other cases, where the infected joint is also being treated by debridement and/or replacement of the prosthetic device in a one- or two-stage arthroplasty exchange, the method may be conveniently performed during the surgery (i.e. the composition may be administered to the joint during the surgery).
The composition may be administered on a single occasion (i.e. a "single shot") or on multiple occasions as may be required.
[140] In some embodiments, the method of the invention may be performed as a combination therapy. For example, the composition comprising the at least two different phage may be administered to the patient as a combination therapy involving the administration of a further active agent or preparation such as, for example, one or more antibiotics (e.g. rifampin and/or a fluoroquinolone such as ciprofloxacin), one or more bactericides, and/or one or more other therapeutic molecules such as a small molecule or biologic that has bactericidal activity.
[141] Where performed as a combination therapy with a further active agent or preparation, the composition may comprise the at least two different phage along with the further active agent or preparation (i.e. as a single composition), or otherwise separate compositions may be used. If administered in separate compositions, the therapeutic phage composition and the further active agent or preparation may be administered simultaneously or sequentially in any order (e.g. within seconds or minutes (e.g. 5 to 6o mins) or even hours (e.g. 2 to 48 hours)).
[142] Although the invention herein has been described with reference to embodiments, it is to be understood that these embodiments, and examples provided herein, are merely illustrative of the principles and applications of the present invention.
It is therefore to be understood that numerous modifications can be made to the illustrative embodiments and examples, and that other arrangements can be devised without departing from the spirit and scope of the present invention as defined by the appended claims. All patent applications, patents, literature and references cited herein are hereby incorporated by reference in their entirety_ EXAMPLES
[143] The invention will now be further illustrated with reference to the following example(s). It will be appreciated that what follows is by way of example only and that modifications to detail may be made while still falling within the scope of the invention.
Example 1:
Selection of phage for inclusion in PJI composition [144] A composition is designed for the treatment of PJI that may be caused by one or more of S. aureus (MRSA and/or MSSA), S. epidermidis, E. fecalis, S.
caprae, and/or S. lugdunensis. Candidate phage for inclusion in the composition are identified from those 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 phage as listed in Table 1 (above).

Formulation of injectable composition [145] In one particular example, an injectable fluid composition is prepared by combining about loll pfu of each of the following phage, APT-PJI-oi, APT-PJI-13 and APT-PJI-15, in a pharmaceutically acceptable diluent (e.g. isotonic saline).
The composition may be provided in a pre-loaded syringe.
Example 2:
Selection of phage for inclusion in PJI composition [146] The composition of Example 1 may be readily modified by adding to the composition, or replacing one or more phage of the composition with, one or more phage targeted to lyse other causative bacteria of an implantable device infection (such as, for example, PJI) (e.g. S. lugdunensis, E. feciurn, S. agalactiae, P. aeruginosa, K.
pneumoniae, E. coli and E. clocae). Examples of some candidate phage for inclusion in such compositions are identified from those 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 are listed below in Table 2.

APT-14-PCT Final 9 Table 2 Bacterial Phage Name GenBank Bacteria Phage ID code host Comments (HER#)* Accession NO.
HER#*
Siphoviridae, A-like 144 (144) 1144 NC oo1416.1 viruses 30 T7 (30) 1024 Podoviridae, species T7 NC_001604.1 E. coil 552 Ebrios (552) 1024 Podoviridae 241 (1392 (241) 1240 Myoviridae NC o23693 F8 Lindberg (10) 1010 Myoviridae DQ163917 P. aeruginosa 369 PP7 (369) 1369 Leviviridae NC 001628 K. pneumoniae 173 1(13 (173) 1173 Myoviridae, phages Myoviridae, T4-like E. dome 67 1 (67) 1067 viruses ni Cl,ni ni Example 3:
Prophetic case study [147] Two months after receiving a ceramic-on-polyethylene replacement hip, a 55 year old male patient reported to his orthopedic surgeon with considerable pain in the joint. He is referred for a CT scan of the affected joint and this confirms joint distention and indicates infection of the periprosthetic tissues. Blood tests also provide further evidence of PJI. Subsequently, a sample of synovial fluid is drawn from the hip joint by joint aspiration (athrocentesis) and the bacteria present in the fluid is cultured in liquid media. Extracted bacterial DNA is then subjected to sequence analysis of the V1-V3 region of the 16S rRNA gene to identify the bacterial species present using a 16S
rRNA sequence database. The patient's PJI is found to be caused by S. aureus and E. fecalis.
[148] The patient is administered, by direct injection into the affected joint, with a single dose of a therapeutic phage composition including about 109 pfu of each of the APT-PJI-oi (targeted to S. aureus) and APT-PJI-15 (targeted to E. fecalis) phage in a pharmaceutical saline. Subsequently, the patient is treated with intravenous (IV) phage (APT-PJI-oi and/or APT-PJ-15) for 10 days. Following the treatment, the patient is closely monitored for clinical response. After 4 weeks, the patient reports that he had little or no remaining pain. The joint might be aspirate to determine whether any infection remains. A further CT scan may be performed if necessary to determine whether the joint is no longer infected.
[149] The invention is not limited to the embodiment herein before described which may be varied in construction and detail without departing from the spirit of the invention. The entire teachings of any patents, patent applications or other publications referred to herein are incorporated by reference herein as if fully set forth herein.

