Method for bacteriophage production

Abstract

Provided is a method for infecting a bacterium, in which a host bacterium is exposed to a phage depolymerase and a bacteriophage. The depolymerase catalyzes degradation of at least one cell-surface or extracellular polysaccharide of the host bacterium, thereby increasing the susceptibility of the host bacterium to infection by the bacteriophage. The method may be used to increase production of the bacteriophage and / or to increase the host range of the bacteriophage. Further provided is use of a phage depolymerase selected from B5, As11, TaPaz, APK2, APK37, and IME200, or a composition comprising the same, for increasing the susceptibility of an A. baumannii to infection by a bacteriophage. The A. baumannii may be an AB030, MRSN 843, MRSN 1183, MRSN 7446, MRSN 15088, MRSN 24008, MRSN 31159, MRSN 31468, or MRSN 32866 strain.

Description

METHOD FOR BACTERIOPHAGE PRODUCTIONFIELD

[0001] The present disclosure relates to the fields of bacteriophage production and phage therapy. Phage therapy may be used to treat bacterial infections, including antibiotic-resistant bacterial infections.BACKGROUND

[0002] Antibiotic resistance is a growing problem worldwide, as bacteria with resistance to multiple antibiotics become increasingly common. New antibiotics are being developed, but researchers are also seeking additional ways to treat bacterial infections, which could be used independently or in combination with antibiotic therapy. One such approach is phage therapy, which involves the use of bacteriophages (also referred to as phages), which are viruses that infect only bacteria, to kill bacterial cells. As discussed in Strathdee et al., 2023, phage therapy was widely used to treat bacterial infections in the 1930s, before penicillin was brought to the market. However, interest in phage therapy waned after antibiotics became widely available, so this technology remains underdeveloped.

[0003] One disadvantage of phage therapy, relative to antibiotic therapy, is host specificity. While antibiotics can offer broad-spectrum killing, phages generally have a limited host range. Some phages are able to infect multiple species within a genus, and some can infect species from different genera, but others have a narrow host range and can infect only single species, or even a low number of isolates from within a single species. A related challenge is the ability to produce phages. Due to host specificity, the production of any particular phage may require a unique bacterial host. This poses logistical challenges to the production of a wide variety of bacteriophages.

[0004] Depolymerases are enzymes produced by some bacteriophages that can break down (degrade) polysaccharides produced by host bacteria, thereby removing a physical barrier to access to host receptors. These polysaccharides can be capsule polysaccharides (CPS), which are attached to the cell surface, or extracellular polysaccharides (EPS), which are secreted into the environment where they may form a protective barrier around the bacterium. CPS and EPS are involved in a wide range of bacterial survival strategies, including immune response evasion, antibiotic resistance, and phage resistance. Examples of CPS-producing bacteria include Acinetobacter baumannii, Streptococcus pneumoniae, Klebsiella pneumoniae, K-antigen producing Escherichia coli, and Neisseria meningitidis. Examples of EPS-producing bacteria include Pseudomonas aeruginosa, These CPS- and EPS-producing bacteria are responsible for significant deaths, and in the case of X. campestris, economic losses associated with crop destruction. Treatment of these bacteria can often be difficult due to antimicrobial resistance.SUMMARY

[0005] The present inventors have found that exposing a bacterium, such as A. baumannii, to a phage depolymerase targeting its capsule type can render the bacterium sensitive to phages to which it was previously resistant, while also improving phage production by the bacterium (increased phage titer).

[0006] Accordingly, there is provided a method for producing a bacteriophage, comprising: a) exposing a host bacterium to a phage depolymerase under conditions that allow the depolymerase to catalyze degradation of at least one cell-surface or extracellular polysaccharide of the host bacterium, thereby increasing the susceptibility of the host bacterium to infection by the bacteriophage, relative to the susceptibility of a control host bacterium that has not been exposed to the depolymerase; b) exposing the host bacterium to the bacteriophage under conditions that allow the bacteriophage to infect the host bacterium; and c) allowing the bacteriophage to replicate within the host bacterium. In an embodiment, the method further comprises d) isolating the bacteriophage from the host bacterium. In embodiments, method steps a) and b) are carried out sequentially or concurrently.

[0007] In an embodiment of the method, prior to step a) the depolymerase is shown to increase production of the bacteriophage by the host bacterium by: i) exposing the host bacterium to a phage depolymerase under conditions suitable for depolymerase activity; ii) exposing the host bacterium to the bacteriophage under conditions suitable for infection of the host bacterium by the bacteriophage; iii) incubating the host bacterium for a period of time under conditions suitable for replication of the bacteriophage within the host bacterium; and iv) determining whether production of the bacteriophage by the host bacterium is increased relative to a control in which the host bacterium is not exposed to the phage depolymerase.

[0008] In an embodiment of the method, the host bacterium is a CPS-producing bacterium. In an embodiment of the method, the host bacterium is Acinetobacter baumannii (A. baumannii). In particular embodiments, the A. baumannii is an AB030, MRSN 843, MRSN 1183, MRSN 7446, MRSN 15088, MRSN 24008, MRSN 31159, MRSN 31468, or MRSN 32866 strain. In a particular embodiment, the A. baumannii is an MRSN 31468 strain.

[0009] In some embodiments of the method, the depolymerase is B5, Asl i, TaPaz, APK2, APK37, or IME200. In particular embodiments, the phage depolymerase is APK2 or IME200.

