Bacteriophage compositions and methods for treating bacterial infections

Abstract

Compositions, devices, kits, and methods for the treatment of Enterobacteriaceae infections are disclosed. Aspects of the disclosure relate to bacteriophage compositions comprising one or more of ES17, ES19, HP3, HP3.1, and HP3.2. Particular aspects of the disclosure relate to compositions comprising (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1. Also disclosed are compositions comprising bacteriophage HP3.2. Additionally, devices and kits comprising such compositions, as well as methods of using such compositions in the treatment and prevention of pathogenic E. coli infections, are also disclosed.

Description

[Technical Field]

[0001] [Sequence Listing] This application contains a sequence listing that has been submitted in XML format and is incorporated herein by reference in its entirety. The XML copy was created on December 2, 2022, is named "BAYMP0340WO2_SL.xml", and is 757,530 bytes in size.

[0002] Aspects of the present invention relate to at least the fields of microbiology and virology. [Background technology]

[0003] Multidrug-resistant (MDR) bacteria infect millions of people worldwide each year. Many infections caused by these bacteria are untreatable due to resistance to last-resort antibiotics. Compounding this crisis is the slow pace of antibiotic development, while resistant strains emerge quickly as new drugs become available. Bacteria belonging to the Enterobacteriaceae family pose an urgent threat of MDR infections, particularly those caused by strains of Escherichia coli. Extraintestinal pathogenic E. coli (ExPEC), a pathotype belonging to the E. coli superfamily, is a natural inhabitant of the human gut microbiome. ExPEC strains are unique in their ability to translocate and cause infections in immunocompromised individuals. Established infections can cause a variety of severe illnesses, including urinary tract infections (UTIs), bacteremia, sepsis, and neonatal meningitis. Novel therapeutic strategies are needed to combat the threat posed by MDR bacteria.

[0004] Bacteriophage (phage) therapy is a promising approach to combating MDR infections. Phages are bacterial-infecting viruses that are ubiquitous in the environment, host-specific, and can effectively infect MDR strains. Importantly, phages have been shown to be safe and effective in animal studies and compassionate use clinical trials. Because phages utilize the host bacterial replication machinery for replication, their mutation rate is directly affected by the host's mutation rate. Therefore, phages have the potential to rapidly adapt and evolve to target bacterial strains. However, because complementary evolutionary rates exist between phages and their hosts, mixed populations of phages and bacteria are caught in an evolutionary arms race. As a result, phage-resistant bacteria are likely to emerge. Summary of the Invention

[0005] There is a need for new and improved methods and compositions for the treatment of bacterial infections, particularly for the treatment of E. coli infections such as ExPEC.

[0006] Aspects of the present disclosure relate to bacteriophage compositions, kits and devices comprising the same, and methods of using the compositions for treating or preventing pathogenic infections. In one aspect, a composition comprising (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1 is disclosed. In one embodiment, the composition comprises bacteriophages ES17, HP3, and HP3.1. In one embodiment, the composition comprises bacteriophages ES19, HP3, and HP3.1. Also disclosed is a composition comprising bacteriophage HP3.2. Additionally, a method for treating or preventing an E. coli infection in an individual is disclosed, comprising administering (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1. In a particular embodiment, a method for treating or preventing an E. coli infection is disclosed, comprising administering bacteriophage HP3.2.

[0007] Embodiments of the present disclosure include bacteriophages, bacteriophage compositions, kits, devices, medical devices, therapeutic devices, polynucleotides, methods of making devices, methods of treating E. coli infections, and methods of preventing E. coli infections. The compositions of the present disclosure may include at least one, two, three, or more of the following components: bacteriophage ES17, bacteriophage ES19, bacteriophage HP3, bacteriophage HP3.1, bacteriophage HP3.2, additional bacteriophages, metals, and pharmaceutical excipients. Any one or more of these components may be excluded in certain embodiments. The methods of the present disclosure may include at least one, two, three, or more of the following steps: detecting an infection in an individual, diagnosing the individual with an E. coli infection, generating a bacteriophage composition, coating a device with the bacteriophage composition, providing the device to the individual, formulating the bacteriophage composition for therapeutic administration, and administering the bacteriophage composition to the individual. Any one or more of these steps may be omitted in certain embodiments.

[0008] In some embodiments, disclosed is a composition comprising (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1. In some embodiments, the composition comprises bacteriophage ES17. In some embodiments, the composition comprises bacteriophage ES19. In some embodiments, the composition further comprises HP3.2. In some embodiments, the amounts of bacteriophages in the composition are substantially equivalent. In some embodiments, the amounts of bacteriophages in the composition are not substantially equivalent. In some embodiments, disclosed is a composition comprising bacteriophage HP3.2. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises one or more metals. In some embodiments, the one or more metals comprise calcium, magnesium, iron, sodium, and / or potassium. In some embodiments, the composition is a liquid formulation. In some embodiments, the composition is a solid formulation. In some embodiments, the composition is contained in a delivery device. Further disclosed in certain embodiments is a method for treating or preventing an E. coli infection comprising administering to an individual a composition of the present disclosure.

[0009] Additionally, disclosed herein in certain embodiments are devices comprising (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1, wherein the bacteriophages are present on, within, and / or around the device. In certain embodiments, the device comprises bacteriophage ES17. In certain embodiments, the device comprises bacteriophage ES19. In certain embodiments, the device further comprises bacteriophage HP3.2. Also disclosed are devices comprising bacteriophage HP3.2, wherein the bacteriophage is present on, within, and / or around the device. In some embodiments, the device is a catheter, driveline, syringe, tubing, implant, defibrillator, artificial joint, pacemaker, screw, rod, disc, intrauterine device, pin, plate, stent, dental device, intraocular lens, shunt, valve, nervous system or neurosurgical device, gastrointestinal device, genitourinary device, catheter cuff, vascular access device, or wound drain. In some embodiments, the device is further defined as having a coating comprising a bacteriophage.

[0010] Also disclosed herein as certain embodiments is a kit comprising (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1, housed in a suitable container. In certain embodiments, the kit comprises bacteriophage ES17. In certain embodiments, the kit comprises bacteriophage ES19. In certain embodiments, the kit further comprises HP3.2. Also disclosed is a kit comprising bacteriophage HP3.2. In certain embodiments, the kit further comprises a device. In certain embodiments, the device comprises a bacteriophage. In certain embodiments, the bacteriophage is present separately from the device.

[0011] Disclosed herein as certain embodiments are methods for treating or preventing an E. coli infection in an individual, comprising administering to the individual (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1. In some embodiments, the method comprises administering bacteriophage ES17. In some embodiments, the method comprises administering bacteriophage ES19. In some embodiments, the method further comprises administering bacteriophage HP3.2 to the individual. In some embodiments, the bacteriophages are contained in the same formulation. In some embodiments, the bacteriophages are not contained in the same formulation. In some embodiments, each bacteriophage is administered to the individual simultaneously. In some embodiments, each bacteriophage is administered to the individual at a different time. In some embodiments, the bacteriophage is administered to the individual intravenously, orally, or via a device. In some embodiments, the bacteriophage is administered to the individual multiple times. In some embodiments, the bacteriophage is administered daily, twice daily, weekly, twice weekly, monthly, or twice monthly. In some embodiments, the bacteriophage is administered twice weekly for 6 to 12 weeks. Also disclosed are methods for treating or preventing E. coli infections, comprising administering bacteriophage HP3.2 to an individual.

[0012] In some embodiments, the individual has an infection in the urinary tract, blood, intestines, abdomen, stomach, lungs, skin, kidneys, prostate, bladder, brain, vaginal tract, heart, liver, spleen, or a combination thereof. In some embodiments, the individual has a catheter-associated urinary tract infection. In some embodiments, the E. coli is multidrug-resistant. In some embodiments, the E. coli is extraintestinal pathogenic E. coli. In some embodiments, the E. coli is ST69, ST73, ST96, or ST131. In some embodiments, the E. coli is ST131. In some embodiments, the individual has a urinary tract infection, neonatal meningitis, bloodstream infection, pneumonia, sepsis, surgical wound infection, skin infection, prostate infection, meningitis, vaginal infection, or a combination thereof. In some embodiments, the individual has diarrhea, stomach cramps, nausea, and / or vomiting. In some embodiments, the individual is immunosuppressed. In some embodiments, the individual has immune cell deficiency, asplenia, splenic dysfunction, nephrotic syndrome, or an autoimmune disease. In some embodiments, the individual is administered a bacteriophage prior to a medical procedure or treatment schedule. In some embodiments, the individual is scheduled to be immunosuppressed. In some embodiments, the individual is undergoing or will undergo chemotherapy. In some embodiments, the individual is taking or will take an immunosuppressant. In some embodiments, the immunosuppressant is a glucocorticoid, a calcineurin inhibitor, an antimetabolite, or an antibody therapy. In some embodiments, the medical procedure involves insertion of a device into the individual. In some embodiments, the source of infection with E. coli is a drink, food, another individual, or the environment. In some embodiments, the environment is groundwater or surface water, water used for crop irrigation, a public water system, a hospital, a school, a nursing home, a petting zoo, a cruise ship, a train, or an airplane. In some embodiments, the individual is at high risk for E. coli infection compared to the general population. In some embodiments, the individual is under the age of 12 or over the age of 65. In certain embodiments, the individual is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years old.In some embodiments, the individual is 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 years of age or older. In some embodiments, the individual consumes undercooked meat, unpasteurized milk, apple juice or cider, or soft cheese made from raw milk. In some embodiments, the individual is taking a medication that suppresses stomach acid (e.g., a proton pump inhibitor).

[0013] Also disclosed herein in certain embodiments is a method for reducing levels of drug-resistant and / or pathogenic E. coli strains in an individual, the method comprising administering to the individual therapeutically effective amounts of (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1. In certain embodiments, the method comprises administering bacteriophage ES17. In certain embodiments, the method comprises administering bacteriophage ES19. In certain embodiments, the method comprises administering to the individual a therapeutically effective amount of bacteriophage HP3.2. Also disclosed in certain embodiments is a method for reducing levels of drug-resistant and / or pathogenic E. coli strains in an individual, the method comprising administering to the individual a therapeutically effective amount of bacteriophage HP3.2. In certain embodiments, the reduction occurs in the intestine of the individual. In certain embodiments, the individual has undergone or will undergo insertion of a device. In some embodiments, the individual is to be placed in an immunosuppressed state. In some embodiments, the E. coli strain is extraintestinal pathogenic E. coli. In some embodiments, the E. coli strain is ST69, ST73, ST96, or ST131. In some embodiments, the E. coli strain is ST131.

[0014] Also described herein, in certain embodiments, is a method of preparing a device, the method comprising exposing the device to (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1. In certain embodiments, the method comprises exposing the device to bacteriophage ES17. In certain embodiments, the method comprises exposing the device to bacteriophage ES19. In certain embodiments, the bacteriophage is disposed on, within, and / or around the device. In certain embodiments, one or more surfaces of the device are coated with the bacteriophage. In certain embodiments, the device is a catheter, driveline, syringe, tubing, implant, defibrillator, artificial joint, pacemaker, screw, rod, disc, intrauterine device, pin, plate, stent, dental device, or intraocular lens. In certain embodiments, the method further comprises delivering the device to an individual in need thereof.

[0015] Throughout this specification, the term "about" is used to indicate that a value includes the inherent variation of error in the measuring or quantitating method.

[0016] The words "a" or "an," when used in conjunction with the term "comprising," can mean "one," but are also consistent with the meanings "one or more," "at least one," and "one or more."

[0017] The phrase "and / or" means "and" or "or." For example, A, B, and / or C includes A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination. In other words, "and / or" functions as an inclusive "or."