Claims (25)

What is Claimed

1. A method of treating an infection caused by one or more bacterium associated with a device implanted within a subject, wherein said method comprises:
(a) identifying at least one identified phage capable of infecting the bacterium, wherein said identified phage is selected from a library comprising at least one phage selected from APT-PJI-oi, APT-PJI-02, APT-PJI-03, APT-PM-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-io, APT-PJI-ii, APT-PM-12, APT-PJI-13, APT-PM-14, APT-PA-15, APT-PM-16, APT-PJI-17, APT-PM-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-PM-33, APT-PM-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PM-41, APT-PJI-42, APT-PM-43, APT-PJI-44, APT-PM-45, APT-PM-46, APT-PM-47, APT-PJI-48, APT-PJI-49, and/or APT-PJI-50, or any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage and which is capable of causing lysis of the bacterium; and (b) administering to said subject a therapeutically effective amount of a composition comprising said identified phage, wherein said composition is effective in treating and/or reducing said infection.

2. A method of identifying a subject suffering or at risk of suffering from a bacterial infection, comprising the steps of:
(a) obtaining a biological sample from the subject;
(b) culturing a bacterium present in the biological sample;
(c) inoculating the cultured bacterium with an identified phage selected from a library comprising at least one phage selected from one or more of APT-PJI-oi, 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-io, APT-PJI-ii, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-Pa-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, ?022- 10- 5 APT-PJI-21, APT-PJI-22, APT-PM-23, APT-PM-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PM-31, APT-PJI-32, APT-PJI-33, APT-PM-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PM-40, APT-PM-41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PM-45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PJI-50, and any other lytic phage that has a genomic sequence with at least 70%
sequence identity to the genomic sequence of any of the foregoing phage and which is capable of causing lysis of the bacterium; and (d) determining whether the cultured bacterium are lysed by the identified phage, wherein when any of the cultured bacterium are lysed by the phage, the subject is determined (1) to be eligible for treatment by bacteriophage for said bacterial infection (2) to be suffering from a bacterial infection; and/or (3) at risk of suffering from a bacterial infection.

3. The method of either claim 1 or 2, wherein said composition comprises at least one identified phage selected from the group consisting of APT-PJI-oi, APT-PJI-02, APT-PJI-03, APT-PM-04, APT-PM-05, APT-PJI-06, APT-PM-07, APT-PJI-08, APT-PM-09, APT-PJI-io, APT-PJI-ii, APT-PM-12, APT-PM-13, APT-PJI-14, APT-PM-15, APT-PM-16, APT-PJI-17, APT-PM-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PM-23, APT-PM-24, APT-PM-25, APT-PJI-26, APT-PM-27, APT-PJI-28, APT-PM-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PM-34, APT-PM-35, APT-PJI-36, APT-PM-37, APT-PJI-38, APT-PM-39, APT-PM-40, APT-PJI-41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PM-45, APT-PJI-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PJI-50, or any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage and which is capable of causing lysis of the bacterium.

