Patent Publication Number: US-2022233615-A1

Title: Bacteriophages for the treatment of tuberculosis

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This patent application claims the benefit of U.S. Provisional Patent Application No. 62/850,942, filed May 21, 2019, which is incorporated by reference. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with Government support under Grant Number GM116884 awarded by the National Institutes of Health. The Government has certain rights in this invention. 
    
    
     INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY 
     Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 339,416 Byte ASCII (Text) file named “749004.TXT,” created on May 20, 2020. 
     BACKGROUND OF THE INVENTION 
     Tuberculosis (TB) is a disease that kills over one million people per year, and it is estimated that the causative agent,  Mycobacterium tuberculosis , infects about one-third of the world&#39;s population. TB can be treated with antibiotics, but the treatment regimen requires a minimum of three drugs taken for six months. Non-compliance to drug therapy has fueled the emergence of antibiotic resistance, including multidrug and extensively drug resistant strains of TB (MDR-TB and XDR-TB). 
     Bacteriophages, viruses that infect bacteria, present an alternative potential therapy for TB. However, identification of phages that efficiently infect and kill clinical isolates of  M. tuberculosis  that could be combined into a broadly used phage composition (or “cocktail”) has remained elusive. Thus, there is a global need for new strategies for TB treatment, for shorter therapy regimens, reduced resistance, and treatment of drug resistant strains. A phage cocktail suitable for use as an anti-tuberculosis therapeutic agent is presented herein. 
     BRIEF SUMMARY OF THE INVENTION 
     An embodiment of the invention provides a composition comprising a combination of two or more (e.g., three, four, or five) phages, wherein the phages are two or more of: 
     (a) phage D29; (b) phage AdephagiaΔ41Δ43; (c) phage FionnbharthΔ47; (d) phage Fred313cpm-1; and (e) phage Muddy HRMN0052-1 ; and a pharmaceutically acceptable carrier. Another embodiment of the invention provides a composition comprising (a) phage D29; (b) phage AdephagiaΔ41Δ43; (c) phage FionnbharthΔ47; (d) phage Fred313cpm-1; and (e) phage MuddyHRM N0052-1 ; and a pharmaceutically acceptable carrier. 
     Another embodiment of the invention provides a method for treating, reducing, or preventing a disease caused by  Mycobacterium tuberculosis  in a mammal comprising administering a pharmaceutical composition comprising a combination of two or more phages wherein the phages are two or more of: (a) phage D29; (b) phage AdephagiaΔ41Δ43; (c) phage FionnbharthΔ47; (d) phage Fred313cpm-1; and (e) phage MuddyHRM N0052-1 ; and a pharmaceutically acceptable carrier, thereby treating, reducing, or preventing the disease in the mammal. 
     Additional embodiments of the invention provide methods for treating, reducing, or preventing tuberculosis, tubercular meningitis and disseminated infections, bone and joint tuberculosis, and antibiotic resistant infections in a mammal, comprising administering a composition comprising a combination of two more phages, wherein the phages are two or more of: (a) phage D29; (b) phage AdephagiaΔ41Δ43; (c) phage FionnbharthΔ47; (d) phage Fred313cpm-1; and (e) phage MuddyHRM N0052-1 ; and a pharmaceutically acceptable carrier, either alone or in combination with an antibiotic. The inventive compositions and methods described herein are suitable for mammals including, but not limited to, humans. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         FIGS. 1A and 1B  are pictures of culture plates spotted with bacterial culture with and without the addition of phage. The key to the bacterial strains is shown to the left of  FIGS. 1A and 1B . H37Rv is the lab strain of  M. tuberculosis  tested; all other strains are clinical isolates. 
         FIG. 1A  is a picture of a culture plate showing spots of bacterial cultures placed onto solid media without the addition of any phage. 
         FIG. 1B  is a picture of a plate showing spots of bacterial cultures as shown in  FIG. 1A , after seeding with 10 9  pfu phage D29 and incubating at 37° C. for six weeks. 
         FIGS. 2A and 2B  are pictures of culture plates spotted with bacterial culture with and without the addition of phage. The key to the bacterial strains is overlayed on the culture plates shown in  FIGS. 2A and 2B . H37Rv is the lab strain of  M. tuberculosis  tested; all other strains are clinical isolates. a=N0145, b=N0136, c=N0004, d=N0072, e=N0052, f=N0054, g=N0153, h=H37Rv, i=N1283, j=N0031, k=N1216, l=N0155, m=N1275, and n=N0157. 
         FIG. 2A  is a picture of a culture plate showing spots of bacterial cultures placed onto solid media without the addition of any phage. 
         FIG. 2B  is a picture of a plate showing spots of bacterial cultures as shown in  FIG. 2A , after seeding with 10 9  pfu phage AdephagiaΔ41Δ43 and incubating at 37° C. for six weeks. 
         FIG. 3A  shows a comparison of the genome maps of phage Adephagia (top) and phage Fionnbharth (bottom). The ruler shows the length of the genome from about 0 Kilobase pairs (kb) to about 7 kb of the genome map of phage Adephagia (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 3B  shows a continuation of the genome maps of phage Adephagia (top) and phage Fionnbharth (bottom) shown in  FIG. 3A . The ruler shows the length of the genome from about 7 kb to about 15 kb of the genome map of phage Adephagia (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 3C  shows a continuation of the genome maps of phage Adephagia (top) and phage Fionnbharth (bottom) shown in  FIG. 3B . The ruler shows the length of the genome from about 15 kb to about 21 kb of the genome map of phage Adephagia (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 3D  shows a continuation of the genome maps of phage Adephagia (top) and phage Fionnbharth (bottom) shown in  FIG. 3C . The ruler shows the length of the genome from about 21 kb to about 29 kb. The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 3E  shows a continuation of the genome maps of phage Adephagia (top) and phage Fionnbharth (bottom) shown in  FIG. 3D . The ruler shows the length of the genome from about 29 kb to about 38 kb of the genome map of phage Adephagia (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 3F  shows a continuation of the genome maps of phage Adephagia (top) and phage Fionnbharth (bottom) shown in  FIG. 3E . The ruler shows the length of the genome from about 38 kb to about 46 kb of the genome map of phage Adephagia (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 3G  shows a continuation of the genome maps of phage Adephagia (top) and phage Fionnbharth (bottom) shown in  FIG. 3F . The ruler shows the length of the genome from about 46 kb to about 53 kb of the genome map of phage Adephagia (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 3H  shows a continuation of the genome maps of phage Adephagia (top) and phage Fionnbharth (bottom) shown in  FIG. 3G . The ruler shows the length of the genome from about 53 kb to about 60 kb of the genome map of phage Adephagia (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIGS. 4A and 4B  are pictures of culture plates spotted with bacterial culture with and without the addition of phage. The key to the bacterial strains is noted on the culture plates of  FIGS. 4A and 4B . H37Rv is the lab strain of  M. tuberculosis  tested; all other strains are clinical isolates. a=N0145, b=N0136, c=N0004, d=N0072, e=N0052, f=N0054, g=N0153, h=H37Rv, i=N1283, j=N0031, k=N1216, l=N0155, m=N1275, and n=N0157. 
         FIG. 4A  is a picture of a culture plate showing spots of bacterial cultures placed onto solid media without the addition of any phage. 
         FIG. 4B  is a picture of a culture plate showing spots of bacterial cultures as shown in  FIG. 4A , after seeding with 10 9  pfu phage FionnbharthΔ47 and incubating at 37° C. for six weeks. 
         FIG. 5A  shows a comparison of the genome maps of phage D29 (top) and phage Fred313cpm-1 (bottom). The ruler shows the length of the genome from about 0 kb to about 7 kb of the genome map of phage D29 (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 5B  shows a continuation of the genome maps of phage D29 (top) and phage Fred313cpm-1 (bottom). The ruler shows the length of the genome from about 7 kb to about 13 kb of the genome map of phage D29 (top) shown in  FIG. 5A . The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 5C  shows a continuation of the genome maps of phage D29 (top) and phage Fred313cpm-1 (bottom) shown in  FIG. 5B . The ruler shows the length of the genome from about 13 kb to about 19 kb of the genome map of phage D29 (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 5D  shows a continuation of the genome maps of phage D29 (top) and phage Fred313cpm-1 (bottom) shown in  FIG. 5C . The ruler shows the length of the genome from about 19 kb to about 27 kb of the genome map of phage D29 (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 5E  shows a continuation of the genome maps of phage D29 (top) and phage Fred313cpm-1 (bottom) shown in  FIG. 5D . The ruler shows the length of the genome from about 27 kb to about 35 kb of the genome map of phage D29 (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 5F  shows a continuation of the genome maps of phage D29 (top) and phage Fred313cpm-1 (bottom) shown in  FIG. 5E . The ruler shows the length of the genome from about 35 kb to about 41 kb of the genome map of phage D29 (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIG. 5G  shows a continuation of the genome maps of phage D29 (top) and phage Fred313cpm-1 (bottom) shown in  FIG. 5F . The ruler shows the length of the genome from about 41 kb to about 49 kb of the genome map of phage D29 (top). The shading between the genomes indicates nucleotide sequence similarity with darker shading reflecting closest similarity and the lightest being the least similar above a threshold E value of 10 −4 . 
         FIGS. 6A-6B  are pictures of culture plates spotted with bacterial culture with and without the addition of phage. The key to the bacterial strains is noted on the culture plates of  FIGS. 6A and 6B . H37Rv is the lab strain of  M. tuberculosis  tested; all other strains are clinical isolates. a=N0145, b=N0136, c=N0004, d=N0072, e=N0052, f=N0054, g=N0153, h=H37Rv, i=N1283, j=N0031, k=N1216, l=N0155, m=N1275, and n=N0157. 
         FIG. 6A  is a picture of a culture plate showing spots of bacterial cultures placed onto solid media without the addition of any phage. 
         FIG. 6B  is a picture of a culture plate showing spots of bacterial cultures as shown in  FIG. 6A , after seeding with 10 9  pfu phage Fred313cpm-1 and incubating at 37° C. for six weeks. 
         FIGS. 7A-7G  are pictures of culture plates taken of a plaque assay performed to test phage MuddyHRMN0052 −1 . Host Range Mutants (HRM) of Muddy are designated HRM-1 to HRM-6. Each plate contains two set of strains, as shown. The key to the bacterial strains is noted below each culture plate. H37Rv is the lab strain of  M. tuberculosis  tested; all other strains are clinical isolates. 
         FIG. 7A  is a picture of a plate showing infection of  M. tuberculosis  strains N1283 (clinical isolate) and H37Rv (lab strain) by Muddy HRM derivatives HRM-1-6 after spotting with each derivative and incubating at 37° C. for six weeks. 
         FIG. 7B  is a picture of a plate showing infection of  M. tuberculosis  clinical isolate strains N1274 and N0155 by Muddy HRM derivatives HRM-1-6 after spotting with each derivative and incubating at 37° C. for six weeks. 
         FIG. 7C  is a picture of a plate showing infection of  M. tuberculosis  clinical isolate strains N0145 and N0157 by Muddy HRM derivatives HRM-1-6 after spotting with each derivative and incubating at 37° C. for six weeks. 
         FIG. 7D  is a picture of a plate showing infection of  M. tuberculosis  clinical isolate strains N0052 and N0072 by Muddy HRM derivatives HRM-1-6 after spotting with each derivative and incubating at 37° C. for six weeks. 
         FIG. 7E  is a picture of a plate showing infection of  M. tuberculosis  clinical isolate strains N0053 and N0054 by Muddy HRM derivatives HRM-1-6 after spotting with each derivative and incubating at 37° C. for six weeks. 
         FIG. 7F  is a picture of a plate showing infection of  M. tuberculosis  clinical isolate strains N0004 and N0136 by Muddy HRM derivatives HRM-1-6 after spotting with each derivative and incubating at 37° C. for six weeks. 
         FIG. 7G  is a picture of a plate showing infection of  M. tuberculosis  clinical isolate strains N1216 and N0031 by Muddy HRM derivatives HRM-1-6 after spotting with each derivative and incubating at 37° C. for six weeks. 