[0010] In embodiments of the method, the A. baumannii is an AB030, MRSN 7446, or MRSN 24008 strain and the depolymerase is B5; or the A. baumannii is an MRSN 31468 strain and the depolymerase is APK2 or IME200.

[0011] In embodiments of the method, the titer of bacteriophage produced is at least 10 times, at least 102times, at least 103times, at least 104times, or at least 105times higher than that produced using a control method in which the depolymerase is omitted.

[0012] In an embodiment, the method is carried out in vitro. In a further embodiment, the method is carried out in liquid culture.

[0013] Another embodiment of the present disclosure is a composition for increasing the susceptibility of an A. baumannii to infection by a bacteriophage, wherein the composition comprises a carrier and an isolated phage depolymerase selected from the group consisting of B5, Asl 1, TaPaz, APK2, APK37, and IME200. In particular embodiments, the A. baumannii is an AB030, MRSN 843, MRSN 1183, MRSN 7446, MRSN 15088, MRSN 24008, MRSN 31159, MRSN 31468, or MRSN 32866 strain. In some embodiments, the carrier is a pharmaceutically acceptable carrier or diluent.

[0014] Another embodiment of the present disclosure is use of a phage depolymerase selected from the group consisting of B5, Asl i, TaPaz, APK2, APK37, and IME200 to increase the susceptibility of an A. baumannii to infection by a bacteriophage. In some embodiments, the A. baumannii is an AB030, MRSN 843, MRSN 1183, MRSN 7446, MRSN 15088, MRSN 24008, MRSN 31159, MRSN 31468, or MRSN 32866 strain.

[0015] Another embodiment is a method for infecting an A. baumannii with a bacteriophage, comprising contacting the A. baumannii with: a) an isolated phage depolymerase selected from B5, Asl i, TaPaz, APK2, APK37, or IME200; and b) the bacteriophage, under conditions that allow for infection of the A. baumannii by the bacteriophage. In embodiments, the A. baumannii is contactedwith the phage depolymerase and bacteriophage consecutively or concurrently. In particular embodiments, the A. baumannii is an AB030, MRSN 843, MRSN 1183, MRSN 7446, MRSN 15088, MRSN 24008, MRSN 31159, MRSN 31468, or MRSN 32866 strain. In an embodiment, the method is carried out in vitro. In another embodiment, the method is carried out in vivo, in a subject infected with the A. baumannii.

[0016] A further embodiment is a method for determining whether a depolymerase can increase infectivity of a bacteriophage towards a host bacterium, the method comprising: a) exposing the host bacterium to a phage depolymerase under conditions suitable for depolymerase activity; b) exposing the host bacterium to the bacteriophage under conditions suitable for infection of the host bacterium by the bacteriophage; c) incubating the host bacterium for a period of time under conditions suitable for replication of the bacteriophage within the host bacterium; and d) determining whether production of the bacteriophage by the host bacterium is increased relative to a control in which the host bacterium is not exposed to the phage depolymerase.

[0017] In an embodiment, steps a) and b) are carried out consecutively or concurrently.

[0018] In embodiments of the methods described herein, the host bacterium is a bacterium that is able to infect a plant or animal. In a particular embodiment, the host bacterium is able to infect an animal, which may be a human or non-human animal.

[0019] A further embodiment is a method for producing a bacteriophage, the method comprising:(a) determining whether a depolymerase can increase infectivity of a bacteriophage towards a host bacterium by: i) exposing the host bacterium to a phage depolymerase under conditions suitable for depolymerase activity; ii) exposing the host bacterium to the bacteriophage under conditions that allow infection of the host bacterium by the bacteriophage; iii) allowing the bacteriophage to replicate within the host bacterium; andiv) determining whether production of the bacteriophage by the host bacterium is increased relative to a control in which the host bacterium is not exposed to the phage depolymerase; and(b) if production of the bacteriophage by the host bacterium is increased relative to the control, v) exposing the host bacterium to the phage depolymerase under conditions that allow the depolymerase to catalyze degradation of at least one cell-surface or extracellular polysaccharide of the host bacterium; vi) exposing the host bacterium to the bacteriophage under conditions that allow the bacteriophage to infect the host bacterium; and vii) allowing the bacteriophage to replicate within the host bacterium.

[0020] In an embodiment, steps i) and ii) are carried out consecutively or concurrently and / or steps v) and vi) are carried out consecutively or concurrently. In an embodiment, the method further comprises isolating the bacteriophage from the host bacterium after step vii).

[0021] In embodiments of the methods described herein, the step of exposing the host bacterium to the bacteriophage under conditions that allow the bacteriophage to infect the host bacterium is carried out in liquid culture.BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 shows the layout of the phage and phage depolymerase treatment plate of an A. baumannii strain under study to determine the concentrations of phages and phage depolymerases that show the highest degree of synergistic interactions to suppress growth of the strain under study.

[0023] FIG. 2 shows the layout of the dilution plate used to determine the efficiency of plating of a phage against a strain of A. baumannii with and without phage depolymerase treatment.

[0024] FIG. 3 shows the top synergistic interaction per phage and phage depolymerase combination based on area under curve assessments of the growth curves. The data was obtained by monitoring a plate as depicted Fig. 1 over an 18-hr time course with ODgoo measured every 20 min.