4. The method of any one of the preceding claims, wherein said composition comprises at least two identified phage selected from the group consisting of APT-PJI-oi, APT-PJI-02, APT-PJI-03, APT-PM-04, APT-PM-05, APT-PJI-06, APT-PM-07, APT-PJI-o 8, APT-PM-09, APT-PM-io, APT-PM-ii, APT-PM-12, APT-PJI-13, APT-PM-14, APT-PM-15, APT-PM-16, APT-PM-17, APT-PM-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PM-23, APT-PM-24, APT-PJI-25, APT-PJI-26, APT-PM-27, APT-PJI-28, APT-PJI-29, APT-PM-30, APT-PM-31, APT-PJI-32, APT-PJI-33, APT-PM-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PM-4o, APT-PM-4i, APT-PM-42, APT-PM-43, APT-PM-44, APT-PM-45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PJI-5o, and any other lytic phage that has a genomic sequence with at least 70%
sequence identity to the genomic sequence of any of the foregoing phage and which is capable of causing lysis of the bacterium.

5.
The method of claim 4, wherein the at least two identified phage are characterized as:
(a) the identified phage cause lysis in the same strain of bacterium;
(b) the identified phage cause lysis in different strains of bacterium;
(c) at least one of the identified phage is selected from the group consisting of APT-PJI-oi, APT-PJI-02, APT-PJI-03, APT-PM-04, APT-PM-05, APT-PJI-06, APT-PM-07, APT-PJI-o 8, APT-PM-09, APT-PM-io, APT-PM-ii, APT-PJI-12, APT-PJI-22, APT-PJI-23, APT-PM-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PM-31, APT-PJI-32, APT-PJI-33, APT-PM-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PM-40, APT-PM-41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PM-45, and APT-PM-46, and any other lytic phage that has a genomic sequence with at least 70%
sequence identity to the genomic sequence of any of the foregoing phage;
(d) at least one of the identified phage is selected from the group consisting of APT-PJI-13, APT-PM-14 and any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage;
(e) at least one of the identified phage is selected from the group consisting of selected from APT-PJI-15, APT-PJI-16, APT-PJI-17, and APT-PJI-18, and any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage;
(f) at least one of the identified phage is selected from the group consisting of selected from APT-PJI-20 and APT-PJI-21 and any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage (g) at least one of the identified phage is selected from the group consisting of APT-PT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PM-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 that has a genomic sequence with at least 70%

sequence identity to the genomic sequence of any of the foregoing phage;
(h) at least one of the identified phage is selected from the group consisting of APT-PJI-47, APT-PM-48, APT-PJI-49, or APT-PJI-50 and any other lytic phage that has a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the foregoing phage, (i) at least one of the identified phage is selected from any of the combination of (a)-(h).

6. The method of any one of the preceding claims, wherein said composition provides a dose of each phage in the range of 105 to 1013 pfu, preferably, wherein said dose of each phage is in the range of 106 to 1012 pfu; 107 to 1011 pfu;
108 to 1011 pfu; 109 tO 1011 pfu; 109 tO 1010 pfu;
or at a dose of approximately 106 pfu, 107 pfu, 108 pfu, 109 pfu, 1010 pfu, 1011 pfu, 1012 pfu, or 1013 pfu.

7. The method of any of the preceding claims, wherein said bacterium is selected from at least one of S. aureus, S. epidermidis, Enterococcus spp., including E.
fecalis and Enterococcus fecium, other coagulase-negative Staphylococcus species, including S. simulans, S. caprae and S. lugdunensis, Streptococcus spp., including Lancefield groups A, B, C and G, S. agalactiae and S. pneumoniae, aerobic gram-negative bacteria including E. coli, other Enterobacteriaceae including Enterobacter clocae, Clostridium spp., Actinomyces spp., Peptostreptococcus spp., Cutibacterium acnes, Klebsiella pneumoniae, P. aeruginosa and Bacteroides fragilis.

8. The method of the preceding claim, wherein the bacterium is selected from two or more different strains of bacterium selected from S. aureus, S. epidermidis, Enterococcus spp., including E. fecalis and Enterococcus fecium, other coagulase-negative Staphylococcus species, including S. simulans, S. caprae and S.
lugdunensis, Streptococcus spp., including Lancefield groups A, B, C and G, S.

agalactiae and S. pneumoniae, aerobic gram-negative bacteria including E.
coli, other Enterobacteriaceae including Enterobacter clocae, Clostridium spp., Actinomyces spp., Peptostreptococcus spp., Cutibacterium acnes, Klebsiella pneumoniae, P. aeruginosa and Bacteroides fragilis.