         FIG. 8  is a schematic showing the geographical distribution of  M. tuberculosis  strains. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     An embodiment of the invention provides a composition comprising a combination of two more phages, wherein the phages are two or more of: 
     (a) phage D29; (b) phage AdephagiaΔ41Δ43; (c) phage FionnbharthΔ47; (d) phage Fred313cpm-1; and (e) phage MuddyHRM N0052-1 ; and a pharmaceutically acceptable carrier. 
     The pharmaceutically acceptable carrier for use in the inventive pharmaceutical composition can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The pharmaceutically acceptable carriers for use in the present invention—for example, vehicles, excipients, and diluents—are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) (i.e., the two or more phages) and one which has no detrimental side effects or toxicity under the conditions of use. The choice of carrier will be determined in part by the particular phages used in the pharmaceutical composition, as well as by the particular method used to administer the two or more phages. 
     The two or more phages for use in the inventive pharmaceutical composition can be any phages which infect a bacterial host, i.e., bacteriophages, which infect  Mycobacterium tuberculosis  ( M. tuberculosis ) bacteria.  M. tuberculosis  is the causative agent of tuberculosis (TB), and other diseases including, but not limited to tubercular meningitis and disseminated infections and bone and joint tuberculosis. Infection of  M. tuberculosis  by the two or more phages causes the biological activity of  M. tuberculosis  to be inhibited, as compared to activity which is observed in the absence of infection by the two or more phages. An embodiment of the invention provides a composition comprising (a) phage D29; (b) phage AdephagiaΔ41Δ43; (c) phage FionnbharthΔ47; (d) phage Fred313cpm-1; and (e) phage MuddyHRM N0052-1 ; and a pharmaceutically acceptable carrier. 
     TB is the cause of death of over a million people per year worldwide, and it is estimated that about one-third of the world&#39;s population are infected by  M. tuberculosis. M. tuberculosis  encompasses limited genetic diversity, and genomic comparisons identify seven major lineages (L1-L7), of which lineages L1-L4 predominate, and span much of the diversity, represent a vast majority of the isolates, and are most geographically widespread ( FIG. 8 , Gagneaux et al.,  Nature Reviews Microbiology , Vol. 16, 202-213 (2018), which is herein incorporated by reference in its entirety). 
     In an embodiment, the invention provides a method of treating, reducing, or preventing a disease caused by  M. tuberculosis  in a mammal comprising administering a pharmaceutical composition comprising a combination of two or more phages and (2) a pharmaceutically acceptable carrier, thereby treating, reducing, or preventing the disease in the mammal. In an embodiment, the invention provides a method of treating, reducing, or preventing tuberculosis in a mammal comprising administering the inventive pharmaceutical composition, thereby treating, reducing, or preventing tuberculosis in the mammal. In an additional embodiment, the invention provides a method of treating, reducing, or preventing tubercular meningitis and disseminated infections, and bone and joint tuberculosis in a mammal comprising administering the inventive composition, thereby treating, reducing, or preventing tubercular meningitis and disseminated infections, and bone and joint tuberculosis, in the mammal. 
     Bacteriophages (which may be referred to as “phages”) are viruses that infect bacterial hosts. As used herein, the term bacteriophage may refer to any bacteriophage or product thereof, including, for example, polypeptides, fragments, variants, or derivatives thereof, including those with genomic insertions, deletions, or any other modifications, isolated from a bacteriophage of the invention, or related to a bacteriophage of the invention, or which has similar properties as a bacteriophage of the invention. The bacteriophages, polypeptide, fragments, variants, and derivatives of the invention exhibit a biological function or activity associated with the bacteriophage from which it was isolated or derived (e.g., antimicrobial or antibacterial activity (e.g., lytic cell killing)). 
     Bacteriophages may serve as therapeutic agents. Phage therapy may include (1) a personalized system in which phages are identified that infect an infectious strain in one specific patient, and (2) a broad-based therapy that can be used to generally treat infections by a particular pathogen. Both approaches suffer from obstacles, including, for example, that clinical isolates of any particular bacterial pathogen can be extremely varied, especially in their phage susceptibility profiles. Phages are often highly specific for their hosts, and often to do not infect all clinical variants. Additionally, phages in laboratory stocks may become unstable over time. 
     Tuberculosis is an attractive target for the therapeutic use of phages. There is a strong clinical need for TB treatment because of widespread infections worldwide and the prevalence of antibiotic resistant strains. Additionally,  M. tuberculosis  clinical isolates are more genetically homogenous than most other bacterial pathogens (Gagneaux et al.,  Nature Reviews Microbiology , Vol. 16, 202-213 (2018), which is herein incorporated by reference in its entirety), increasing the prospects of identifying phages whose infectibility spans most clinical strains. Further, mycobacteria do not contain lipopolysaccharide (LPS), which is highly toxic, and therefore a contaminant of certain phage preparations. 