[0025] FIG. 4 shows the percent inhibition of bacterial growth when the bacterium is treated with phage, phage depolymerase, or both phage and phage depolymerase, compared to an untreated control.

[0026] FIG. 5 shows the endpoint titer of phages SB3 and SB5 after propagation in liquid cultureusing the specified bacterial strain, in both the presence (white bars) and absence (black bars) of the specified phage depolymerase. P-values are as follows: **, 0.02; ***, 0.002; ****, 0.0002.

[0027] FIG. 6 shows the endpoint titer of phages after propagation in liquid culture using the specified bacterial strain, in both the presence (black bars) and absence (hatched bars) of the specified phage depolymerase. P-values are as follows: **, 0.02; ***, 0.002; ****, 0.0002.

[0028] FIG. 7 shows the efficiency of plating for phages against a specified bacterial strain, in the presence (black bars) or absence (hatched bars) of the specified depolymerase at 200 pg / ml. P-values are as follows: **, 0.02; ***, 0.002; ****, 0.0002.DETAILED DESCRIPTIONThe following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure.Definitions

[0029] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0030] The term “about” as used herein may be used to take into account experimental error, measurement error, and variations that would be expected by a person having ordinary skill in the art. For example, “about” may mean plus or minus 10%, or plus or minus 5%, of the indicated value to which reference is being made.

[0031] As used herein the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Similarly, the terms “bacterium” and “bacteriophage” may be used to refer to a strain, type, or culture of bacterium or bacteriophage, rather than to a single bacterial cell or a single viral particle.

[0032] The phrase "and / or", as used herein, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion,i . e . , "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified.

[0033] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of’ or "exactly one of or, when used in the claims, "consisting of will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either”, "one of’, "only one of’, or "exactly one of."

[0034] As used herein, all transitional phrases such as "comprising”, "including”, "carrying”, "having”, "containing”, "involving”, "holding”, "composed of’, and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of’ and "consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively.

[0035] As used herein, the phrase "at least one”, in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0036] The terms “phage” and “bacteriophage” are used interchangeably herein, and they refer to a virus that infects and replicates within a bacterium.

[0037] As used herein, the terms “phage depolymerase” and “depolymerase” are used interchangeably herein to refer to a depolymerase enzyme derived from a bacteriophage; naturally, synthetically, or via recombinant means. Phage depolymerases are enzymes that act on bacterial cellsurface polysaccharides, including capsular polysaccharides (CPS) and lipopolysaccharides (LPS), to unmask phage binding sites, facilitating phage diffusion and adsorption. Phage depolymerases may also act on extracellular polysaccharides (EPS), which may also be referred to asexopolysaccharides, that are secreted by bacteria. Phage depolymerases have been shown to sensitize bacteria to antibiotic treatments and boost innate immune responses (Wang et al., 2024).

[0038] As used herein, the term “isolated phage depolymerase” refers to a depolymerase enzyme derived from a bacteriophage that is not in its natural association with the source bacteriophage. An isolated phage depolymerase may be produced naturally or through recombinant means, and separated from other components present in the cell in which the depolymerase was produced. A phage depolymerase may also be considered to be “isolated” if it is produced using cell-free protein synthesis or other synthetic means. Absolute purity is not required for a phage depolymerase to be considered “isolated” and an “isolated phage depolymerase” may be included in a composition with other components (such as buffers, diluents, stabilizing agents, or antibiotics) while still being considered “isolated”. An “isolated phage depolymerase” may be mixed with, or included together in a composition with, its source bacteriophage or another bacteriophage and still be considered “isolated”.

[0039] As used herein, the act of “isolating a bacteriophage” from a host bacterium refers to the act of removing the bacteriophage from the host bacterium or components thereof and / or removing the bacteriophage from the medium in which the host bacterium is grown. For example, the bacterium may be lysed to release the bacteriophage and the bacteriophage may be collected from the cell lysate, orthe bacteriophage may be collected from amedium in which the bacterium is grown. Absolute purity is not required for a bacteriophage to be considered isolated or purified. Rather, the bacteriophage need only be separated from a portion of cellular and / or chemical components present at the time of its production. For example, a bacteriophage may be isolated by precipitation, filtration, and / or centrifugation, or by any other suitable method(s) known in the art.

[0040] As used herein, the term “host bacterium” refers to a bacterium that is a host for a given bacteriophage. Whether a bacterium is a host for a given bacteriophage may be known in the art, or it may be determined empirically by attempting to infect the bacterium with the bacteriophage and determining whether the bacterium has been infected. Methods for infecting bacteria with bacteriophages and methods for determining whether bacteria have been infected by bacteriophages are well known in the art. In the methods described herein, the selection of a suitable “host bacterium” for use in the method may be based on its known or empirically determined susceptibility to infection by the bacteriophage employed in the method.

[0041] As used herein, the term “subject” refers to any organism (such as a plant or animal) that may be infected by a bacterium. The subject may, for example, be a human, companion animal (suchas a cat, dog, or rodent), a livestock animal (such as a fowl, horse, goat, sheep, pig, or bovine), or a crop species.