[0018] The words "comprising" (and its forms "comprise" and "comprises"), "having" (and its forms "have" and "has"), "including" (and its forms "includes" and "include") or "containing" (and its forms "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0019] The compositions and methods of use disclosed herein may be described as "comprises," "consist essentially of," or "consist of." The phrase "consisting essentially of" is limited to certain materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0020] "Individual," "subject," and "patient" are used interchangeably and can refer to a human or non-human.

[0021] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in the "use" claim format, i.e., the "use of" a compound, composition, or agent described herein to achieve or carry out the stated therapeutic, diagnostic, or physiological purpose or effect.

[0022] It is expressly contemplated that any limitations discussed with respect to one embodiment of the present invention may also be applied to any other embodiment of the present invention. Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to make or utilize any composition of the present invention. Any embodiment discussed with respect to one aspect of this disclosure may also be applied to other aspects, and vice versa. For example, any step of a method described herein may be applied to any other method. Furthermore, any method described herein may exclude any step or combination of steps. Aspects of an embodiment described in an example are embodiments that can be implemented in the context of other examples or embodiments discussed elsewhere in this specification, such as the summary, detailed description, claims, and brief description of the drawings.

[0023] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, and that various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art.

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0025] [Figures 1A-1F](Figure 1A) Mice were orally gavaged with 1 × 109 CFU of ExPEC JJ1901 on day 0, and colonization was monitored by plating fecal pellets on selective medium (LB + chloramphenicol) and counting colonies. From day 1 until day 5, each group received one of the following: daily phage administration (1 × 109 PFU), phage in water (1 × 109 PFU / mL), or antibiotic (ABX), ampicillin 2 mg / mL in water. On day 6, mice were euthanized, and organs were homogenized and plated for CFU or PFU levels (plaque assay). (Figure 1B) Intestinal (fecal) colonization by ExPEC. Colored points represent mean values. ExPEC + phage gavage group: ExPEC1U (G); ExPEC + phage in water group: ExPEC1U (W); ExPEC + antibiotic in water group: ExPEC1ABX (W). (Figure 1C) Phage levels in intestinal tissue and contents. (Figure 1D) ExPEC levels in intestinal tissue and contents. N = 8–10. Open boxes indicate individual mice, and bars indicate the mean. # indicates values ​​missing due to histological analysis. (Figure 1E) ExPEC levels after 4.5 hours of incubation (shaking, 37°C) in LB medium, fecal medium, or cecal medium with or without phage (MOI 10). (Figure 1F) PFU counts of phage HP3 after 4.5 hours of incubation in different media. Open boxes indicate independent cultures (N = 3), and bars indicate the mean ± standard deviation (SD). NS: not significant; “: not detected. One-way ANOVA was used for statistical analysis. [Figures 2A-2C](Figure 2A) Cecal medium (CM) was prepared and either heat-treated at 100°F for 20 minutes (HT CM) or centrifuged at 6,000 × g for 5 minutes and filtered through a 0.22 mm filter (FT) before being used for a 4.5-hour growth assay. (Figure 2B) Cecal medium was centrifuged at high speed (9,000 × g for 5 minutes) to separate the pellet (insoluble [Ins]) and supernatant (soluble [Sol]). Ins was resuspended in saline and treated with or without NAC (N-acetylcysteine, 5 mg / mL). The soluble component was treated with or without mucus (porcine stomach mucin, 1% [w / v]). ExPEC levels are shown after the 4.5-hour assay. (Figure 2C) ExPEC levels after a 4.5-hour assay screening with phage in the presence of mucin (1.5% [w / v] in LB medium). A multiplication factor (MOI) of 10 was used in all experiments. Open boxes indicate independent cultures. Bars indicate mean values ​​(±standard deviation [SD]). N = 3–9. “: Not detected. One-way ANOVA was used for statistical analysis. [Figures 3A-3E](Figure 3A) Transmission electron microscope (TEM) image of phage ES17. (Figure 3B) Mucin (0%, 0.5%, 1.5% [w / v]) was added to ExPEC culture medium and incubated at room temperature (RT) with shaking (255 rpm) for 10 minutes. The culture medium was centrifuged (6,000 × g for 5 minutes) and gently washed with PBS. Adsorption curves (MOI: 1) were constructed using culture medium in the logarithmic growth phase and sampled every 5 minutes for 10 minutes. Mucin-coated ExPECs are shown in the TEM images. White bar: 1 mm. (Figure 3C) Adsorption of phage ES17. (Figure 3D) Adsorption of phage HP3. The adsorption constant (K) and adsorption rate (%Ads) for 10 minutes in the presence of 0% and 1.5% mucin are shown above each curve. Regression lines were generated from the average values ​​of remaining phage at the indicated time points. Open boxes indicate individual values ​​obtained from separate cultures. Dashed line: 0% mucin, red line: 0.5% mucin, blue line: 1.5% mucin. Two biological replicates were performed for the 0% and 1.5% mucin conditions. (Figure 3E) ELISA using wells coated with 1.5% mucin. Different phages were incubated on the coated wells overnight, then washed and assayed. Open boxes represent biological replicates. The x-axis shows the different concentrations of phage incubated on the wells. N=6. Bars represent mean ± standard deviation (SD). In Figures 3C and 3D, simple regression analysis was used to determine whether the slopes were significantly different from zero. Two-way ANOVA was used for statistical analysis in Figure 3E. [Figure 4A-4B]ES17 binds to the surface of human intestinal enteroids (HIEMs). Differentiated HIEMs were incubated with phage (1 × 108 PFU / mL) for 1 hour in a humidified incubator at 37°C with 5% CO2. The HIEMs were then washed, fixed with Clark's fixative, and stained. Phage (Alexa Fluor 488; green), intestinal epithelial cells (DAPI; blue), and Muc2 (Alexa Fluor 594; red) were imaged. Image magnification: ×60. Orthogonal cross-sectional images (XY) are shown at the bottom. (Figure 4A) No phage added (i), phage ES17 added (ii), and phage HP3 added (iii). (Figure 4B) To selectively remove HSPG from glycosaminoglycan (GAG) chains, enteroid cultures were pretreated with heparinase III (HepIII; 2 U / mL) for 2 hours in a humidified incubator at 37°C with 5% CO2 and then incubated with or without phage according to the method described above. No phage (i and ii), phage ES17 (iii), phage HP3 (iv), HepIII-treated HIEMs with ES17 (v), and HepIII-treated HIEMs with HP3 (vi). Image magnification: ×60. (vii) Quantification of particle (phage) numbers per well using FIJI software. Mean values ​​± standard deviation (SD) are shown (N = 1–2). Open boxes indicate independent cultures. Statistical analysis was performed using one-way ANOVA. Figure 4B was generated using BioRender software. [Figures 5A-5D](Figure 5A) ExPEC levels (MOI: 10) after a 4.5-hour growth assay in cecal or LB medium. Bars indicate the mean, and open boxes indicate independent cultures. N = 3. (Figure 5B) Mice were orally gavaged with ExPEC JJ1901 (1 × 10 CFU) on day 0. From days 1 to 5, mice were gavaged daily with phage (HP3 or ES17, 1 × 10 PFU) or left untreated. On day 6, mice were euthanized, and organs were homogenized and plated to measure CFU levels or subjected to plaque assay to measure PFU levels. (Figure 5C) Phage levels in intestinal tissue and contents. (Figure 5D) ExPEC levels in intestinal tissue and contents. N = 10 mice per group. Bars indicate the mean ± standard deviation (SD), and open boxes indicate individual mice. ": Not detected." One-way ANOVA was used for statistical analysis. [Figure 6] Model diagram: (1) Mucins derived from the intestinal mucus layer inhibit phage infection, (2) phage ES17 binds to mucins and uses other intestinal glycans as receptors to infect and kill bacteria embedded in the mucus, and (3) phages such as ES17 bind to heparan sulfate glycans to coat the intestinal epithelium and can be used to protect against infection by invasive pathogens. [Figures 7A-7B] Resistance to φHP3 was induced using three clinical ExPEC isolates—JJ2050, JJ2528, and JJ2547. (Figure 7A) Resistant isolates were isolated by either culture or mouse culture. In both methods, the parent strains were exposed to selective pressure in the presence of φHP3. (Figure 7B) Isolate titers were measured in liquid culture after 4.5 hours of incubation in LB medium with or without φHP3. Panel B is grouped by parent strain: JJ2050 (i), JJ2528 (ii), and JJ2547 (iii). P values ​​were determined by Student's t test or Mann-Whitney test, as appropriate. *, P 0.05; **, P 0.01; ***, P 0.001. Bars indicate mean titers. Each data point represents the average of three parallel technical replicates. [Figure 8]Isolate titers were determined by liquid culture after 4.5 hours of incubation in LB medium (black) or human urine (yellow) with or without φHP3. Each panel is grouped by parent strain: JJ2050 (i), JJ2528 (ii), and JJ2547 (iii). P values ​​were calculated by Student's t test or Mann-Whitney test, as appropriate. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Each data point represents the mean of three parallel technical replicates. [Figure 9A-9B] (Figure 9A) Human whole blood was separated by centrifugation, and the plasma was heat-treated to inactivate complement. Whole blood cells (WBCs) were resuspended in heat-treated plasma (HIP), serum mimicking solution (BSM), or a 1:1 mixture of HIP and BSM. (Figure 9B) After 24 h of incubation in different blood media, the titers of JJ2547 and its resistant strains were measured. The media used were as follows: whole blood (WB, dark red) (i), HIP blood (HIPB, red) (ii), HIP / BSM mixed blood (HIPB / BSM, orange) (iii), BSM (BSM1, light orange) (iv), or BSM without blood cells (BSM, white) (v). P values ​​were calculated by Student's t-test or Mann-Whitney test, as appropriate. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Each data point represents the mean of three parallel technical replicates. [Figures 10A-10D]Two representative resistant strains, JJ2528-8 ("resistant strain 8," dark gray, ompA disruption) and JJ2528-12 ("resistant strain 12," black, waaC disruption), and their parental strain (light gray) were tested for virulence in a mouse sepsis model. (Figure 10A) Swiss-Webster mice were infected with logarithmically growing phage-untreated culture fluid (3.5 × 10 CFU) suspended in PBS. Animals were observed for 72 hours and then euthanized unless moribund. Livers and spleens were homogenized and plated for bacterial burden measurement. (Figure 10B) Animals infected with the parental strain had a survival rate of only 20%, whereas animals infected with the resistant strain showed a 100% survival rate. (Figure 10C) Infected animals were observed and assessed for health scores based on four indices in accordance with the NIH Advisory Committee on Animal Research guidelines. A score of 4 or higher was considered moribund and required euthanasia. (Figure 10D) After euthanasia, the livers and spleens of infected animals were removed, weighed, homogenized, and plated on LB agar to assess bacterial burden. Titers per gram are shown. P values ​​for Figure 10B were calculated by the log-rank (Mantel-Cox) test. P values ​​for Figure 10D were calculated by Student's t test or Mann-Whitney test, as appropriate. *, P < 0.05; **, P < 0.01; ***, P < 0.001. [Figures 11A-11C] The genome sequences of all 21 resistant strains and their parent strains were analyzed. (Figure 11A) Alignment of JJ2528 with the resistant strains revealed multiple truncations in the waa operon, hldE, and ompA. The dashed lines in the panels indicate downstream single nucleotide polymorphisms (SNPs) in ompA. (Figure 11B) The structure of LPS (lipopolysaccharide) and the names of the enzymes involved in each bond formation are shown. Several steps in the LD-Hep construction process are also shown on the left. [Figure 12A-B](Figure 12A) Evolution of φHP3 was performed using the resistant strain JJ2528-12 as a target. φHP3 was adapted to the target in a continuous-flow bioreactor using a method similar to the Appelman method. (Figure 12B) Isolate titers were measured in liquid culture after 4.5 hours of incubation in LB medium in the presence or absence of evolved φHP3.1 (top panel). The titers of φHP3.1 against these isolates were also measured using a standard phage spot assay (bottom panel). Figure 12B is grouped by parent strain: JJ2050 (i), JJ2528 (ii), and JJ2547 (iii). P values ​​were calculated by Student's t test or Mann-Whitney test, as appropriate. *, P<0.05; **, P<0.01; ***, P<0.001. Each data point represents the mean of three parallel technical replicates. [Figure 13A-B] (Figure 13A) Sequencing revealed a key missense mutation in the binding spike of φHP3.1 (highlighted in red). (Figure 13B) Protein modeling revealed that the amino acid substitution was located in the binding region of the tail protein, based on the tail spike structure of φT4. [Figures 14A-14C]Two φHP3-resistant strains, JJ2528-5 and JJ2528-12, were used to select φHP3.1-resistant isolates using the culture-derived method described in Figure 7B. (Figure 14A) Phage (φHP3 and φHP3.1) titers were measured by spot assay against the parental, primary, and secondary resistant strains. (Figure 14B) Isolate titers were measured in liquid culture after 4.5 hours of incubation in LB medium (black) or urine (yellow) in the presence or absence of φHP3 or φHP3.1. (Figure 14C) The secondary resistant strain was subjected to whole-genome sequencing and aligned with the parental and primary resistant strains. The secondary resistant strain retained the truncation of the waa operon and also acquired a truncation of ompA. The dashed lines in the panels indicate downstream SNPs in ompA. P values ​​in Figures 14A and 14B were calculated by Student's t-test or Mann-Whitney test, as appropriate. *, P<0.05, **, P<0.01, ***, P<0.001. Each data point represents the mean of three parallel technical replicates. [Figure 15] Phage-resistant isolates are shown. A total of 21 φHP3-resistant isolates were obtained from three parent strains and two selection methods. Each isolate and its origin are described. [Figure 16] Figure 1 shows the results of phage screening for resistant isolates. Isolates were screened by spot assay against φHP3, φHP3.1, φEC1 (similar to φHP3), and φES17 (dissimilar to φHP3). Phage titers are shown where applicable. Green boxes indicate plaque formation, and red boxes indicate resistance to the phage. The plaque formation efficiency of φHP3.1 is shown relative to that of φHP3 against the parent strain ExPEC. [Figure 17A-B] (Figure 17A) Growth (measured by OD600) of E. coli treated with a phage cocktail (HP3 and ES17). (Figure 17B) Growth of E. coli treated with a phage cocktail (φHP3, φHP3.1, and φHP3.2). [Figure 18] Colony forming units (CFU) of E. coli on human catheters either untreated or treated with the indicated phage cocktails are shown. [Figure 19] 1 shows molecular models for the protein products of the tail fiber genes of φHP3, φHP3.1, and φHP3.2. φHP3.1 and φHP3.2 each contain mutations in the tail fiber gene, and the mutant proteins are thought to interact with a receptor in E. coli. [Figure 20] This figure shows the results of an analysis of mice infected with E. coli and treated with HP3 phage or a phage cocktail containing HP3, HP3.1, and ES17. Standard deviations and means are shown. N=10 per group. Statistical significance was determined using two-way ANOVA. ***P<0.001, ****P<0.0001. [Figure 21] This study demonstrates the efficacy of a phage cocktail (HP3, HP3.1, and ES17) in reducing bacterial levels in mice infected with pathogenic Escherichia coli (E. coli). Mice were infected with the pathogenic pandemic strain ST131 E. coli, ExPEC strain 2050 (1 × 10 CFU). After 24 hours, mice were subcutaneously injected with 1 × 10 PFU (total) of phages (3, 3.1, and ES17) or left untreated. After another 24 hours, mice were necropsied and bacterial levels were measured by organ swabbing. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present disclosure is based, at least in part, on the discovery and development of novel bacteriophages and bacteriophage combinations for the treatment and prevention of E. coli infections, including multidrug-resistant (MDR) E. coli infections. In this regard, in certain embodiments, disclosed herein are bacteriophage compositions comprising one or more of ES17, ES19, HP3, HP3.1, and HP3.2. Particular aspects of the disclosure relate to compositions comprising (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1. Also disclosed are compositions comprising bacteriophage HP3.2. Additionally, devices and kits comprising such compositions, as well as methods of using the compositions in the treatment and prevention of pathogenic E. coli infections, are also disclosed.