     Phages for use in the present invention can be obtained in the field, or from any collection of individual bacteriophage isolates. Bacteriophages which are suitable for the inventive composition and methods comprise any bacteriophage, polypeptide, or fragment, variant, or derivative thereof, including those with genomic insertions, deletions, or any other modification, collected from the field, or from any accessible collection of individual bacteriophage isolates, such as those provided herein. An exemplary collection of bacteriophage isolates was collected in the large multi-institutional Science Education Alliance Phage Hunters Advancing Research and Education (SEA-PHAGES) program. The SEA-PHAGES program collection includes 10,000 bacteriophage isolates which were isolated using  Mycobacterium smegmatis  mc2155 as a host. The genomes of a portion of the isolates have been sequenced. The sequences may be accessed at the website for the Actinobacteriophage Database, phagesdb.org. Additional bacteriophage sequences and strains which are suitable for use in the inventive compounds and methods are available from the American Type Culture Collection (ATCC), which may be accessed at the website for ATCC, atcc.org. Additional bacteriophage sequences and strains which are suitable for use in the inventive compounds and methods are archived at the University of Pittsburgh, Department of Biological Sciences (Pittsburgh, Pa.). 
     Previously, a subset of the isolates collected in the SEA-PHAGES program were tested to determine which will efficiently infect  M. tuberculosis  mc27000 (an avirulent derivative of the lab strain  M. tuberculosis  H37Rv), and it was shown that many representative phages of clusters/subclusters A2, A3, and K infect this strain (Jacobs-Sera et al.,  Virology,  20; 434(2): 187-201 (December 2012), which is herein incorporated by reference in its entirety). It was also previously shown that although some phages do not efficiently infect  M. tuberculosis , that some infect at a reduced plating efficiency, and that mutants can be readily isolated that plate with similar efficiencies on  M. tuberculosis  mc27000 and  M. smegmatis  mc 2 155. 
     Phages infecting actinobacterial hosts can be grouped into clusters according to their sequence relationships, and some clusters can be further divided into subclusters. The largest cluster is Cluster A, which is divided into 20 subclusters (A1, A2, A3, etc). Because the overall sequence diversity is large, phages within either clusters or subclusters may differ in a substantial number of their genes. Phages that are genomically different from each other are more likely to differ in their overall biology than phages grouped in the same cluster, and those that differ genomically are the least likely to share resistance mechanisms. Accordingly, when selecting phages for therapeutic use, more than one phage is used in combination (i.e., a therapeutic phage “cocktail”), and the phages should be selected such that resistance to one phage does not confer resistance to other phages in the cocktail. Therefore, it is advisable to use phages that span considerable genomic diversity. 
     Exemplary phages include, but are not limited to: phage D29, phage AdephagiaΔ41Δ43, phage FionnbharthΔ47, phage Fred313cpm-1, and phage MuddyHRM N0052-1  and any product thereof, including, for example, polypeptides, or fragments, variants, or derivatives thereof, including those with genomic insertions, deletions, or any other modification. Further exemplary phages are those isolated from, related to, or similar to phage D29, phage AdephagiaΔ41Δ43, phage FionnbharthΔ47, phage Fred313cpm-1, and phage MuddyHRM N0052-1 . Such phages replicate lytically, either naturally or though engineering, have broad host ranges among  M. tuberculosis  clinical isolates, and diversity which suggests that they are unlikely to share common resistance mechanisms. Additional descriptive information is provided below. 
     Phage D29 (SEQ ID NO: 1). Phage D29 is known to infect  M. tuberculosis . (Jacobs-Sera et al.,  Virology,  20; 434(2): 187-201 (December 2012), which is herein incorporated by reference in its entirety). D29 is grouped through genomic comparisons into Subcluster A2 (Hatfull et al.,  J. Mol. Biol.,  397(1): 119-143, (Mar. 19, 2010), which is herein incorporated by reference in its entirety). D29 is a lytic phage and does not form lysogens, although genomic analysis shows that it is a derivative of a temperate parent (Ford et al.,  J. Mol. Biol.,  279: 143-164 (1998); Dedrick et al.,  BMC Microbiology  17:225 (2017), which are herein incorporated by reference in their entirety). 
     Phage AdephagiaΔ41Δ43 (SEQ ID NO: 2). Mycobacteriophage Adephagia was isolated as part of the SEA-PHAGES program and is archived at the University of Pittsburgh, Department of Biological Sciences (Pittsburgh, Pa.). Genomic characterization showed that Adephagia can be grouped into Subcluster K1 (Pope et al.,  PLoS ONE  6(10): e26750 (2011), which is herein incorporated by reference in its entirety). Adephagia is a temperate phage, but its repressor gene has been identified and a deletion derivative was constructed (Adephagia Δ43) in which the repressor gene is deleted (Petrova et al.,  Microbiology,  161(Pt 8): 1539-1551 (August 2015), which is herein incorporated by reference in its entirety). A second derivative was constructed (Adephagia Δ41Δ43) in which the integrase gene was also deleted (Petrova et al., 2015). Adephagia was shown to infect  M. tuberculosis  mc 2 7000, a property shared by many other Cluster K phages. 
     Phage FionnbharthΔ47(SEQ ID NO: 3). Phage Fionnbharth was isolated as part of the SEA-PHAGES program. The genome was sequenced and the phage is grouped in Subcluster K4 (Pope et al.,  PLoS ONE  6(10): e26750 (2011), which is herein incorporated by reference in its entirety). Fionnbharth efficiently infects  M. tuberculosis  mc 2 7000 (Jacobs-Sera et al.,  Virology,  20; 434(2): 187-201 (December 2012), which is herein incorporated by reference in its entirety). Although Adephagia and Fionnbharth are in the same cluster (Cluster K), they differ in many parts of their genomes ( FIGS. 3A-3H ). Without wishing to be bound by theory, it is plausible that resistance to one may not necessarily confer resistance to the other. Preliminary experiments with  Mycobacterium smegmatis  mc 2 155 as a model system suggest that resistance to one of these phages may not confer resistance to the other. 
     Phage Fred313cpm-1 (SEQ ID NO: 4). Phage Fred313 was isolated as part of the SEA-PHAGES program. The genome was sequenced and the phage is grouped in Subcluster A3 (Butela et al.,  Genome Announcements,  5(43) e01182-17 (October 2017) DOI: 10.1128/genomeA.01182-17 2017, which is herein incorporated by reference in its entirety). Fred313 is a different genomic subcluster to D29 (which is in subcluster A2). Without wishing to be bound by theory, Fred313 and D29 have sufficient differences (including different tail genes) such that they may have distinct resistance profiles ( FIGS. 5A-5G ). 