[0042] As used herein, the term “recombinant”, when used in reference to a polypeptide or protein, refers to a polypeptide or protein that is produced by recombinant techniques, wherein generally DNA or RNA encoding the expressed polypeptide is introduced into a host cell, or inserted into a suitable expression vector that is in turn introduced into a host cell, to allow expression of the recombinant polypeptide, which may then fold, dimerize or multimerize, and / or be subject to posttranslational processing or modification to form a mature protein. Recombinant polypeptides may include amino acid sequences from two or more sources, such as different proteins or different domains of a single protein. Such recombinant polypeptides may be referred to as fusion polypeptides, fusion proteins, or fusion constructs. Recombinant polypeptides may also include one or more synthetic amino acid sequences.

[0043] As used herein, the term “carrier” refers to a carrier that may be included in a composition together with a bacterium, bacteriophage, and / or phage depolymerase, without having any significant deleterious impact on the viability of the bacterium, the ability of the bacteriophage to infect a host bacterium, or the activity of the phage depolymerase. Suitable carriers include, for example, diluents, bacterial growth media, and pharmaceutically acceptable carriers, as described below.

[0044] As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier that is non-toxic. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and combinations thereof. Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffering agents that enhance shelf life or effectiveness.

[0045] An experimental “control” is an experiment performed under the same conditions as a test experiment, with the exception that a single variable is altered between the two experiments. A “control bacterium” is of the same species, strain, and isolate as the test bacterium, and it is exposed to the same conditions as the test bacterium, with the exception of a single identified variable (e.g., the test bacterium may be exposed to a phage depolymerase, while the control bacterium is not exposed to the phage depolymerase but is otherwise subjected to all the same conditions as the test bacterium). Similarly, a control method is carried out under the same conditions, and using the same reagents and / or components, as a test method, with the exception of a single variable (e.g., omission of a phage depolymerase from the control method).

[0046] A method may be described herein as being carried out “in vivo” or “in vitro”. In the context of the current disclosure, an “in vivo” method is carried out in a host organism infected by the bacterium, whereas an “in vitro” method is performed outside of any host organism for the bacterium, for example in a bacterial culture.

[0047] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.Details

[0048] As demonstrated herein, phage depolymerases can increase the susceptibility of bacteria to infection by bacteriophages to which the bacteria would otherwise be partially or fully resistant. This may allow a single bacterial strain, treated with a phage depolymerase, to be used for production of a variety of different bacteriophages, including bacteriophages that would otherwise not infect the bacterium or have low infectivity towards the bacterium. Further, as demonstrated herein, treatment of a bacterium with a phage depolymerase can result in substantially higher phage titers when the bacterium is used for phage production. This approach has significant advantages, as it allows for higher levels of phage production, while also allowing for bacterial host strains to be used more universally to produce a variety of different bacteriophages.

[0049] In some embodiments, the bacterium is an Acinetobacter baumannii (A. baumannii). In particular embodiments, the A. baumannii is an AB030, MRSN 843, MRSN 1183, MRSN 7446, MRSN 15088, MRSN 24008, MRSN 31159, MRSN 31468, or MRSN 32866 strain and the phage depolymerase is B5, Asl 1, TaPaz, APK2, APK37, or IME200. Any and all combinations of these strains and phage depolymerases are contemplated and included within the scope of the present disclosure, as if each combination had been individually recited.

[0050] An embodiment of the present disclosure is a method for producing a bacteriophage, comprising: a) exposing a host bacterium to a phage depolymerase under conditions that allow the depolymerase to catalyze degradation of at least one cell-surface or extracellular polysaccharide of the host bacterium, thereby increasing the susceptibility of the host bacterium to infection by the bacteriophage, relative to the susceptibility of a control host bacterium that has not been exposed to the phage depolymerase; b) exposing the host bacterium to the bacteriophage under conditions that allow the bacteriophage to infect the host bacterium; and c) allowing the bacteriophage to replicate within the host bacterium. Steps a) and b) may be carried out sequentially or concurrently and the method may further comprise isolating the produced bacteriophage from the host bacterium.Allowing the bacteriophage to replicate within the host bacterium may comprise incubating the host bacterium for a period of time under conditions suitable to allow for replication of the bacteriophage within the host bacterium. In certain embodiments, the incubation is carried out in liquid culture.

[0051] Another embodiment of the present disclosure is a method for determining whether a depolymerase can increase infectivity of a bacteriophage towards a host bacterium, the method comprising: a) exposing the host bacterium to a phage depolymerase under conditions suitable for depolymerase activity; b) exposing the host bacterium to the bacteriophage under conditions suitable for infection of the host bacterium by the bacteriophage; c) incubating the host bacterium for a period of time under conditions suitable for replication of the bacteriophage within the host bacterium; and d) determining whether production of the bacteriophage by the host bacterium is increased and / or growth of the bacterium is reduced relative to a control in which the host bacterium is not exposed to the phage depolymerase. If the depolymerase is found to increase infectivity of the bacteriophage towards the bacterium, then the bacterium and depolymerase may subsequently be used to produce the bacteriophage, as described in the preceding paragraph.

[0052] The host bacteria employed in the method described herein should be selected host bacteria that produce cell-surface or extracellular polysaccharides that impede infection by the phages, and the depolymerases employed in the methods described herein should be selected depolymerases that are able to catalyze said cell-surface or extracellular polysaccharides, such that partial or complete degradation of the cell-surface or extracellular polysaccharides increases infectivity of the phages for the host bacteria. Suitable combinations of phages, depolymerases, and host bacteria may be empirically determined, as will be understood to one skilled in the art.