[0027] I. Bacteriophage Compositions Aspects of the present disclosure relate to compositions comprising one or more bacteriophages. As used herein, a "bacteriophage composition" refers to any composition comprising one or more bacteriophages (also referred to as "phages"). The bacteriophages of the present disclosure may be lytic phages. The bacteriophages of the present disclosure may be phages capable of infecting one or more pathogenic bacteria. The bacteriophages of the present disclosure may be phages capable of infecting one or more bacteria belonging to the Enterobacteriaceae family. In certain embodiments, the bacteriophages of the present disclosure are phages capable of infecting Escherichia coli (E. coli), such as multidrug-resistant (MDR) E. coli and / or extraintestinal pathogenic E. coli (ExPEC). The bacteriophage composition can include, for example, bacteriophage generated using the methods or systems described in WO 2020 / 264096, entitled "Systems and Methods For Generating Bacteriophages Adapted To Infect A Target Bacterial Strain," which is incorporated herein by reference in its entirety.

[0028] Bacteriophage compositions of the present disclosure may include one or more bacteriophages, for example, capable of lysing pathogenic bacteria. Bacteriophage compositions of the present disclosure may include at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different bacteriophages. In some embodiments, bacteriophage compositions of the present disclosure include one or more of ES17, ES19, HP3, HP3.1, and HP3.2. In some embodiments, bacteriophage compositions of the present disclosure include ES17. In some embodiments, bacteriophage compositions of the present disclosure include ES19. In some embodiments, bacteriophage compositions of the present disclosure include HP3. In some embodiments, bacteriophage compositions of the present disclosure include HP3. In some embodiments, bacteriophage compositions of the present disclosure include HP3.2. In some embodiments, the bacteriophage composition comprises two or more of ES17, ES19, HP3, HP3.1, and HP3.2. In some embodiments, the bacteriophage composition comprises three or more of ES17, ES19, HP3, HP3.1, and HP3.2. In some embodiments, the bacteriophage composition comprises four or more of ES17, ES19, HP3, HP3.1, and HP3.2. In some embodiments, the bacteriophage composition comprises (a) ES17 or ES19, (b) HP3, and (c) HP3.1. In some embodiments, the bacteriophage composition comprises ES17, HP3, and HP3.1. In some embodiments, the bacteriophage composition comprises ES19, HP3, and HP3.1. In some embodiments, the bacteriophage composition comprises ES17, HP3, HP3.1, and HP3.2. In some embodiments, the bacteriophage composition comprises ES19, HP3, HP3.1, and HP3.2. In some embodiments, the bacteriophage composition comprises ES17, ES19, HP3, HP3.1, and HP3.2.

[0029] The bacteriophage compositions of the present disclosure may include one or more additional bacteriophages (e.g., additional bacteriophages capable of lysing pathogenic E. coli) in addition to one or more of ES17, ES19, HP3, HP3.1, and HP3.2. Additional bacteriophages contemplated herein include, but are not limited to, bacteriophages EC1, CF2, ES12, ES21, and ES26. These bacteriophages are described, for example, in Gibson SB, Green SI, Liu CG, et al. Constructing and Characterizing Bacteriophage Libraries for Phage Therapy of Human Infections. Front Microbiol. 2019;10:2537, which is incorporated herein by reference in its entirety.

[0030] The bacteriophage compositions of the present disclosure may include one or more metals in addition to one or more bacteriophages. The one or more metals may include, for example, calcium, magnesium, iron, sodium, and / or potassium. Bacteriophage compositions containing two or more different bacteriophages may vary in the amount of each bacteriophage. For example, a composition may contain substantially the same amount of each bacteriophage. Alternatively, a composition may contain substantially different amounts of each bacteriophage.

[0031] The bacteriophage composition may comprise at least, at most, or about 10 for each of one or more bacteriophages in the composition. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12In certain embodiments, the bacteriophage composition may contain at least, at most, or about 10 plaque-forming units (PFU) for ES17. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 In one embodiment, the bacteriophage composition comprises at least, at most, or about 10 PFU for ES19. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 In one embodiment, the bacteriophage composition comprises at least, at most, or about 10 PFU for HP3. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 In one embodiment, the bacteriophage composition comprises at least, at most, or about 10 PFU for HP3.1. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 In one embodiment, the bacteriophage composition comprises at least, at most, or about 10 PFU for HP3.2. 3 , 10 4 , 10 5 , 106 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 Including PFU.

[0032] (A.ES17) Embodiments of the present disclosure include bacteriophage ES17 (also referred to interchangeably herein as "Escherichia phage ES17," "φES17," or "ES17"), compositions, kits, and devices comprising ES17, and methods of use thereof. The genomic sequence of ES17 is described by GenBank® Accession No. MN508615 and is provided as SEQ ID NO:3.

[0033] (B.ES19) Embodiments of the present disclosure include bacteriophage ES19 (also referred to interchangeably herein as "Escherichia phage ES19," "φES19," or "ES19"), compositions, kits, and devices comprising ES19, and methods of use thereof. The genomic sequence of ES19 is described by GenBank® Accession No. MN508616 and is provided as SEQ ID NO:7.

[0034] (C.HP3) Aspects of the present disclosure include bacteriophage HP3 (also referred to interchangeably herein as "Escherichia phage HP3," "φHP3," or "HP3"), compositions, kits, and devices comprising HP3, and methods of use thereof. The genomic sequence of HP3 is described by GenBank® Accession No. NC_041920 and is provided as SEQ ID NO:4.

[0035] (D.HP3.1) Aspects of the present disclosure include bacteriophage HP3.1 (also referred to interchangeably herein as "Escherichia phage HP3," "φHP3.1," or "HP3.1"), compositions, kits, and devices comprising HP3.1, and methods of using the same. The genomic sequence of HP3.1 is described by GenBank® Accession No. OK275722.1 and is provided as SEQ ID NO:5.

[0036] (E.HP3.2) Aspects of the present disclosure include bacteriophage HP3.2 (also referred to interchangeably herein as "Escherichia phage HP3," "φHP3.2," or "HP3.2"), compositions, kits, and devices comprising HP3.2, and methods of use thereof. The genomic sequence of HP3.2 is provided as SEQ ID NO:6.

[0037] [II. Treatment and prevention of bacterial infections] Aspects of the present disclosure relate to methods for treating and preventing bacterial infections in individuals. In some embodiments, disclosed are methods for treating or preventing an E. coli infection in an individual. In certain aspects, the present disclosure provides methods for treating or preventing an E. coli infection in an individual, comprising administering to the individual an effective amount of one or more bacteriophages, including those disclosed herein. Bacteriophages useful in such treatment methods include those described herein, such as ES17, ES19, HP3, HP3.1, and / or HP3.2. Accordingly, in some embodiments, disclosed herein are methods for treating or preventing an E. coli infection in an individual, comprising administering to the individual an effective amount of ES17, ES19, HP3, HP3.1, and / or HP3.2. In some embodiments, the method comprises administering (a) ES17 or ES19, (b) HP3, and (c) HP3.1. In some embodiments, the method comprises administering ES17, HP3, and HP3.1. In some embodiments, the method comprises administering ES17, HP3, and HP3.2. In some embodiments, the method comprises administering HP3.2. In some embodiments, the method comprises administering ES17, HP3, HP3.1, and HP3.2. In some embodiments, the method comprises administering ES19, HP3, HP3.1, and HP3.2. In some embodiments, the method comprises administering ES17, ES19, HP3, HP3.1, and HP3.2.