     Phage MuddyHRM N0052-1  (SEQ ID NO: 5). Phage Muddy was isolated is archived at the University of Pittsburgh, Department of Biological Sciences (Pittsburgh, Pa.), and is one of only two members of Cluster AB; its genome sequence is known (Pope et al.,  PLoS ONE  6(10): e26750 (2011), which is herein incorporated by reference in its entirety). Muddy infects  M. tuberculosis  mc 2 7000. 
     All of the phages that infect mycobacterial hosts are morphologically siphoviral, and over 50% are temperate, forming stable lysogens that are immune to superinfection (Hatfull,  Microbiol Spectr.,  6(5): 10.1128/microbiolspec.GPP3-0026-(October 2018), which is herein incorporated by reference in its entirety). Temperate phages are not suitable for therapeutic use because temperate phages have the ability to integrate their DNA into the bacterial host genome (i.e., they are lysogens). Lysogens is not useful for phage therapy because once the genome is integrated, the bacterial cell becomes immune to other similar phages. Lytic derivatives in which the repressor gene has been altered or deleted are suitable for phage therapy, and can be isolated or engineered using BRED technology (Marinelli et al.,  FEMS Microbiology Letters,  344(2), 166-172 (July 2013), which is herein incorporated by reference in its entirety). 
     All of the phages known to infect  M. tuberculosis  have genomes with defined termini and thus use a cos-type DNA packaging system. Such phages usually do not mediate generalized transduction at any readily detectable frequency. Avoiding generalized transduction is a desirable property for a therapeutic phage. Additionally, genomic characterization of the phages that infect  M. tuberculosis  has not yet identified toxic genes such as Shiga-like toxins. 
     TB can be treated with antibiotics, however, current therapeutic regimens require a minimum of three drugs taken for six months. Non-compliance with therapy has fueled the emergence of antibiotic resistance, including multidrug and extensively drug resistant strains (e.g., MDR-TB and XDR-TB). Accordingly, antibiotic resistance is a global concern for the treatment of  M. tuberculosis  infections. Antibiotic drugs typically used for TB therapy include, e.g., isoniazid, ethambutol, pyrazinamide, rifampicin, streptomycin, amikacin, kanamycin, ciprofloxacin, delamanid, and bedaquiline or any combination thereof. Resistance occurs independently within  M. tuberculosis  lineages, and typically involves mutations within target genes for the antibiotics used. For example, resistance to rifampicin is solely due to rpoB mutations. Therefore, without wishing to be bound by theory, there is not anticipated to be systematic differences in phage susceptibility related to drug resistance profiles. Further without wishing to be bound, it is believed that using the inventive composition in combination with antibiotics such as those listed above, as a treatment for diseases caused by  M. tuberculosis  and related antibiotic resistant infections, may reduce the current treatment time of six months, and may also reduce the incidence of antibiotic resistance. 
     In an embodiment, the invention provides a method of treating, reducing, or preventing a disease caused by  M. tuberculosis , tuberculosis, tubercular meningitis and disseminated infections, and bone and joint tuberculosis, and antibiotic resistant infections such as pulmonary tuberculosis, comprising administering a pharmaceutical composition comprising a combination of two or more (e.g., three, four, or five) phages comprising two or more of: (a) phage D29; (b) phage AdephagiaΔ41Δ43; (c) phage FionnbharthΔ47; (d) phage Fred313cpm-1; and (e) phage MuddyHRM N0052-1 , in combination with an antibiotic. Suitable antibiotics for use in combination with the inventive composition include, but are not limited to: isoniazid, ethambutol, pyrazinamide, rifampicin, streptomycin, amikacin, kanamycin, ciprofloxacin, delamanid, and bedaquiline, or any combination thereof. Without wishing to be bound, it is believed that treatment of the diseases listed herein, with the inventive composition in combination with one or more antibiotics, would reduce the total length of treatment, as compared to treatment with antibiotics in the absence of the inventive composition. 
     In this respect, the length of time for a typical therapeutic regimen for the treatment of TB with one or more antibiotics, in the absence of the inventive composition, is about six months, as noted above. In an embodiment, the invention provides a method of treating, reducing, or preventing a disease caused by  M. tuberculosis , tuberculosis, tubercular meningitis and disseminated infections, bone and joint tuberculosis, and antibiotic resistant infections such as pulmonary tuberculosis, comprising administering a pharmaceutical composition comprising a combination of two or more phages comprising two more of: (a) phage D29; (b) phage AdephagiaΔ41Δ43; (c) phage FionnbharthΔ47; (d) phage Fred313cpm-1; and (e) phage MuddyHRM N0052-1 , in combination with an antibiotic, wherein the length of treatment is reduced as compared to the length of treatment with an antibiotic alone. In embodiments, the length of the inventive therapeutic method may comprise, 6 months, 5 months, 4 months, 3 months, 2 months, or one month. 
     As noted above, about one-third of the world is infected with  M. tuberculosis , although a large percentage of carriers have latent (asymptomatic), not active, TB. In latent TB, the bacterium establishes itself in the body at relatively small numbers, but are never cleared from the carrier&#39;s body. There is a 10% chance of activation of latent TB infection into an active TB infection over the course of a TB carrier&#39;s lifetime. Patients suffering from immunocompromising conditions such as e.g., AIDS or HIV, or who take immunocompromising drugs, have an increased chance that such latent infections will activate into TB disease. Without wishing to be bound by theory, it is plausible that the inventive bacteriophage composition described herein could be used to treat latent  M. tuberculosis  infections by eliminating the bacteria from latent infections and thus eliminating the risks arising from the potential for activation. 
     In an embodiment, the invention provides a method of treating, reducing, or preventing activation of a latent disease caused by  M. tuberculosis , comprising administering a pharmaceutical composition comprising a combination of two or more phages comprising two more of: (a) phage D29; (b) phage AdephagiaΔ41Δ43; (c) phage FionnbharthΔ47; (d) phage Fred313cpm-1; and (e) phage MuddyHRM N0052-1 , 