[0053] The production method may lead to increased bacteriophage production relative to a control method in which the phage depolymerase is omitted. The bacteriophage titer may be increased by at least 10 times, at least 102times, at least 103times, at least 104times, at least 105times, or at least 106times, relative to titer obtained using the control method.

[0054] The production method will typically be carried out in vitro, with the host bacterium being grown in culture. In an embodiment, the host bacterium is grown in liquid culture.

[0055] Phage depolymerases described herein may be included in a composition for sensitizing A. baumannii to infection by a bacteriophage. The composition may be used for the purpose of phage production or for therapeutic purposes, e.g., in combination with a bacteriophage to treat a subject infected with A. baumannii. The composition may also be used for sanitation purposes, e.g., toincrease the susceptibility of an A. baumannii present on an abiotic surface to infection by a bacteriophage.

[0056] Accordingly, further provided is a method for infecting an A. baumannii with a bacteriophage as described herein, the method comprising contacting, either consecutively or concurrently, the A. baumannii with (a) an isolated phage depolymerase as described herein and (b) the bacteriophage. The method may be carried out in vivo, e.g., to treat an A. baumannii infection in a subject, or it may be carried out in vitro, e.g., to infect A. baumannii in culture or on an abiotic surface, for the purpose of killing at least a portion of the A. baumannii population and / or for the purpose of producing the bacteriophage using the A. baumannii as a bacterial host.Examples

[0057] The following non-limiting examples are illustrative of the present disclosure.

[0058] The specific phage depolymerases, bacteria, and phages tested in the examples demonstrate that phage depolymerases can increase the susceptibility of a bacterium to infection by a bacteriophage and also substantially increase the titer of bacteriophage produced by a bacterium. This finding is expected to apply broadly to any bacteriophage that is impeded from infecting a bacterium by the presence of a cell-surface or extracellular polysaccharide that can be degraded by a phage depolymerase. A person of ordinary skill in the art will be familiar with the types of bacteria that produce cell-surface or extracellular polysaccharides. Further, only routine experimentation is required to test a phage depolymerase in combination with any particular bacteriophage and bacterium of interest to determine whether the phage depolymerase will enable increased production of the bacteriophage by the bacterium of interest.Example 1: Effect of Mixtures of Bacteriophages and Phage Depolymerases on the Growth of Host Bacterial Cells

[0059] Synergy Test

[0060] Panels of bacteriophages and phage depolymerases were tested to identify their effect on the growth of a variety of A. baumannii strains. The following strains were used: AB030 (WGS GenBank Accession No. CP009257.1), BEI-2 (strain MRSN 843, obtained through BEI Resources, NIAID, NIH; WGS GenBank Accession No. VHDU00000000.1), BEI-8 (strain MRSN 1183, obtained through BEI Resources, NIAID, NIH; WGS GenBank Accession No. VHHD00000000. 1), BEI-30 (strain MRSN 7446, obtained through BEI Resources, NIAID, NIH; WGS GenBank Accession No. VHEB00000000), BEI-50 (strain MRSN 15088, obtained through BEI Resources,NIAID, NIH; WGS GenBank Accession No. VHGT00000000), BEI-61 (strain MRSN 24008, obtained through BEI Resources, NIAID, NIH; WGS GenBank Accession No. VHGH00000000), BEI-72 (strain MRSN 31159, obtained through BEI Resources, NIAID, NIH; WGS GenBank Accession No. VHFV00000000), BEI-75 (strain MRSN 31468, obtained through BEI Resources, NIAID, NIH; WGS GenBank Accession No. VHFS00000000), and BEI-89 (strain MRSN 32866, obtained through BEI Resources, NIAID, NIH; WGS GenBank Accession No. VHFE00000000). Any and all sequences described herein by reference to GenBank Accession Numbers are herein incorporated by reference in their entirety.

[0061] The strains were grown overnight on LLB agar, then diluted in LLB to an ODgoo of 0.025- 0.03 and incubated with shaking at 37°C until an ODgoo of 0.150 ± 0.005 was reached. While the cells were incubating, non-tissue culture treated 96-well plates were prepared with varying amounts of bacteriophage and phage depolymerase in each well. The bacteriophages SB1, SB2, SB3, SB4.1, SB4.2, SB5, and SB6 (all isolated from sewage samples in Ottawa, Ontario, Canada) were each tested at MOIs of 101, IO0,10’1,10‘2,10‘3,10‘4,10‘5, and 0; in combination with the phage depolymerases B5 (GenBank Accession No. ASN73455.2, tailspike protein from Acinetobacter phage vB_AbaP_B5), Asl i (GenBank Accession No. AQN32697.1, tailspike protein from Acinetobacter phage vB AbaP ASl l), TaPaz (GenBank Accession No. QVW53860.1, tailspike protein II from Acinetobacter phage TaPaz), APK2 (GenBank Accession No. AZU99242.1, tailspike protein from Acinetobacter phage vB_AbaP_APK2), APK37 (GenBank Accession No. AZU99445.1, tailspike protein from Acinetobacter phage vB_AbaP_APK37), and IME200 (GenBank Accession No. ALJ97635.1, tail fiber protein from Acinetobacter phage IME-200), which were each tested at concentrations of 50, 12.5, 3.1, 0.8, 0.2, and 0 ng / ml. The plate layout used for each phage depolymerase tested is shown in Figure 1.