[0038] In some embodiments, multiple bacteriophages (e.g., two or more of ES17, ES19, HP3, HP3.1, and HP3.2) are administered to an individual in the same formulation. Alternatively, multiple bacteriophages may be administered to an individual in different formulations (e.g., two, three, or more formulations). Multiple bacteriophages may be administered to an individual at the same time or at different times.

[0039] Multiple bacteriophages may be administered to an individual substantially simultaneously, for example, via a single composition. For example, ES17, HP3, and HP3.1 may be administered simultaneously as a single composition to an individual with a pathogenic E. coli infection. As another example, ES19, HP3, and HP3.1 may be administered simultaneously as a single composition to an individual with a pathogenic E. coli infection. As yet another example, ES17, HP3, HP3.1, and HP3.2 may be administered simultaneously as a single composition to an individual with a pathogenic E. coli infection. As a further example, ES19, HP3, HP3.1, and HP3.2 may be administered simultaneously as a single composition to an individual with a pathogenic E. coli infection.

[0040] Multiple bacteriophages may be administered to an individual sequentially in any order. For example, an individual with a pathogenic E. coli infection may be administered ES17, followed by HP3, followed by HP3.1. Exemplary administration sequences for ES17, HP3, and HP3.1 include: ES17, HP3, HP3.1; HP3, ES17, HP3.1; HP3.1, ES17, HP3; ES17, HP3.1, HP3; HP3, HP3.1, ES17; and HP3.1, HP3, ES17. Exemplary administration sequences for ES19, HP3, and HP3.1 include: ES19, HP3, HP3.1; HP3, ES19, HP3.1; HP3.1, ES19, HP3; ES19, HP3.1, HP3; HP3, HP3.1, ES19; and HP3.1, HP3, ES19. Exemplary administration sequences for ES17, HP3, HP3.1 and HP3.2 include: ES17, HP3, HP3.1, HP3.2; HP3, ES17, HP3.1, HP3.2; HP3.1, ES17, HP3, HP3.2; ES17, HP3.1, HP3, HP3.2; HP3, HP3.1, ES17, HP3.2; HP3.1, HP3, ES17, HP3.2; HP3.2, HP3, ES17, HP3.1; HP3, HP3.2, ES17, HP3.1; ES17, HP3.2, HP3, HP3.1; HP3.2, ES17, HP3.1; HP3, ES17, HP3.2, HP3, HP3.1; HP3.2, ES17, HP3, HP3.1; HP3, ES17, HP3.2, HP3.1; ES17, HP3 , HP3.2, HP3.1;ES17, HP3.1, HP3.2, HP3;HP3.1, ES17, HP3.2, HP3;HP3.2, ES17, HP3.1, HP3;ES17, HP3.2, HP3.1, HP3;HP3.1, HP3.2, ES17, HP3;HP3.2, HP3.1, ES17, HP3;HP3.2, HP3.1, HP3, ES17;HP3, HP3.2, HP3.1, ES17;HP3, HP3.2, HP3.1, ES17;HP3, HP3.2, HP3, HP3.1, ES17;HP3.2, HP3, HP3.1, ES17;HP3.1, HP3, HP3.2, ES17; and HP3, HP3.1, HP3.2, ES17.Exemplary administration sequences for ES19, HP3, HP3.1 and HP3.2 include: ES19, HP3, HP3.1, HP3.2; HP3, ES19, HP3.1, HP3.2; HP3.1, ES19, HP3, HP3.2; ES19, HP3.1, HP3, HP3.2; HP3, HP3.1, ES19, HP3.2; HP3.1, HP3, ES19, HP3.2; HP3.2, HP3, ES19, HP3.1; HP3, HP3.2, ES19, HP3.1; ES19, HP3.2, HP3, HP3.1; HP3.2, ES19, HP3.2, HP3, HP3.1; HP3.2, ES19, HP3, HP3.1; HP3, ES19, HP3.2, HP3.1; ES19, HP3 , HP3.2, HP3.1;ES19, HP3.1, HP3.2, HP3;HP3.1, ES19, HP3.2, HP3;HP3.2, ES19, HP3.1, HP3;ES19, HP3.2, HP3.1, HP3;HP3.1, HP3.2, ES19, HP3;HP3.2, HP3.1, ES19, HP3;HP3.2, HP3.1, HP3, ES19;HP3.1, HP3.2, HP3, ES19;HP3, HP3.2, HP3.1, ES19;HP3.2, HP3, HP3.1, ES19;HP3.1, HP3, HP3.2, ES19; and HP3, HP3.1, HP3.2, ES19. One or more of the above administration sequences may be excluded from embodiments of the present disclosure.

[0041] The bacteriophages of the present disclosure may be administered to an individual once or multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more times). The bacteriophages may be administered in an appropriate treatment regimen and for an appropriate period of time for effective treatment or prevention of pathogenic E. coli infection. For example, the bacteriophage composition may be administered to an individual 1, 2, 3, or 4 (or more) times per day; 1, 2, 3, 4, 5, 6, or 7 (or more) times per week; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or more) times per month. The bacteriophage composition may be administered for at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days (or more); 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 weeks (or more); or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (or more). In some embodiments, the bacteriophage is administered twice a week for between 6 and 12 weeks.

[0042] An individual receiving treatment according to the present disclosure may have, for example, an infection in one or more of the urinary tract, blood, intestines, abdomen, stomach, lungs, skin, kidneys, prostate, bladder, brain, vaginal tract, heart, liver, and spleen. In some embodiments, the individual has a urinary tract infection. In some embodiments, the individual has a catheter-associated urinary tract infection. The individual may have one or more of a urinary tract infection, neonatal meningitis, bloodstream infection, pneumonia, sepsis, surgical wound infection, skin infection, prostate infection, meningitis, and vaginal infection. The individual may have one or more symptoms of a pathogenic infection, such as, for example, diarrhea, stomach cramps, nausea, and / or vomiting. In some embodiments, the individual does not have any symptoms of a pathogenic infection.

[0043] In some embodiments, the individual receiving treatment according to the present disclosure has an E. coli infection. In some embodiments, the individual has been diagnosed with an E. coli infection. In some embodiments, the individual has one or more symptoms of an E. coli infection (e.g., including diarrhea, stomach cramps, nausea, and / or vomiting). In some embodiments, the individual does not have any symptoms of an E. coli infection. In some embodiments, the E. coli is multidrug-resistant (i.e., multidrug-resistant E. coli). In some embodiments, the E. coli is extraintestinal pathogenic E. coli. Extraintestinal pathogenic E. coli is described, for example, in Smith JL, Fratamico PM, Gunther NW. Extraintestinal pathogenic Escherichia coli. Foodborne Pathog Dis. 2007;4(2):134-163, incorporated herein by reference in its entirety. The individual may have an E. coli infection from a variety of sources. For example, in some embodiments, E. coli originates from beverages, edible materials (e.g., undercooked meat, unpasteurized milk, apple juice, or cider; or soft cheese made from raw milk), other individuals, or the environment (e.g., groundwater or surface water, water used to irrigate crops, public water systems, hospitals, schools, nursing homes, petting zoos, cruise ships, trains, or airplanes).

[0044] The individual receiving the treatment of the present disclosure may be an immunosuppressed individual. The immunosuppressed individual may be, for example, an individual with immune cell deficiency, asplenia, impaired splenic function, nephrotic syndrome, or an autoimmune disease. The immunosuppressed individual may be an individual exposed to an immunosuppressive condition, for example, an individual exposed to a chemotherapeutic or immunosuppressive agent (e.g., a glucocorticoid, a calcineurin inhibitor, an antimetabolite, a gastric acid suppressant (e.g., a proton pump inhibitor), or antibody therapy). Thus, in certain embodiments, a bacteriophage of the present disclosure may be administered to an individual before, during, and / or after exposure to an immunosuppressive condition. For example, a bacteriophage of the present disclosure (e.g., ES17, ES19, HP3, HP3.1, and / or HP3.2) may be administered to an individual with cancer undergoing chemotherapy treatment to treat or prevent pathogenic E. coli infection.

[0045] [III. Equipment] Also contemplated herein, in certain embodiments, are devices (e.g., medical devices) comprising one or more bacteriophages or bacteriophage compositions of the present disclosure. The devices of the present disclosure may comprise one or more of ES17, ES19, HP3, HP3.1, and HP3.2. In certain embodiments, the devices comprise (a) ES17 or ES19, (b) HP3, and (c) HP3.1. In certain embodiments, the devices comprise ES17, HP3, and HP3.1. In certain embodiments, the devices comprise ES19, HP3, and HP3.1. In certain embodiments, the devices comprise HP3.2. In certain embodiments, the devices comprise ES17, HP3, HP3.1, and HP3.2. In certain embodiments, the devices comprise ES19, HP3, HP3.1, and HP3.2. As used herein, when a device is described as "comprising" a bacteriophage or bacteriophage composition, it refers to a bacteriophage or bacteriophage composition present within, on, around, or attached to the device. In some embodiments, the device of the present disclosure is a medical device. In such cases, a medical device containing a bacteriophage capable of treating or preventing a pathogenic infection, such as an E. coli infection, may be desirable. Various medical devices are known in the art and are contemplated in this disclosure. Examples of devices contemplated herein include, but are not limited to, catheters, drivelines, syringes, tubing, implants, defibrillators, artificial joints, pacemakers, screws, rods, discs, intrauterine devices, pins, plates, stents, dental devices, intraocular lenses, shunts, valves, neurological or neurosurgical devices, gastrointestinal devices, genitourinary devices, catheter cuffs, vascular access devices, and wound drains. In some embodiments, the device is a stent. In some embodiments, the device is a catheter. In one embodiment, the device is an implant.

[0046] One aspect of the present disclosure relates to a method of manufacturing a device, comprising exposing the device to a bacteriophage composition of the present disclosure (e.g., a bacteriophage composition comprising ES17, HP3, and HP3.1). Disclosed methods include disposing the bacteriophage composition on, around, or within the device, and coating the device (e.g., coating one or more surfaces of the device) with the bacteriophage composition. Further disclosed are embodiments that include delivering the device to an individual after such manufacturing.

[0047] IV. Pharmaceutical Compositions The compositions or agents used in the methods of the present disclosure, e.g., bacteriophages (e.g., ES17, ES19, HP3, HP3.1, and / or HP3.2), may be contained in a suitable pharmaceutically acceptable carrier. In certain embodiments, the carrier is selected to be non-toxic, biocompatible, and not adversely affect the biological activity of the agent. In some aspects of the disclosure, the agents are formulated for local delivery (i.e., delivery to a specific site in the body) or systemic delivery in solid, semi-solid, gel, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, inhalants, and injections, allowing for oral, parenteral, or surgical administration. Certain aspects of the disclosure also contemplate local administration of the compositions by coating medical devices, etc.

[0048] Suitable carriers for parenteral administration by injection, infusion, or perfusion, and for topical administration, include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solution, dextrose solution, Hank's solution, or propanediol. Sterile, fixed oils may also be used as solvents or suspending media. Any biocompatible oil, including synthetic monoglycerides or diglycerides, can be used for this purpose. Additionally, fatty acids such as oleic acid are also used in the preparation of injectables. The carrier and agent may be formulated as a liquid, suspension, polymeric or non-polymeric gel, paste, or ointment.

[0049] Carriers may also include delivery vehicles to sustain (i.e., extend, delay, or modulate) drug delivery or to enhance the delivery, uptake, stability, or pharmacokinetics of therapeutic agents, including, but not limited to, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels, and polymeric micelles.

[0050] In certain embodiments, the actual amount of composition administered to a patient or subject will depend on physical and physiological factors, such as body weight, severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, patient characteristics, and the route of administration. The professional responsible for administration will, in any event, determine the concentration of active ingredient(s) in the composition and the appropriate dose for that subject.