     The terms “treat,” and “prevent” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive compositions and methods can provide any amount of any level of treatment or prevention of disease caused by  M. tuberculosis  in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease caused by  M. tuberculosis , e.g., tuberculosis, being treated or prevented. Also, for purposes herein, “prevention” can encompass delaying the onset of a disease caused by  M. tuberculosis , or a symptom or condition thereof. With respect to the inventive methods, the disease may be any disease caused by  M. tuberculosis , including any of the types of diseases caused by or associated with  M. tuberculosis  and any of the conditions or treatments discussed herein. 
     For purposes of the invention, the amount or dose of the inventive composition administered should be sufficient to effect the desired biological response, e.g., a therapeutic or prophylactic response, in the mammal over a clinically reasonable time frame. The dose will be determined by the efficacy of the particular composition and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated. The dose of the inventive composition also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular phage or combination thereof. 
     Typically, the attending physician will decide the dosage of the inventive composition with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, composition to be administered, route of administration, and the severity of the condition being treated. The dose, or dosage, of a pharmaceutical composition of the present invention may be appropriately determined by considering the dosage form, method of administration, patient age and body weight, symptoms of the patient, or severity of the condition. 
     Generally, the daily dose for an adult can be, e.g., between 0.1 ml to 10,000 ml at once or in several portions. In embodiments, the dose comprises between 1×10 1  and 1×10 9  plaque forming units (pfu) per dose (e.g., 1×10 1  pfu, 1×10 2  pfu, 1×10 3  pfu, 1×10 4  pfu, 1×10 5  pfu, 1×10 6  pfu, 1×10 7  pfu, 1×10 8  pfu, 1×10 9  pfu, 1×10 10  pfu, or any ranges therebetween) of bacteriophage. These examples are not limiting. These doses, or dosages, may vary, depending on the patient body weight and age, and the method of administration; however, selection of suitable dose, or dosage, is well within the purview of those skilled in the art. 
     Similarly, the dosing period may be appropriately determined depending on the therapeutic progress. In embodiments, the dosing period may comprise less than one year, less than 9 months, less than 8 months, less than 7 months, less than 6 months, less than 5 months, less than 4 months, less than 3 months, less than 2 months, or one month. In embodiments, the dosing period may comprise three doses per day, two doses per day, or one dose per day for the length of the dosing period. 
     As used herein, the term “mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swine (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human. The mammal can be non-diseased, a mammal afflicted with a disease caused by  M. tuberculosis , or a mammal predisposed to having a disease caused by  M. tuberculosis.    
     In an embodiment, the invention provides administering the inventive composition comprising a combination of two more phages, wherein the phages are two or more of: (a) phage D29; (b) phage AdephagiaΔ41Δ43; (c) phage FionnbharthΔ47; (d) phage Fred313cpm-1; and (e) phage MuddyHRM N0052-1  to the mammal intravenously, intramuscularly, subcutaneously, or intraperitoneally, or as an aerosol, using an inhalation device. The following formulations for administration are exemplary and are in no way limiting. More than one route can be used to administer the inventive composition, and in certain instances, a particular route can provide a more immediate and more effective response than another route. 
     Intravenous, intramuscular, subcutaneous, or intraperitoneal formulations may include any suitable carrier. For example, formulations suitable for intravenous e.g., as a bolus or by continuous infusion over a period of time, intramuscular, subcutaneous, or intraperitoneal administration may comprise sterile aqueous solutions of the inventive composition with solutions which are preferably isotonic with the blood of the recipient. Such formulations may be prepared by dissolving the inventive composition in water or other suitable physiologically acceptable solvent containing physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine, and the like, and having a buffered pH compatible with physiological conditions to produce solution (e.g., an aqueous solution or solution of the inventive composition (i.e., bacteriophages) in a suitable physiologically acceptable solvent), and rendering said solution sterile. 
     Alternatively and additionally, an effective amount of the pharmaceutical composition described herein, via an inhalation route. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, the inventive compositions described herein can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder. 
     The following example further illustrates the invention but, of course, should not be construed as in any way limiting its scope. 
     Example 1 
     This example demonstrates the process for identifying the genomically distinct phages that infect and kill all of the tested  M. tuberculosis  isolates tested. 
     A set of clinical isolates of  M. tuberculosis  for testing phage susceptibility profiles was obtained (Table 1) from the Department of Immunology and Infectious Diseases, School of Public Health TB Research Program, Harvard University (Boston, Mass.). The set of strains provided in Table 1 contains 2-4 isolates of each of the six lineages (L1-L6). All of the strains were cultured for testing, although several strains (N1176, N1063, N1272, N0091, N1202, and N1177) representing lineages L6 and L7 have not yet been propagated. Identification of phages that broadly infect the L1-L4 lineages provides a resource for evaluating the clinical utility of phage interventions for TB. Lab strain H37Rv, a member of L4, was also tested. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   
                   