[0062] Prior to use, the phage depolymerases and bacteriophages were diluted in LLB-S to the desired concentrations, as described in the previous paragraph, and 25 pl of each was added per well into the 96-well plates. Finally, 50 pl of host bacteria, at an ODgoo of 0.150 ± 0.005, was added to each well and the plates were incubated in a Biotek LogPhase™ 600 microbiology reader at 37°C / 800 rpm for 18 h, with reads performed in 20-minute intervals. In parallel, 2 x 20 pl of the host bacteria, at the ODgoo of 0.150 ± 0.005, was transferred to 2 wells of a titer plate containing 180 pl SM. These cells were used to determine the inoculum after the plates were loaded into the Biotek LogPhase™ 600 microbiology reader. A lOx serial dilution of these cells was performed and 3 x 5 pl were dropped onto LLB agar plates and grown overnight at room temperature, followed by counting of the CFUs.

[0063] The reads from the Biotek LogPhase™ 600 microbiology reader were imported into GraphPad™ and each growth curve was analyzed for Area Under Curve (AUC). Figure 3 shows the AUC data, organized based on the strain under study and the phage depolymerase used. Each panel represents the phage depolymerase concentration with the highest level of host growth suppression observed in the experiment. The percent growth inhibition for each test was calculated relative to a respective zero (0) treatment (i.e., no phage or phage depolymerase) control well, where % inhibition = (AU Co - AUCtest) / AUCo. The growth inhibition data is provided in Figure 4 and Table 1. These data demonstrate increased growth inhibition in the presence of phage depolymerase and phage for a number of different A. baumannii strains and phages, relative to treatment with either the phage depolymerase or the phage alone.

[0064] Table 1: Percent growth inhibition for bacteria treated with different combinations of phage and phage depolymerase (depol / phage), phage depolymerase only (depol / -), or phage only (- / phage). Six different phages were tested against various combinations of bacteria and depolymerase.HOST / DEPOL. BACTERIOPHAGE-Zphage 1% 0% 62% 6% 0% 84% 2%

[0065] Endpoint Titer Determination

[0066] Based on the readout from the Biotek LogPhase™ 600 microbiology reader, wells were selected that had good inhibition of growth (with different MOIs if possible). The contents of these wells were collected, as well as their respective 0 depolymerase control well, and transferred into low-bind MCF tubes. The cultures were then centrifuged and the bacteriophage-containing supernatant was collected. The bacteriophage lysates were then subjected to a 10 x dilution series. The resulting suspensions were applied in 3 x 5 pl drops onto TALLB plates impregnated with the AB5075cmstrain of A. baumannii and incubated at 37°C overnight. The AB5075cmstrain is the bacterial host of the phage panel and is a capsule mutant. This assessment was conducted for phagesSB3 and SB5 against strain BEI-30 and BEI-61 with depolymerase B5, and against strain BEI-75 with depolymerases APK2 and IME200 (Figure 5).

[0067] Efficiency of Liquid Propagation (EoLP) in the Presence of Depolymerase

[0068] Fresh bacterial subcultures in biological triplicates were prepared, then transferred into a 24-well plate, with or without 50 ng / ml depolymerase (3 technical replicate s / condition). AB5075cmwas included as a positive control for phage propagation and as a negative control for depolymerase sensitivity. The infectability of this strain is not expected to be affected by a depolymerase that targets a capsule polysaccharide, since it does not have a capsule. Phages were then added to each well (MOI=1.0) and incubated overnight with shaking at 37°C. Lysates were centrifuged at 12,000 x g for 10 minutes, then the supernatants were removed and serially diluted ten-fold to determine the phage titer, using the host strain AB5075cmon LLB overlay plates. PFUs were counted the following day and the endpoint titer of the phage lysates produced with and without depolymerase were compared. Figure 5 shows the endpoint titer of phages SB3 or SB5 when propagated in the presence or absence of depolymerase (B5, APK2, or IME200) on a test strain of A. haumannii (BEI-30, BEI- 61, or BEI-75). Figure 6 shows the endpoint titer of a phage (SB1, SB2, SB3, SB4. 1, SB5, or SB6) when propagated in the presence or absence of a depolymerase (B5, TaPaz, APK37, or APK2) on a test strain of A. baumannii (BEI-50, BEI-72, BEI-75, AB030, BEI-30, or BEI-89).

[0069] Efficiency of Plating (EOP) Synergy Test

[0070] Bacteriophage was added in 10 pl aliquots to 90 pl of LLB-S or 90 pl of LLB-S with 200 pg / ml of phage depolymerase in a 96-well microtiter plate. The bacteriophage was then subjected to a lOx serial dilution series (10-1to IO"6) in LLB-S or LLB-S broth with 200 pg / mL depolymerase. Initial experiments had the resulting suspensions applied in 3 x 5 pl drops onto TALLB plates impregnated with an overnight culture of the strain of A. baumannii under study generated using a single colony and incubated at 37°C overnight. The plate layout used is shown in Figure 2 and the preliminary results are shown in Table 2.