[0051] Solutions of the pharmaceutical compositions may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0052] In some embodiments, pharmaceutical compositions are advantageously administered as injectable compositions, either as liquid solutions or suspensions. They can also be prepared in solid forms suitable for dissolving or suspending in liquid prior to injection. These preparations may also be emulsified. Typical compositions for such purposes include a pharmaceutically acceptable carrier. For example, the composition may contain up to 10 milligrams, 25 milligrams, 50 milligrams, or up to about 100 milligrams of human serum albumin per milliliter of phosphate-buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients (including salts, preservatives, buffers, etc.).

[0053] Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters (such as ethyl oleate). Aqueous carriers include parenteral carriers such as water, alcoholic / aqueous solutions, saline, sodium chloride, Ringer's dextrose, and the like. Intravenous carriers include fluid and nutrient replenishers. Preservatives include antibacterial agents, antifungal agents, antioxidants, chelating agents, and inert gases. The pH and precise concentration of each component in the pharmaceutical composition are adjusted according to well-known parameters.

[0054] Additional formulations are suitable for oral administration. Oral formulations include typical excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders.

[0055] In further embodiments, the pharmaceutical composition may comprise a classical pharmaceutical formulation. Administration of the pharmaceutical composition according to certain embodiments can be via any common route, as long as the target tissue is accessible via that route. This includes oral, nasal, buccal, rectal, vaginal, or transdermal administration. Alternatively, administration may be via in situ, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. The composition is typically administered as a pharmaceutically acceptable composition containing a physiologically acceptable carrier, buffer, or other excipient. Aerosol administration can be used to treat pulmonary diseases. The volume of the aerosol may be about 0.01 ml to 0.5 ml.

[0056] Precise amounts of pharmaceutical compositions depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dosage include the patient's physical and clinical condition, the route of administration, the therapeutic objectives (e.g., relief of symptoms or cure), and the efficacy, stability, and toxicity of the therapeutic agent.

[0057] (A. Other drugs) To enhance the therapeutic effect of certain aspects of this embodiment, it is contemplated that other drugs may be used in combination. These additional drugs include drugs with antibacterial properties (e.g., antibiotics). For example, one or more of the therapeutic phages of the present disclosure can be used in combination with one or more antibiotics. A variety of antibiotics are recognized in the art and are contemplated herein, including, for example, ceftazidime, ciprofloxacin, kanamycin, colistin (polymyxin E), trimethoprim, cefepime, sulfamethoxazole, levofloxacin, and polymyxin B.

[0058] [V.Kit] Certain aspects of the present disclosure also relate to kits comprising compositions or compositions for practicing the methods disclosed herein. In certain embodiments, the kits can be used to evaluate one or more biomarkers. In certain embodiments, the kits can include, at least include, or at most include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000, or more probes, primers or primer sets, synthetic molecules, or inhibitors, or any number from any range or combination thereof. In certain embodiments, kits for assessing biomarker activity in cells are included.

[0059] The kit may contain components individually packaged or disposed within tubes, bottles, vials, syringes or other suitable container means.

[0060] Individual components may be provided in the kit in a concentrated form. In some embodiments, components may be provided individually at the same concentration as they would be in a mixed solution with other components. Component concentrations may be provided at 1x, 2x, 5x, 10x, or 20x or greater.

[0061] In some embodiments, kits of the present disclosure include one or more bacteriophages housed in a suitable container. The kits may include, at least include, at most include, or exactly include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different bacteriophages. In some embodiments, kits of the present disclosure include one, two, three, four, or all of ES17, ES19, HP3, HP3.1, and HP3.2. One or more phages may be excluded from certain embodiments. In some embodiments, kits of the present disclosure include ES17, HP3, and HP3.1. In some embodiments, kits of the present disclosure include ES19, HP3, and HP3.1. In some embodiments, kits of the present disclosure include HP3.2. In some embodiments, kits of the present disclosure include ES17, HP3, HP3.1, and HP3.2. In some embodiments, kits of the present disclosure include ES19, HP3, HP3.1, and HP3.2. In some embodiments, a kit of the present disclosure includes ES17, HP3, HP3.1, and HP3.2. The phage may be contained in a suitable container. The kit of the present disclosure may further include a device. The kit may include a device having one or more bacteriophages of the present disclosure disposed on, within, or around the device. The kit may include a device separate from the one or more bacteriophages. [Example]

[0062] The following examples are included to demonstrate specific embodiments of the present invention. It should be understood by those of skill in the art that the techniques disclosed in the examples below are techniques discovered by the inventors to function well in the practice of the invention and can be considered to constitute aspects of embodiments of the invention. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made to the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0063] Example 1: Targeting mammalian glycans to promote phage phagocytosis in the gastrointestinal tract The gastrointestinal tract presents a barrier to phage therapy. Previous studies have reported that phage HP3, a lytic myovirus isolated from an environmental reservoir of ExPEC, reduces ST131 bacteremia and disease severity in a mouse infection model. Given that the human gastrointestinal tract is the primary reservoir of ExPEC ST131, we investigated whether phage HP3 could act prophylactically to reduce or eliminate the intestinal ExPEC burden. To test this, mice were orally gavaged with the ExPEC ST131 clinical isolate JJ1901 and then treated with phage or antibiotics, as shown in Figure 1A. Stable bacterial colonization was observed in the untreated group throughout the experimental period (6 days) (Figure 1B). When mice were administered phage HP3 via water or daily oral administration, ExPEC levels at the end of the experiment were indistinguishable from those in the untreated control group. An increase in CFU was observed on day 2, but remained constant from day 3 onwards in all groups. In the antibiotic-treated group, no ExPEC were detected at any time point. Interestingly, phage HP3 was still detected and active on day 4 when feces from phage-treated mice were applied to an ExPEC overlay plate and a plaque assay was performed. This indicates that the reduction in ExPEC was not due to insufficient phage delivery to the intestinal environment or phage inactivation. Furthermore, despite the administration of high concentrations of phage between days 1 and 4, the ExPEC removal effect was not superior to that observed when phage was added to water, indicating that a dose-dependent effect of phage was not observed in this experiment. Furthermore, on day 6, the final day of the experiment, a maximum of 10 5Although phage PFU / g were detected in mouse intestinal tissues (including the cecum and colon), there was little elimination of ExPEC compared to untreated controls (Figures 1C and 1D). Importantly, antibiotic treatment significantly reduced the number and diversity of operational taxonomic units (OTUs) based on the Shannon diversity index, which considers species richness and distribution (P = 0.007, P = 0.009). Furthermore, principal component analysis (PCA) of beta diversity revealed that the antibiotic group clustered distinctly from the untreated and phage groups, suggesting that antibiotics had a stronger effect on the gut microbiome than phages. Finally, we evaluated the bactericidal activity of phages in modified cecal medium (CM), which is prepared by homogenizing cecal contents collected from mice immediately after euthanasia in sterile saline and centrifuging to remove large particles. This medium is designed to mimic the complexity of the mammalian intestinal lumen by containing feces, microbiome, mucus, and small molecules and proteins likely to be present in the intestinal lumen. While phage HP3 completely killed ExPEC in LB medium (approximately 9-order reduction, no viable organisms detected) and nearly killed fecal slurry from the same mice (approximately 8-order reduction), it showed little or no bactericidal effect in CM. This is shown in Figures 1E and 1F. 6 ~10 7.5 These results are consistent with those from the ExPEC colonization model and suggest that factors that inhibit this lytic phage exist in the mammalian gastrointestinal tract.

[0064] The inhibitory component is mucin. We sought to understand why phage HP3 was ineffective in this intestinal microenvironment. Next, we investigated whether the inhibition was related to the presence of viable bacterial flora in CM. However, eliminating the microbiota through widespread use of antibiotics that inhibit protein synthesis, cell wall synthesis, and DNA synthesis did not enhance phage-mediated killing of ExPEC (although these antibiotics effectively killed antibiotic-susceptible commensal E. coli added to CM). Without a clear understanding of the inhibitory factor, we decided to test a more potent treatment that could restore phage-mediated killing in CM. First, we heat-treated CM (HT CM) (see Figure 2A). Interestingly, heat treatment resulted in a 0.6-log improvement in phage HP3-mediated killing of ExPEC (P<0.0001). Similarly, when CM was filtered (0.22 μm filter) (FT CM), there were no detectable levels of ExPEC in the medium after treatment with phage HP3 (see Figure 2A). This observation extended to another phage, EC1, that showed inhibition in CM (P < 0.0001). Therefore, this inhibitory component appears to be large in size (retained by the 0.22 μm filter) and heat-inactivated.

[0065] We hypothesized that intestinal mucins might fit these characteristics because they are highly cohesive and sticky (captured by filters) and, as proteins, are denatured by heat. Mucins are glycoproteins present throughout the gastrointestinal tract, forming a layer between intestinal epithelial cells (IECs) and commensal or pathogenic microbiota. Mucins can also function as receptors for microorganisms. To verify whether mucins inhibit phage-mediated killing, we devised a method to separate CM into a soluble fraction (S CM) and an insoluble fraction (INS CM) by ultracentrifugation (see Figure 2B). We hypothesized that INS CM, which typically contains large intestinal mucins that inhibit phage-mediated killing, would lack these properties in S CM. Indeed, untreated CM and INS CM inhibited phage-mediated killing more strongly than S CM (see Figure 2B). To further directly test the hypothesis that mucin is an inhibitory factor, we added the expectorant N-acetylcysteine ​​(NAC) to INS CM and porcine stomach mucin (1.5% [wt / vol]) to S CM. Indeed, the addition of NAC to INS CM reduced phage killing activity against ExPEC by 5 logs, restoring it to the same killing level as in S CM (P < 0.0001). Even more convincingly, the addition of mucin to S CM inhibited phage killing to the level seen with CM alone (not statistically significant, P = 0.970), supporting this hypothesis. These results indicate that intestinal mucin is the inhibitor of phage killing in cecal-derived media.

[0066] In this context, E. coli is known to utilize mucin as a carbon source. We hypothesized that NAC treatment in mice colonized with ExPEC would liberate mucin for bacterial utilization, leading to bacterial growth. Consequently, this could serve as a system to test whether the reduction in mucin aggregation enhances the ExPEC-killing activity of phage HP3. Two weeks of NAC administration increased ExPEC levels in the mouse small intestine, and phage HP3 reduced ExPEC levels, although this was not statistically significant. A similar trend was observed in the large intestine. The poorer effect in the large intestine compared to the small intestine may be due in part to the thicker mucus layer in the large intestine, which reduces the effectiveness of NAC in breaking down this mucus. Furthermore, NAC is known to be rapidly absorbed by small intestinal tissue, which may result in its loss of efficacy in the distal colon.

[0067] Discovery of a phage whose activity is enhanced by mucin. We hypothesized that human sewage or animal feces might contain phages that have evolved to target hosts in mucin-rich environments such as the intestinal tract. We screened a phage library and phages recently isolated from these environments for enhanced activity in LB medium containing 1.5% mucin (Figure 2C). This concentration was confirmed to inhibit the activity of phage HP3 in soluble cecal medium and to exhibit a strong inhibitory effect in LB medium for up to 8 hours. Surprisingly, only one phage, designated phage ES17 (GenBank® accession number: MN508615), significantly reduced bacteria (Figure 2C) (P<0.0001, approximately 1-log reduction). Phage ES17 was active in the presence of mucin, but its effectiveness was significantly lower (0.3 log) than that of phages HP3, J2W, Ult1, Shp1, or M1S, which completely killed ExPEC to undetectable levels in LB medium alone. However, it still retained its activity in the presence of mucin. Consistent with these data, we compared the lytic activity of phages HP3 and ES17 against ExPEC at mucin concentrations ranging from 0% to 2%. Phage HP3 was highly effective at mucin concentrations of 0.5% but was completely inhibited at higher concentrations. On the other hand, phage ES17 exhibited the highest activity at mucin concentrations (0.5%-1%) at which HP3 was inactive, and its activity decreased at lower or higher concentrations. Taken together, these results suggest that phage ES17 possesses unique properties that allow it to efficiently lytically kill ExPEC even in the presence of mucin.