                 Country of 
                 Place of birth 
               
               
                   
                 Strain 
                 Lineage 
                 Isolation 
                 of patient 
               
               
                   
                   
               
             
            
               
                   
                 N0157 
                 L1 
                 USA 
                 Philippines 
               
               
                   
                 N0072 
                 L1 
                 USA 
                 India 
               
               
                   
                 N0153 
                 L1 
                 USA 
                 Vietnam 
               
               
                   
                 N0145 
                 L2 
                 USA 
                 China 
               
               
                   
                 N0052 
                 L2 
                 USA 
                 China 
               
               
                   
                 N0031 
                 L2 
                 USA 
                 China 
               
               
                   
                 N0155 
                 L2 
                 USA 
                 China 
               
               
                   
                 N0004 
                 L3 
                 USA 
                 India 
               
               
                   
                 N1274 
                 L3 
                 Germany 
                 Afghanistan 
               
               
                   
                 N0054 
                 L3 
                 USA 
                 Ethiopia 
               
               
                   
                 N1216 
                 L4 
                 Ghana 
                 Ghana 
               
               
                   
                 N0136 
                 L4 
                 USA 
                 USA 
               
               
                   
                 N1283 
                 L4 
                 Germany 
                 Germany 
               
               
                   
                 N1176 
                 L5 
                 Ghana 
                 Ghana 
               
               
                   
                 N1063 
                 L5 
                 Switzerland 
                 Liberia 
               
               
                   
                 N1272 
                 L5 
                 Ghana 
                 Ghana 
               
               
                   
                 N0091 
                 L6 
                 The Gambia 
                 Gambia 
               
               
                   
                 N1202 
                 L6 
                 Ghana 
                 Ghana 
               
               
                   
                   
               
            
           
         
       