9. The method of any one of the preceding claims, wherein the bacterium is selected from S. aureus, S. epidermidis, E. fecalis, S. caprae, and/or S. lugdunensis.

10. The method of the preceding claim, wherein the composition comprises at least two phage having different lytic specificities capable of infecting at least two of S.
aureus, S. epidermidis, E. fecalis, S. caprae, and/or S. lugdunensis.

11. The method of the preceding claim, wherein the composition comprises at least three phage haying different lytic specificities, each phage capable of infecting at least one of S. aureus, S. epidermidis, E. fecalis, S. caprae, and/or S.
lug dunensis .

12. The method of any one of the preceding claims, wherein the said other lytic phage has a genomic sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the genomic sequence of any of the foregoing phage.

13. The method of any one of the preceding claims, wherein the composition comprises:
(a) identified phage matched to the strains of bacterium known to be present in a geographic location;

(b) phage selected from APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PM-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PM-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-PM-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49, and/or APT-PJI-50.

14. The method of any one of the preceding claims, wherein the device is:
(a) permanently implanted in the subject;
(b) temporarily implanted in the subject;
(c) removable; and/or (d) is selected from a prosthetic joint, a left-ventricular assist device (LVAD), a stent, a metal rod, an in-dwelling catheter, spinal hardware and/or instrumentation, and/or bone hardware and/or instrumentation.

15. The method of any one of the preceding claims, wherein the infection is selected from: a prosthetic joint infection (PJI), a chronic bacterial infection, an acute bacterial infection, a refractory infection, an infection associated with a biofilm, an infection associated with an implantable device.

16. The method of any one of the preceding claims, wherein the composition is administered:
(a) by IV injection;
(b) by direct injection to the site of infection;
(c) an intra-articular injection;
(d) an IM injection;
(e) prophylactically;
(f) prior to surgery;
(g) in lieu of surgery;
(h) during surgery;
(i) on a single occasion (i.e., as a "single shot"); and/or (j) as a therapeutic course over 2 weeks or more.

'022- 10- 5

17. The method of any one of the preceding claims, wherein lysis is measured by a change in:
(a) growth inhibition;
(b) optical density;
(c) metabolic output;
(d) photometry (e.g., fluorescence, absorption, and transmission assays);
and/or (e) plaque formation.

18. The method of the preceding claim, wherein the change in photometry is measured using an additive that causes and/or enhances the photometric signal detection, preferably wherein said additive is tetrazolium dye.

19. The method of any one of the preceding claims, wherein the biological sample is obtained from:
(a) synovial fluid;
(b) an area surrounding an implantable device;
(c) a site of infection;
(d) an intra-operative sample;
(e) a swab of the device;
(f) a biofflm;
(g) a fistula; and/or (h) an aspiration of a site of infection.

20. The method of any one of the preceding claims, wherein the bacterial infection is:
(a) multi-drug resistant;
(b) clinically refractory to antimicrobial treatment;
(c) clinically refractory to antimicrobial treatment due to biofilm production;
and/or (d) clinically refractory due to the subject's inability to tolerate antimicrobials due to adverse reactions.

21. The method of any one of the preceding claims, wherein the subject is suffering from a hardware related infection.

22. The method of any of the preceding claims, wherein the subject is suffering from a prosthetic joint infection (PJI).

23. The method of any one of the preceding claims, wherein the library comprises phage pre-screened to exclude phage comprising undesirable and/or toxic characteristics.

24. The method of the preceding claim, wherein the excluded phage comprising undesirable and/or toxic characteristics are selected from toxin genes or other bacterial virulence factors, phages which possess lysogenic properties and/or carry lysogeny genes, phages which transduce bacterial virulence factor genes or antibiotic resistance genes, phages which carry any antibiotic-resistance genes or can confer antibiotic resistance to bacterial strains, and phages which elicit an inappropriate immune response and/or provoke a strong allergenic response in a mammalian system.

25. The method of claim 23 or 24, wherein said undesirable and/or toxic characteristics is exclude by genome editing.