[0068] Phage ES17 is a C3-type phage whose activity is enhanced by mucin. We sought to elucidate the molecular mechanism by which phage ES17's ability to locate and lyse host bacteria in mucin is enhanced. Phage ES17 is a double-stranded DNA (dsDNA) virus classified in the order Caudovirales, family Podoviridae, and genus Kuravirus. PhiEco32, a Kuravirus phage, shows high genetic similarity to ES17. Kuravirus phages have an unusual morphology—an elongated C3-type capsid, a short tail fiber, and a small genome. ES17 shares these morphological characteristics with a capsid length of approximately 100 nm (Figure 3A). ES17 has a small genome consisting of 75,007 base pairs and 123 predicted open reading frames (ORFs) (MN508615).

[0069] To clarify why this phage differs from other phages that lack activity in mucin-rich environments, we investigated the ability of phages ES17 and HP3 to adsorb to their respective E. coli hosts. Previous data showed that 98% of HP3 was adsorbed within 10 minutes, while only 32% of ES17 was adsorbed within that time frame. Furthermore, no significant differences were observed between ES17 and HP3 in one-step growth curve parameters, such as burst size (ES17, 36; HP3, 60) and incubation period (ES17, 32 min; HP3, 22.5 min). We hypothesized that the addition of mucin might improve the adsorption of phage ES17 and inhibit the adsorption of phage HP3. For this experiment, we utilized a modified adsorption assay using ExPEC and mucin. Briefly, bacteria were incubated in different concentrations of mucin (0%–1.5%), pelleted, and washed to remove the mucin (Figure 3B). Next, we performed standard adsorption assays using phages ES17 and HP3. Interestingly, phage ES17 showed no adsorption to ExPEC within 10 min in the absence of mucin, but preincubation with 1.5% mucin increased the adsorption rate to 98% (Figure 3C). In contrast, phage HP3 showed 98% adsorption in the absence of mucin, but this decreased to 84% in the presence of mucin (Figure 3D). Finally, we applied an enzyme-linked immunosorbent assay (ELISA)-like technique to examine whether phage ES17 prefers to bind to mucin-coated surfaces, a hypothesis consistent with our data. Indeed, when mucin was immobilized on an ELISA plate, phage ES17 bound to the mucin surface at a higher level than phage HP3 (Figure 3E) (P = 0.041 and P = 0.002, respectively). These different approaches suggest that phage ES17 has a mucin-binding property that may enhance its ability to infect E. coli in mucin-rich environments.

[0070] Phage ES17 binds to human heparan sulfate proteoglycans. Compared to other coliphages, phage ES17 has an enhanced ability to find its bacterial host in environments where carbohydrates are the major chemical component (such as cecal medium and mucin-rich broth, as mentioned above). BLAST analysis revealed that the putative tail fiber protein of ES17 (ES17-TFP) showed high similarity (64% similarity, E-value 0) to that of another lytic podophage, T7-like bacteriophage LM33_P1 (YP_009324518.1). LM33_P1 also targets ST131 strains. The tail fiber of T7-like phages has been shown to possess an endosialidase that targets surface sugars, such as capsule-forming polysaccharides. BLAST analysis revealed that ES17-TFP contains a putative pectinesterase domain (E-value: 7.45e203, 369 bp). This domain has only been found in four other phages (myPSH1131, myPSH2311, vB_EcoS_Golestand, and LM33_P1), of which only myPSH1131, like ES17, has this domain within the tail fiber protein.

[0071] Structural analysis of the modeled ES17-TFP showed high similarity to the phage K5 lyase-binding domain (E value = 4e212). Phage K5 binds to the K5 capsule polysaccharide and functions as a K5 polysaccharide lyase. The K5 E. coli capsule is composed of repeating disaccharides identical to the precursors of heparin and heparan sulfate (HS), which are linear polysaccharides present in glycosaminoglycans (heparan sulfate proteoglycans [HSPGs]). These proteoglycans are present on mammalian cells and in mucus. Furthermore, mucins (α-linked glucosamine or N-acetyl-D-glucosamine) with structures similar to heparan sulfate / heparin are also present in the intestinal tract and are contained in porcine gastric mucin (PGM).

[0072] We hypothesized that the enhanced activity of ES17 might be due to its ability to bind mammalian polysaccharides present on glycoproteins, as other research groups have found with different phage types. However, none of these groups had identified heparan sulfate proteoglycans, glycoproteins ubiquitously present in the basement membrane and surface of various cell types, as a potential receptor for this interaction. This mechanism could potentially allow phages to localize to the host and explain the enhanced activity in mucin-rich environments. We sought to further expand on these observations and identify the precise carbohydrates that may mediate the hypothesized activity. To test this idea, we cloned and purified ES17-TFP (Figure S4E) and assessed its ability to bind to a glycan array containing porcine gastric mucin (PGM), glycosaminoglycans (GAGs), and a variety of synthetic and naturally occurring glycans created by the Functional Glycan Consortium (CFG), including over 860 unique glycan structures. For a wide range of mammalian glycans, including those purified from porcine gastric mucin, relative fluorescence units (RFU), an indicator of binding, were either absent or very low. However, surprisingly, binding to heparan sulfate-containing GAGs (identification numbers [ID nos.] 64–173) increased RFU by several orders of magnitude (RFU > 2000), whereas no increase was observed for structurally similar hyaluronan (ID nos. 1–20) or chondroitin sulfate (ID nos. 21–63) (Figure S4Fiii). The finding that purified ES17-TFP binds to human heparan sulfate proteoglycans provides a potential mechanism to explain why phage ES17 exhibits enhanced activity in the intestinal environment.

[0073] ES17 Binds to the Surface of Human Intestinal Enteroids. Human intestinal enteroids (HIEs) are an organ-like, higher-order culture system that has emerged as an alternative approach to modeling the human intestine. They can be cultured as 3D structures with lumens and crypt / villus axes, or as 2D monolayers, which facilitate host-pathogen interactions. These culture systems are useful because they express a variety of glycans found in the human intestine, including mucins and proteoglycans. Human intestinal enteroid monolayers (HIEMs) were derived from colonic epithelial stem cells by culturing them in high-Wnt medium for 5 days. Phages ES17 or HP3 were added to confluent HIEMs for 1 hour. After extensive washing, the phage was visualized by immunofluorescence microscopy using antibodies raised against the respective phages. When phage HP3 was immobilized on the slide alone, the antibody showed strong signal and specificity, but little or no detectable phage HP3 was observed on the surface of intestinal epithelial cells (IECs) in HIEMs (Figure 4A). On the other hand, phage ES17 (green) was uniformly bound to apical IECs, not only in areas where prominent Muc2 (red) localization was observed in the mucus, but also in areas where Muc2 staining was absent (Figure 4A). To determine whether phage ES17 binds to IECs in HIEMs via heparan sulfate, we pretreated HIEMs with heparinase III to enzymatically remove HSPGs and then assessed phage binding. Indeed, the amount of bound ES17 was significantly reduced both qualitatively and quantitatively in heparinase-treated HIEMs (Figure 4B) (P < 0.0001). This finding is consistent with the specific binding of purified tail fiber protein to HSPGs. The localization of ES17 to both the mucus layer and the IEC surface is due to binding to HSPGs, suggesting that the phage is precisely positioned to locate bacterial targets in the intestinal microenvironment. To demonstrate that ES17 binding to HIEMs still allows bacterial infection, we used entero-aggregating Escherichia coli (EAEC), a diarrhea-causing pathogen that, unlike ExPEC, adheres to HIEMs in a tightly cohesive mesh.In this experiment, we pretreated HIEMs with phage ES17, washed them as described above, and then infected them with EAEC strain 042. To visualize the cells and bacteria, HIEMs were fixed and stained with Giemsa-Wright staining as previously described. In infected HIEMs, strong bacterial adhesion to the cells, exhibiting an agglutinating phenotype, was observed (Figure 4S hii, red arrows). In contrast, phage-coated HIEMs significantly reduced EAEC on the organoid surface (P = 0.007), indicating that bound ES17 remained infective to this important biofilm-forming pathogen.

[0074] Phage ES17 kills ExPEC in the mammalian intestine. Phage ES17 exhibited enhanced lytic activity in the presence of mucin, demonstrating its superiority over multiple phages screened in a mucin-rich simulated intestinal environment. Furthermore, its binding to human organotypic IECs via HSPGs led us to investigate whether this phage could overcome the intestinal-induced suppression of lytic activity against ExPEC observed with phage HP3. First, we examined whether phage ES17 was effective in cecal culture. In this environment, phage ES17 demonstrated a 2.5-log improvement in ExPEC elimination efficiency compared to phage HP3 (Figure 5A) (P<0.0001). This experiment did not affect the OTU number or Shannon diversity index, suggesting that phage ES17 selectively eliminated only the targeted ExPEC strains. Next, we examined the effect of phage ES17 on ExPEC in the mouse intestine. Animals were colonized with ExPEC as in Figure 1A and then treated with phage ES17 or HP3 (Figure 5B). In addition, considering the characteristics of phages that move slowly through the gastrointestinal tract, we increased the phage dose to 10 s, aiming to enhance the effect of ExPEC elimination, especially in the proximal part. 9 10 from PFU 10 Examination of the small and large intestine on day 6 revealed that all groups had 10 PFU / g of intestinal tissue. 6 ~10 8High phage levels of PFU were confirmed (Fig. 5C). In animals treated with phage HP3, there were no detectable CFU in the small intestinal tissue (Fig. 5D), but the levels in the cecum were indistinguishable from those of untreated controls. This clearance effect and improved phage levels in the intestine were consistent with the 10% phage concentration used in Fig. 1. 9 Compared to PFU, it is 10, which is one digit higher. 10 This may be due to the daily administration of PFU of phage. In contrast, all but one of the animals treated with phage ES17 had no detectable ExPEC in either the small or large intestine. This suggests that this lytic phage has the unique ability to target ExPEC even in a complex mucosal environment such as the large intestine.

[0075] Materials and Methods Bacterial strains and phages ExPEC ST131 isolate JJ1901 was used in all ExPEC infection experiments, except for JJ2528. Both isolates were previously obtained from Jim Johnson (University of Minnesota). Commensal E. coli ECN was isolated from a human fecal sample. Prior to infection, all strains were grown overnight at 37°C from single colonies smeared on LB agar plates.

[0076] Phages HP3, ES12, ES17, ES19, ES21, and ES26 have been previously described and characterized. Phages 6914, 6915, and 6939 were recently isolated from sewage. All phages described were isolated by single plaque isolation from the environment according to previously described methods.

[0077] (Mouse infection experiment) For the infection model, BALB / c mice (Jackson Laboratories, Bar Harbor, ME) of mixed ages (6–10 months) and sexes were used. Mice were housed in a specific pathogen-free (SPF) environment at the Taub Facility, Center for Comparative Medicine (CCM) at Baylor College of Medicine. All procedures performed on mice complied with the relevant guidelines and regulations of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine (Protocol No. AN-6372). For the infection experiment, mice were housed in a biohazard facility with sterile food and water. Mice were housed individually during the colonization experiment, and bedding was replaced with autoclaved Techboard liners for daily fecal collection. The sample size for the colonization experiment was determined based on previous mouse colonization experiments. Mice were inoculated with ExPEC strain JJ1901 at 10 9 The rodents were administered orally intragastrically at a dose of 100 CFU. The health status of the rodents was confirmed by daily observation for signs of pain or illness. Bacterial colonization (feces and intestines) was assessed by homogenization followed by plating on LB agar (containing chloramphenicol) and counting colonies to selectively detect the chloramphenicol-resistant strain JJ1901. A 6-day time course was performed with 10 9 This was established based on a previous study showing stable colonization with ExPEC after a single oral dose of CFU ( 87 ).