     
     To identify phages that infect these strains, the focus of testing was on the types of phages that were previously shown to infect  M. tuberculosis  mc 2 7000 (Jacobs-Sera et al.,  Virology,  20; 434(2): 187-201 (December 2012), which is herein incorporated by reference in its entirety), together with a screen for additional phages. These phages were examined in further detail to identify those that infect the clinical isolates. The resulting phages were then further manipulated as appropriate, either to convert from temperate to lytic phages, or to mutationally expand their host range. The goal was to identify 4-5 genomically distinct phages that infect and kill all of the isolates tested. The methods and resulting phages are described below. 
     Methods 
     The following methods were used for each phage described below. 
     A lab strain of  M. tuberculosis  H37Rv, as well as the isolates listed in Table 1, were grown in Middlebrook 7H9 media with OADC (oleic acid, dextrose, catalase), and 0.05% Tween80 for 3-6 weeks at 37° C., with shaking. These  M. tuberculosis  strains grow with a doubling time of approximately 24 hours, with isolated colonies visible on solid medium in 4-6 weeks. For plaque assays,  M. tuberculosis  cultures were sonicated briefly in a cup-horn sonicator (Q-sonica 700, Qsonica, Newtown, Conn.) at 30% amplitude with 15 sec on and 10 sec off until visibly dispersed. 
     These studies confirmed several general features among these phages: (1) some of the phages infect only some of the clinical isolates, (2) some phages infect all or most of the clinical isolates; (3) some of the phages do not efficiently infect the isolates, but host range mutants can be isolated that do (e.g. Muddy, described below), (4) some of the host range mutants infect all of the clinical isolates, but some do not (e.g. different mutants of phage Muddy, described below), and (5) many of the phages that infect  M. tuberculosis  are temperate, but lytic derivatives can be identified or constructed (e.g. Fionnbharth and Fred313cpm-1, described below). The five bacteriophages described herein were identified as candidates for components of a therapeutic cocktail. 
     Phage D29 
     Thirteen  M. tuberculosis  clinical isolates (Table 1) as well as the lab strain  M. tuberculosis  H37Rv were grown and spotted onto solid media seeded with 10 9  plaque forming units of phage D29 (SEQ ID NO: 1) ( FIG. 1B ), or without phage ( FIG. 1A ), and grown at 37° C. for three weeks. As shown, Phage D29 prevented growth of all of the  M. tuberculosis  strains. 
     Phage AdephagiaΔ41Δ43 
       M. tuberculosis  strains were plated on solid media. One plate was grown without phage ( FIG. 2B ), and the other was seeded with 10 9  pfu phage AdephagiaΔ41Δ43 (SEQ ID NO: 2), and efficient killing was observed ( FIG. 2B ). Specifically, four 100-fold serial dilutions of bacterial cultures were spotted (right to left) and the plates incubated at 37° C. for six weeks. For some strains (e.g. N1274,  FIG. 2B ) some residual spots were observed, although they did not appear to continue to grow after prolonged incubation. Some colonies of strain N0136 were observed that may correspond to resistant mutants ( FIG. 2B ). 
     Phage FionnbharthΔ47 
     As noted above, Fionnbharth is a temperate phage and thus not suitable for therapeutic use as-is. Thus, BRED engineering technology was used (Marinelli et al.,  FEMS Microbiology Letters,  344(2), 166-172 (July 2013), which is herein incorporated by reference in its entirety) to construct a derivative in which the repressor gene (47) is deleted (SEQ ID NO: 3). Four 100-fold serial dilutions of bacterial cultures were spotted (right to left,  FIGS. 4A and 4B ) and the plates were incubated at 37° C. for six weeks. As shown in  FIGS. 4A  (no phage) and  4 B, this lytic derivative (FionnbharthΔ47) efficiently kills  M. tuberculosis  clinical isolates. 
     Phage Fred313cpm-1 
     Fred313 is a temperate phage and forms turbid plaques on lawns of  M. smegmatis  mc 2 155. To develop a lytic derivative, a clear plaque variant (Fred313cpm-1), was isolated, purified, and sequenced (SEQ ID NO: 4). Fred313cpm-1 contains a 2,904 bp deletion (coordinates 43,561-46,464) that removes all of the repressor gene (71). Fred313cpm-1 also contains single nucleotide polymorphisms at G2278A, G22996A and 48934ΔA that are likely not related to the clear plaque phenotype. 
     Survival of  M. tuberculosis  on solid media seeded with 10 9  pfu phage Fred313cpm-1 is shown in  FIG. 6B  ( M. tuberculosis  without phage is shown in  FIG. 6A ). Four 100-fold serial dilutions of bacterial cultures were spotted (right to left) and the plates were incubated at 37° C. for six weeks.  FIG. 6B , as compared to  FIG. 6A , show that phage Fred313cpm-1 kills all tested strains efficiently. 
     Phage #5: MuddyHRM N0052 -1 
     Muddy is known to infect  M. tuberculosis  mc 2 7000 and therefore it was tested on the  M. tuberculosis  clinical isolates listed in Table 1. Reduced efficiencies of plating on all or most of the strains tested was observed. To recover derivatives with clinical utility, phage particles were recovered from plaques observed at low frequency on each of the other strains (designated HRM-1-HRM-6). 
     HRM-1 contains a DNA substitution of a G to an A at coordinate 21643 (i.e. G21643A). The mutation lies within Muddy gene 24, and confers a substitution of a glutamic acid residue with a lysine residue as position 680 of the protein (i.e. E680K). 
     HRM-6 likely is a mixture of two mutants. The first mutant has a DNA substitution of a G to a T at coordinate 21064 (i.e. G21064T). The mutation lies within Muddy gene 24, and confers a substitution of a glycine residue with a tryptophan residue as position 487 of the protein (i.e. G487W). The second mutant has a DNA substitution of an A to an G at coordinate 21427 (i.e. A21427G). The mutation lies within Muddy gene 24, and confers a substitution of an alanine residue with a glycine residue as position 608 of the protein (i.e. T608A). 
     While not wishing to be bound by any particular theory, the mutations in gene 24 are believed to expand the host range of Muddy to include other clinical isolates of  M. tuberculosis.    
     These were propagated on  M. smegmatis  mc 2 155, and tested in a plaque assay on the other strains. The results are shown in  FIGS. 7A-7G . Each plate contained two set of strains. Muddy HRM recovered from different  M. tuberculosis  hosts were serially diluted 10 2 -fold and added to cells as an overlay on solid media. Muddy HRM-1 and HRM-6 infect all strains; HRM-5 does not infect strain N0155 ( FIG. 7B ). It was found that some of the HRM derivatives of Muddy infect some but not all of the strains. For example, HRM-5, which was recovered by its ability to form a plaque on strain N0072 (lineage-1) does not infect strain N0155 ( FIG. 7B ). In contrast, HRM-1 and HRM-6, which were recovered from strains N0157 ( FIG. 7C ) and N0052 ( FIG. 7D ), respectively, infect all of the strains tested. These derivatives represent good candidates for therapeutic evaluation. 
     This example demonstrates the process for identifying the genomically distinct phages that infect and kill all of the  M. tuberculosis  isolates tested. These phages are good candidates for inclusion in a phage cocktail for the treatment of diseases caused by  M. tuberculosis.    
     All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. 
     The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. 
     Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.