[0071] Table 2: Preliminary results, efficiency of plating for each phage with depolymerase, relative to media control, against the designated host.x = no infection, ns = not significant

[0072] Subsequent EoP experiments were carried out using five colonies per biological replicate, generating fresh cultures with which to impregnate the TALLB overlay. The experiments were repeated in biological and technical triplicate and the data is shown in Figure 7. The phages tested in the subsequent experiments were SB1, SB2, SB3, SB4.1, SB5, and SB6; the depolymerases tested were B5, APK37, APK2, and TaPaz; and the A. baumannii tested were AB030, BEI-30, BEI-89, BEI-72, BEI-75, and BEI-50. The SB4.2 phage was left out of the subsequent EoP assay since sequencing of the phages revealed that SB4.2 is a variant of SB4.1. Further, BEI-8 was left out of the subsequent study due to its resistance to the phage panel. Additionally, depolymerase IME200 was found to have the same activity profile as APK2; thus, it was left out of the final experiment. Finally, BEI-61 was dropped from the subsequent experiment to enable inclusion of BEI-50 with depolymerase TaPaz, expanding the panel to include a K47 capsular type.

[0073] In the subsequent experiment, phages SB1, SB2, SB5, and SB6 showed different EoP effects from the initial screening trial with AB030 and depolymerase B5. Phages SB1, SB2, and SB6 display increased EoP in the subsequent experiment (Figure 6), compared to no infectivity, or insignificant impact (SB2) in the preliminary trial (Table 2). Further, phage SB5 showed no activity against AB030 in the preliminary trial, and the subsequent trial showed it is capable of infecting, though it does not synergize with B5 to infect AB030. These changes appear to be due to differences in the host culturing method. For the initial experiments, bacterial cultures were generated from a single colony to produce an overnight culture, whereas subsequent experiments used 5 colonies to produce a freshly grown culture. Without wishing to be bound by theory, it is hypothesized that expression of phage receptor(s) by the host bacterium may be impacted by the host bacterium’s phase of growth (stationary versus exponential phase).

[0074] The synergistic effects observed between the depolymerase, phage, and host bacterium are specific to the particular phage-host bacterium-depolymerase combination employed in the experimental conditions.

[0075] Without being bound by theory, it is believed that the differences in the concentrations of depolymerase and phages required for effective activity demonstrated herein are attributable, at least in part, to the relative thickness of the capsule surrounding the target bacterial strains. Strains possessing thicker capsules may require higher concentrations of depolymerase to sufficiently degrade the capsule and render the bacterium susceptible to phage infection. Furthermore, the nature of the phage receptor utilized for host recognition, and the level at which such receptor is expressed on the bacterial surface, are additional factors that may influence the phage concentration necessary to achieve the desired effect.

[0076] In certain instances, specific capsular types do not appear to impede the activity of some phages within the tested panel. This is evident when no significant change is observed in the EoP or EoLP values for particular phages on defined host bacterial strains in the presence of the depolymerase. For example, phages SB1, SB2, and SB4.1 are able to infect bacterial strain BEI-50, which possesses the K47 capsular type, with comparable efficiency regardless of the presence or absence of the depolymerase TaPaz. This observation suggests that the K47 capsular type does not act as a barrier to infection for these specific phages. In contrast, phages SB3, SB5, and SB6 exhibit enhanced activity against strain BEI-50 when combined with depolymerase TaPaz, indicating that the K47 capsular type represents an impediment to these phages in the absence of depolymerase. Without being bound by theory, it is believed that variations in tail fiber composition and structure among the phages in the tested panel influence the manner in which each phage interacts with distinct capsular types, thereby accounting for the differences observed. Additionally, differences in host susceptibility were noted for certain strains of Acinetobacter when cultured in liquid media as compared to solid media. For example, strain BEI-8 is susceptible to phages SB1, SB3, SB4.1, SB4.2, and SB6 when grown in liquid culture, but exhibits resistance to these phages when grown on solid medium (Figures 3 and 4, and Table 2). Without wishing to be bound by theory, it is hypothesized that the observed difference in host susceptibility may be due to differences in phagereceptor expression by the host bacterium when grown in, or on, liquid versus solid medium. From the experiments described herein, it appears that liquid culture may be preferable. However, it’s possible that certain bacterial strains may show higher susceptibility to infection by phages when grown on solid medium. The optimal growth conditions for any particular bacterium may be empirically determined.

[0077] For any particular combination of phage and host bacterium, the effectiveness of a given depolymerase may be empirically determined. If a CPS or EPS produced by a given host bacterium does not impede infection by a given phage, then the addition of a depolymerase that degrades the CPS or EPS is not expected to increase infectivity of the phage for that host bacterium. However, if a CPS or EPS produced by the host bacterium is a barrier to infection by a given phage, then the addition of a depolymerase that degrades the CPS or EPS may increase infectivity of the phage, resulting in reduced bacterial growth and / or increased phage production by the host bacterium. Using no more than ordinary skill in the art, specific combinations of phages, depolymerases, and host bacteria suitable for use in the methods described herein may be empirically determined, for example using methods such as those described in the preceding examples.

[0078] The preceding examples have been provided to illustrate various aspects of the invention and are non-limiting. The scope of the claims is not limited to specific details provided in the examples, rather the claims are to be given the broadest interpretation consistent with the teachings of the specification and drawings as a whole.References:Strathdee, Steffanie A., et al. "Phage therapy: From biological mechanisms to future directions." Cell 186.1 (2023): 17-31.Wang, Honglan, et al. "Translating bacteriophage -derived depolymerases into antibacterial therapeutics: challenges and prospects." Acta Pharmaceutica Sinica B 14.1 (2024): 155-169.