[0078] Purified phages were suspended in 3% (w / v) sodium bicarbonate (NaHCO3) and administered ad libitum or by intragastric administration with 5% (w / v) sucrose in water. All groups were provided with sucrose and NaHCO3 in water for consistency. The antibiotic ampicillin (1 g / 500 mL) was administered in water. Phage colonization was quantified by serial plating of ExPEC strains using a double-layer agar overlay method after dilution of the homogenate. Phage confirmation was performed by observing plaque morphology compared with the phage inoculated into mice. Phages present in the normal murine intestinal flora did not form plaques against ExPEC strains.

[0079] (In vitro cecum model) A modified cecal assay was used for the experiments. Specifically, cecal contents were collected from recently euthanized mice and homogenized at a 1:5 dilution (mg / mL) in sterile 0.09% NaCl solution. The homogenate was centrifuged (2,000 × g, 30 seconds) to remove large particles. The supernatant was used in a phage killing assay (conventional method) at a multiplicity of infection (MOI) of 10 at 37°C with shaking (255 rpm) for 4.5 hours. All cecal and mucin experiments were performed using independent bacterial cultures grown from different colonies streaked on agar plates. These were considered biological replicates. For FS CM, cecal supernatants were centrifuged (6,000 × g, 5 minutes) and then filtered through a 0.22 μm syringe filter. For HT CM, the supernatant was heated in a water bath at 100°F for 20 minutes and then cooled to room temperature (RT) for infection. Insoluble CM and soluble CM (supernatant) were separated after high-speed centrifugation of CM (9,000 × g, 5 min). The insoluble pellet was resuspended in sterile 0.09% NaCl solution for infection (IN CM). Various concentrations of porcine gastric mucin type II (PGM; Sigma-Aldrich) diluted in phosphate-buffered saline (PBS) were used in the mucin assay. The mucolytic agent N-acetylcysteine ​​(NAC; Sigma-Aldrich, 5 mg / mL) was used diluted in PBS in the demucus assay.

[0080] (Mucin coating adsorption and imaging) The assay for adsorption curves was performed at a multiplicity of infection (MOI) of 1 using mid-logarithmic cultures independently grown from different colonies on agar media (which were considered biological replicates), following the method previously used by the inventors for the characterization of phages ES17 and HP3. Prior to adsorption, PGM (0%, 0.5%, and 1.5% [wt / vol]) was added to the bacterial culture for 10 min at room temperature (RT) with shaking (255 rpm). The culture was centrifuged (6,000 × g, 5 min) and gently washed with phosphate-buffered saline (PBS). The adsorption rate constant (Ks) was calculated from the relationship between the natural logarithmic slope of the adsorption curve and the bacterial concentration. Time points were collected every 5 min to accurately test different conditions simultaneously.

[0081] (Mucin-binding ELISA assay) For the ELISA, we used clear-walled Immulon 2 HB 96-well microtiter plates (Immunochemistry Technologies, part number 227). PGM (200 mL of 1 mg / mL) was added to the microtiter plate and incubated overnight at 4°C. The next day, mucin was removed, and each well was washed twice with PBS. Phages were added to each well for 1 hour, followed by three washes with PBST (PBS containing 0.1% Tween 20). The wells were blocked with bovine serum albumin (BSA) and then incubated overnight at 4°C with antibodies against the phage. After a washing step, peroxidase (HRP)-conjugated antibodies were added for 1 hour. To assess phage binding, 3,3',5,5'-tetramethylbenzidine (TMB) solution was added until the wells turned pale blue, after which stop solution (2 M H2SO4) was added. Absorbance was measured at 450 nm. In this experiment, each well was considered a biological replicate.

[0082] (HIEM infection and imaging) Human enteroid monolayers (HIEM) were differentiated for 5 days (>90% confluence) according to the method described previously (54). In each experiment, each well containing HIEM was considered a biological replicate. HIEM were cultured in differentiation medium containing phage (1 × 10 8 The sections were incubated with 1000 PFU / mL of HIV-1 virus in a humidified incubator at 37°C with 5% CO2 for 1 hour and then washed with PBS. HIEMs were fixed with Clark's solution for 10 minutes to preserve the mucin layer. HIEMs were permeabilized and blocked with 5% BSA in PBS containing 0.1% Triton X-100 for 30 minutes at room temperature (RT). Mucin was detected using an antibody against Muc2 (1:200) (Abcam), and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (300 nM) for 5 minutes at room temperature. Antibodies against phages HP3 and ES17 were produced at Pacific Immunology by immunizing rabbits with whole virus (phage). The 13-week antibody production protocol included four immunizations and collection of antisera.

[0083] To selectively remove HSPG from GAG chains, enteroid cultures were pretreated with heparinase III (Sigma, 2 U / mL) for 2 hours (described in Reference 89). Phages were then added. Images were acquired using a Zeiss LSM 510 confocal microscope. Images were uniformly adjusted for brightness and contrast using FIJI software version 2.0.0. All images were uniformly adjusted for brightness and contrast. The number of particles per well was calculated by particle analysis.

[0084] (statistics) Statistical analysis was performed using PRISM 8 software. ATIMA (Agile Toolkit for Incisive Microbial Analyses) was used to analyze the microbiome 16S data. For figures containing log-transformed data or when the number of groups exceeded two, significant differences were determined by one-way analysis of variance (ANOVA) (Figures 1–5). Two-way ANOVA was performed when necessary (Figure 3E). Tukey's post hoc test was applied for multiple comparison analysis. For untransformed data, a Shapiro-Wilk test was performed to confirm normality, and if normality was confirmed, one-way ANOVA was performed. Significant differences were determined when P was 0.05 or less. Unless otherwise noted, all graphs show the mean and standard deviation.

[0085] Example 2: Phage counteradaptation against antiviral- and antibiotic-resistant extraintestinal pathogenic E. coli (Selection of phage-resistant strains) φHP3 is an extensively characterized lytic phage and an effective therapeutic agent in a mouse sepsis model. It was also successfully administered under a single-use investigational new drug application (IND) in two patients with E. coli infection. Three clinically derived extraintestinal pathogenic E. coli (ExPEC) strains, namely JJ2050, JJ2528, and JJ2547, were selected as multidrug-resistant (MDR) pathogens capable of causing disease in this model. All of these strains belong to sequence type (ST) 131 and were isolated from different patients. When treating mice with ExPEC-induced sepsis, the inventors found that the phage reduced bacterial load in most animals, although relatively high bacterial levels persisted in some individuals. The inventors hypothesized that in these cases, phage-resistant bacteria ("resisters") emerged during treatment. To test this hypothesis, we isolated phage-resistant bacteria against φHP3 using two different methods (Figure 7A). The first method was culture-based selection, in which overnight cultured bacteria were streaked onto phage-coated LB agar plates. The second method was animal-based selection, in which resistant bacteria were recovered from phage-treated animals using our mouse sepsis model. Specifically, each ST131 strain was injected intraperitoneally (IP) into mice, followed by an IP injection of phage one hour later. The next day, bacteria were recovered from the liver and spleen of euthanized mice.

[0086] Isolates obtained from both methods were tested for phage resistance by two methods: (1) co-cultivation in liquid culture and (2) phage spot assay. In the co-cultivation assay, isolates were cultured in LB medium with or without phage for 4.5 h. The three parental ST131 strains were easily killed by φHP3 (Figure 7B). However, isolates that survived co-cultivation selection did not show any susceptibility to phage killing (Figure 7B). Furthermore, while the parental strains produced high titers of phage in the spot assay, the resistant strains did not produce any visible phage. These results indicate that these isolates had acquired complete resistance to φHP3. A total of 21 independent resistant strains were isolated through the two isolation methods, and an overview of these is shown in Figure 15. The majority of the resistant strains had smaller colony sizes compared to their wild-type ancestors.

[0087] Resistance is associated with reduced fitness in the host microenvironment. Resistance to phages can be accompanied by reduced fitness under certain environmental conditions. Of particular interest is the finding of a loss of virulence in the host. Such a loss has been observed in bacterial pathogens of fish and moths, demonstrated in a model using A. baumannii, and predicted in humans for V. cholera. To assess the virulence of the isolated resistant strains, we tested their growth ability in media mimicking the host microenvironment. Because ExPEC strains cause urinary tract infections and bacteremia, human urine and blood were used in this experiment. In urine, the majority of resistant strains showed comparable growth compared to Luria medium (LB); these resistant strains also showed comparable growth to their respective wild-type (WT) ancestors (Figure 8). Interestingly, all resistant strains (6 of 9) that showed reduced growth in urine were isolated from mouse models. Furthermore, φHP3 retained efficacy against wild-type isolates but was ineffective against the resistant strains. Notably, for reasons currently unknown, six growth-attenuated resistant animal-derived strains exhibited enhanced growth in the presence of φHP3 (Figure 8; yellow bars, +). This growth-enhancing effect was not reproduced in phage buffer alone (data not shown). Overall, these data suggest that the majority of resistant animal-derived strains (but not culture-derived strains) experienced a fitness trade-off that compromised their ability to grow well in human urine.

[0088] To assess whether resistant ExPEC strains exhibited reduced fitness in blood, we first assessed the growth and survival of the isolates in untreated human whole blood (WB) (Figure 9A). While wild-type JJ2547 grew well in WB after 24 hours, the resistant strains showed significantly reduced survival regardless of the isolation method (Figure 9B). We hypothesized that the fitness reduction of the resistant strains was due to the complement system in serum. To neutralize the complement system, we heat-treated plasma components and remixed them with blood cell components. This mixture was called "heat-inactivated plasma blood (HIPB)" (Figure 9A). However, the resistant strains were significantly attenuated even in HIPB, exhibiting an average 7-log reduction in survival (Figure 9B). We next hypothesized that the fitness reduction of the resistant strains might be due to a loss of nutrient uptake. To mimic the nutrient environment of blood, we used serum mimic defined medium (BSM). The survival of the resistant strain was improved in a 1:1 mixture of HIPB and BSM (HIPB / BSM) (Figure 9B); however, interestingly, survival was poor in a medium in which hemocyte components were suspended in BSM (BSM+) (Figure 9B). When cultured in BSM alone, the growth of the resistant strain was comparable to that of the parent strain (Figure 9B). This result suggests that there are factors unfavorable to the survival of the resistant strain in either the hemocyte or plasma components. This trend was also replicated with other wild-type and resistant strains. Interestingly, the parent strain JJ2528 showed a clear growth defect in blood; this was somewhat surprising considering that this strain is highly virulent in a mouse bacteremia model. Overall, these data suggest that the resistant strains have a significant loss of fitness in human blood to overcome phage infection, independent of the complement system and nutrient uptake.

[0089] Phage-resistant strains exhibit reduced virulence in a systemic infection model. Based on the reduced fitness observed in human blood and urine for some animal-derived resistant strains, we evaluated the virulence of resistant strains in a mouse bacteremia model (Figure 10A). In this study, we selected two representative strains (both of animal origin based on colony morphology) from the resistant strain list and compared their virulence and bacterial burden after infection with the parental strain. Surprisingly, all mice infected with the resistant strain survived the experimental period, whereas the survival rate for the parental strain was only 20% (Figure 10B). Furthermore, mice infected with the resistant strain had lower disease severity scores at all time points during the experimental period (Figure 10C) and an average reduction in bacterial burden in organs of approximately 4.5 logs (Figure 10D). These data indicate that the resistant strains suffer reduced fitness in a mouse systemic infection model, consistent with the reduced survival observed in a host-mimicking environment.