Claims

WHAT IS CLAIMED IS:

1. A method for producing a bacteriophage, comprising: a) exposing a host bacterium to a phage depolymerase under conditions that allow the depolymerase to catalyze degradation of at least one cell-surface or extracellular polysaccharide of the host bacterium, thereby increasing the susceptibility of the host bacterium to infection by the bacteriophage, relative to the susceptibility of a control host bacterium that has not been exposed to the depolymerase; b) exposing the host bacterium to the bacteriophage under conditions that allow the bacteriophage to infect the host bacterium; and c) allowing the bacteriophage to replicate within the host bacterium.

2. The method of claim 1, further comprising: d) isolating the bacteriophage from the host bacterium.

3. The method of claim 1 or 2, wherein steps a) and b) are carried out sequentially or concurrently.

4. The method of any one of claims 1 to 3, wherein the host bacterium is a capsule polysaccharide (CPS)-producing bacterium.

5. The method of any one of claims 1 to 4, wherein prior to step a) the depolymerase is shown to increase production of the bacteriophage by the host bacterium by: i) exposing the host bacterium to a phage depolymerase under conditions suitable for depolymerase activity; ii) exposing the host bacterium to the bacteriophage under conditions suitable for infection of the host bacterium by the bacteriophage;iii) incubating the host bacterium for a period of time under conditions suitable for replication of the bacteriophage within the host bacterium; and iv) determining whether production of the bacteriophage by the host bacterium is increased relative to a control in which the host bacterium is not exposed to the phage depolymerase.

6. The method of any one of claims 1 to 5, wherein the host bacterium is an Acinetobacter baumannii (A. baumannii).

7. The method of claim 6, wherein the depolymerase is B5, Asl i, TaPaz, APK2, APK37, or IME200.

8. The method of claim 6 or 7, wherein the A. baumannii is an AB030, MRSN 843, MRSN 1183, MRSN 7446, MRSN 15088, MRSN 24008, MRSN 31159, MRSN 31468, or MRSN 32866 strain.

9. The method of claim 8, wherein the phage depolymerase is APK2 or IME200.

10. The method of any one of claims 1 to 9, wherein the titer of bacteriophage produced by the method is at least 10 times, at least 102times, at least 103times, at least 104, or at least 105times higher than that produced using a control method in which the depolymerase is omitted.

11. The method of any one of claims 1 to 10, wherein the method is carried out in vitro.

12. A method for determining whether a depolymerase can increase infectivity of a bacteriophage towards a host bacterium, the method comprising: a) exposing the host bacterium to a phage depolymerase under conditions suitable for depolymerase activity; b) exposing the host bacterium to the bacteriophage under conditions suitable for infection of the host bacterium by the bacteriophage;c) incubating the host bacterium for a period of time under conditions suitable for replication of the bacteriophage within the host bacterium; and d) determining whether production of the bacteriophage by the host bacterium is increased relative to a control in which the host bacterium is not exposed to the phage depolymerase.

13. The method of claim 12, wherein steps a) and b) are carried out consecutively or concurrently.

14. The method of claim 12 or 13, wherein the host bacterium is a bacterium that is able to infect an animal.

15. The method of any one of claims 1 to 14, wherein the method is carried out in liquid culture.

16. A composition for increasing the susceptibility of an A. baumannii to infection by a bacteriophage, wherein the composition comprises a carrier and an isolated phage depolymerase selected from the group consisting of B5, Asl 1, TaPaz, APK2, APK37, and IME200.

17. The composition of claim 16, wherein the A. baumannii is an AB030, MRSN 843, MRSN 1183, MRSN 7446, MRSN 15088, MRSN 24008, MRSN 31159, MRSN 31468, or MRSN 32866 strain.

18. The composition of claim 16 or 17, wherein the carrier is a pharmaceutically acceptable carrier or diluent.

19. Use of a phage depolymerase selected from the group consisting of B5, Asl i, TaPaz, APK2, APK37, and IME200 to increase the susceptibility of an A. baumannii to infection by a bacteriophage.

20. The use of claim 19, wherein the A. baumannii is an AB030, MRSN 843, MRSN 1183, MRSN 7446, MRSN 15088, MRSN 24008, MRSN 31159, MRSN 31468, or MRSN 32866 strain.

21. A method for infecting an A. baumannii with a bacteriophage, comprising contacting the A. baumannii with:a) an isolated phage depolymerase selected from B5, Asl i, TaPaz, APK2, APK37, or IME200; and b) the bacteriophage, under conditions that allow for infection of the A. baumannii by the bacteriophage.

22. The method of claim 21, wherein the A. baumannii is contacted with the phage depolymerase and the bacteriophage consecutively or concurrently.

23. The method of claim 21 or 22, wherein they!, baumannii is an AB030, MRSN 843, MRSN 1183, MRSN 7446, MRSN 15088, MRSN 24008, MRSN 31159, MRSN 31468, or MRSN 32866 strain.

24. The method of any one of claims 21 to 23, wherein the method is carried out in vitro.

25. The method of any one of claims 21 to 23, wherein the method is carried out in vivo, in a subject infected with they!, baumannii.

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