[0090] The resistance mechanism is related to mutations in bacterial surface components. To clarify the mechanisms underlying ExPEC resistance to φHP3 and the reasons for its reduced virulence, we performed whole-genome sequencing on all 21 resistant strains and aligned the assembled genomes with those of the parent strain (Fig. 11A). Notably, 15 of the 21 strains, regardless of the parent strain or isolation method, had varying degrees of gene truncation in a single operon, the waa (or rfa) system, involved in lipopolysaccharide (LPS) assembly. Among these 15 strains, one gene, waaC, was truncated or deleted in all mutants. waaC is responsible for attaching the second glycan (L-glycero-D-manno-heptulose, LD-Hep) to the first glycan (1-deoxy-D-manno-oct-2-ulosonic acid, KDO) to form the inner core of LPS (Fig. 11B). This mutation appears to result in the loss of most of the inner and outer cores, as well as the O antigen. Four of the remaining six strains had truncations in hldE, which is responsible for two steps in LD-Hep synthesis (Figure 10B). This mutation appears to phenotypically result in a truncated LPS molecule, similar to the waaC truncation. This is consistent with a report by Mutalik et al. that E. coli strains tend to develop mutations in the waa operon or LD-Hep synthesis when they acquire resistance to coliphage. A similar phenomenon has also been reported in Pseudomonas aeruginosa isolates. These findings suggest that resistance of ST131 E. coli to φHP3 is a conserved mechanism mediated by truncation of the surface LPS.

[0091] Of the remaining two resistant strains, one had a truncation in ompA, which encodes outer membrane protein (OMP) A. OMPA forms pores in the bacterial outer membrane and is responsible for nutrient uptake. Both of the remaining two mutant strains had a single nucleotide polymorphism (SNP) immediately downstream of the ompA gene. We hypothesize that this region is a ρ-independent terminator region, and this modification may affect transcript polyadenylation, resulting in reduced transcript stability. These two resistant strains also had a truncation in the wzy O-antigen polymerase gene, which may be unrelated to the phage resistance observed in this study. An overview of the mutations in each of the 21 resistant strains and a structural representation showing their location in the LPS molecule are shown in Figure 11C. These findings suggest that the resistant ST131 strains acquired resistance to φHP3 via loss of LPS (19 of 21 strains) or OmpA (2 of 21 strains). Because both of these components are present on the surface of E. coli, our data suggest that these two components may constitute the primary and / or secondary receptors for phage HP3. Another hypothesis is that loss of these genes may disrupt the integrity of the E. coli surface structure, thereby preventing proper attachment or adsorption of HP3.

[0092] Directed evolution leads to the emergence of resistant phages. A somewhat unexpected finding from these studies was that resistant ST131 strains emerged at high frequency in vitro and during infection, and all 21 independent resistant strains converged on one or two key mutations in either LPS or OmpA. Although two mutants tested in the mouse sepsis model (one LPS, the other OmpA) showed clear reductions in virulence (Fig. 10A–10D), it is noteworthy that they were isolated in infected animals upon phage challenge of the parent strain and were present at high enough levels to be isolated (Fig. 13). The consistency of these results suggests the development of highly effective "two-pronged" phage cocktails—that is, specifically tailored cocktails combining phages that promote the emergence of resistant strains with reduced virulence in the host with phages that "predictively" target such resistance mechanisms.

[0093] To test this hypothesis, we first screened two well-characterized phages (φES17 and φEC1) for bactericidal activity against resistant ST131 strains. Surprisingly, these phages, although effective against all three parent strains, did not exhibit bactericidal activity against all resistant strains (Figure 16). These phages are genetically and morphologically distinct from HP3, which may suggest the existence of a common resistance mechanism against coliphages (E. coli phages) in ST131. Furthermore, screening of sewage samples for plaque formation of resistant strains on lawns did not detect any promising lytic phages. We hypothesized that the original phage φHP3 might undergo adaptive evolution, enabling it to reinfect resistant strains. To test this, we constructed an automated bacteria-phage bioreactor. This device constantly circulates a fresh supply of phages cultured on the original bacterial host (parent strain) into a chamber containing resistant (target) bacteria (Figure 12A).

[0094] To confirm whether this approach would yield phage derivatives capable of infecting resistant strains, we cocultured the parent strain JJ2528 with phage φHP3 and circulated the resulting phage product into a chamber containing the resistant strain JJ2528-12. Samples were taken from the chamber after 5, 21, 24, and 37 hours and tested against both the host and target strains in spot assays. Unexpectedly, in the sample taken after 5 hours, clear lytic plaques (clearing) were observed against both JJ2528-12 and the parent strain, which increased over time. One of these plaques was isolated, propagated in a new host, and then purified. This new phage, designated φHP3.1, exhibited bactericidal activity in liquid culture assays (and plaque-forming ability in spot assays) against most resistant strains derived from JJ2528, whether animal- or culture-derived (Figure 12B). Furthermore, φHP3.1 was able to infect not only the target resistant strains used in the directed evolution experiment, but also all LPS-deleted resistant strains, while retaining efficacy against all three parental strains (Fig. 12B; Fig. 16). Interestingly, the two ompA-resistant strains retained partial (SNP only, JJ2528-9) or complete (SNP and gene truncation, JJ2050-3) resistance to the evolved phages.

[0095] To verify that φHP3.1 is a derivative of φHP3 and identify genetic changes associated with its broad activity against resistant strains, we performed whole-genome sequencing of this purified phage. Bioinformatics comparison of φHP3.1 and φHP3 revealed only two single nucleotide polymorphisms (SNPs) across the entire 176,000 base pairs. The first SNP was located in the gene encoding the spike protein and was a change from lysine-tyrosine 464 to arginine-histidine 464 (Figure 13A), which may correspond to the bacterial host-binding site (Figure 13B). The second SNP was a missense mutation in the long-tail fiber gene, resulting in a change from glutamine 9 to arginine 9. In both changes, an amino acid residue with a nonpolar side chain was replaced with a positively charged residue at the tip of the spike gene. This suggests that phage φHP3.1 may be able to reinfect ST131-resistant strains by enhancing its interaction with the host surface through compensatory mutations, resulting in electrostatic charge modifications that promote binding or adsorption.

[0096] Compensatory mutations in ST131 ExPEC allow resistant strains to regain resistance to evolved φHP3.1. Phages and their bacterial hosts are involved in a cycle of coevolution. We investigated whether these resistant strains have the ability to re-evolve resistance to the new phage φHP3.1. Using the culture-based selection method shown in Figure 7A, we identified three ExPEC isolates capable of growing in the presence of φHP3.1: two strains derived from JJ2528-5 (JJ2528-5.1 and JJ2528-5.2) and one strain derived from JJ2528-12 (JJ2528-12.1). Animal-derived φHP3.1-resistant strains could not be induced due to their low virulence in an infection challenge model (Figure 10). The newly resistant strains were confirmed by their resistance to lytic activity in both phage spot assays (Figure 14A) and liquid culture assays (Figure 14B). These secondary resistant strains retained the ability to grow in human urine (Figure 14B). Whole-genome sequencing of these resistant strains revealed that they retained the hldE and waaC truncations from their respective parent strains. Interestingly, consistent with the results described above, all three strains acquired novel truncations in the ompA gene similar to that of JJ2025-3 (Figure 14C). This finding strongly suggests that OmpA is a partial receptor for phage φHP3 and the primary receptor for the evolved form φHP3.1. The ompA mutations observed in two of the 21 primary resistant strains may reflect the evolutionary dynamics between phage and ST131 as a result of phage selective pressure on ST131 strains during this second stage of the initial screening.

[0097] Example 3: Activity of a phage cocktail containing HP3, HP3.1, and HP3.2 E. coli was cultured with a cocktail containing phages ES17 and HP3. As shown in Figure 17A, the phage cocktail initially inhibited bacterial growth, but the effect was overcome by resistant strains (as indicated by an increase in OD600 within 24 hours after treatment in some experiments). E. coli was then cultured with a cocktail containing phages HP3, HP3.1, and HP3.2. As shown in Figure 17B, under these conditions, no regrowth of E. coli was observed between 8 and 24 hours after treatment.

[0098] Next, E. coli biofilms were formed on human catheters, which were then treated with a cocktail containing ES17, HP3, HP3.1, and HP3.2 (φCocktail-R). As shown in Figure 18, treatment with φCocktail-R inhibited biofilm formation and prevented the emergence of resistance 24 hours after treatment.

[0099] Example 4: Activity of a phage cocktail containing HP3, HP3.1, and ES17 Mice (Swiss-Webster strain, female, 6-8 weeks old) were inoculated with 1 × 10 E. coli JJ2528, a clinical isolate. 8 CFU, 1 × 10 purified phage (HP3) or a cocktail of phages (HP3, HP3.1, and ES17) were added to the cells by the intraperitoneal (IP) route. After 1 hour, 1 × 10 purified phage (HP3) or a cocktail of phages (HP3, HP3.1, and ES17) were added to the cells by the intraperitoneal (IP) route. 9 After overnight infection, mice were euthanized, major organs were homogenized, and residual CFU were measured on agar plates.

[0100] As shown in Figure 20, mice treated with the phage cocktail containing HP3, HP3.1, and ES17 had significantly reduced E. coli CFUs in the lungs, spleen, kidneys, and liver.

[0101] Example 5: Efficacy of phage cocktail in a sepsis model The subject matter of the present invention was investigated by evaluating the performance of a phage cocktail compared to the prototype phage HP3. In the first study (Figure 20), a cocktail of three phages (3, the anti-resistant phage 3.1, and the biofilm and mucosal-binding phage ES17) was evaluated for its effectiveness in reducing bacterial levels in a mouse sepsis model compared to the parental and prototype phage HP3. Mouse hosts were challenged with ExPEC strain 2050 at 1 x 10 8 Mice were infected with HP3 phage and 24 hours later, the cocktail was administered subcutaneously (SC). The cocktail was superior to the HP3 phage alone in reducing bacterial levels in all four organs evaluated. Figure 21 evaluates the disease index of the cocktail compared to untreated controls, showing a significant reduction in disease in the cocktail-treated animals. This also extended to bacterial levels within the organs.

[0102] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of specific embodiments, it will be apparent to those skilled in the art that various changes can be made in the steps or sequence of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents which are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the claims.

[0103] [References] The following references, to the extent that they provide exemplary matter supplementary to procedural or other details set forth herein, are specifically incorporated herein by reference.

[0104] Alavidze, Z. et al. Silk route to the acceptance and re-implementation of bacteriophage therapy. Biotechnol. J. 11, 595-600 (2016). Altamirano, F. G. et al. Bacteriophage-resistant Acinetobacter baumannii are resensitized to antimicrobials. Nat. Microbiol. (2021) doi:10.1038 / s41564-020-00830-7. Aslam, S. et al. Lessons Learned From the First 10 Consecutive Cases of Intravenous Bacteriophage Therapy to Treat Multidrug-Resistant Bacterial Infections at a Single Center in the United States. Open Forum Infect. Dis. 7, (2020). Blanquart, F. & Gandon, S. Time-shift experiments and patterns of adaptation across time and space. Ecol. Lett. 16, 31-38 (2013). Brockhurst, M. A. & Koskella, B. Experimental coevolution of species interactions. Trends Ecol. Evol. 28, 367-375 (2013). Brussow, H. Phage therapy: The Escherichia coli experience. Microbiology 151, 2133-2140 (2005). Buckling, A. & Rainey, P. B. . Antagonistic coevolution between a bacterium and a bacteriophage. Proc. R. Soc. B Biol. Sci. 269, 931-936 (2002). Capparelli, R. et al. Bacteriophage-resistant Staphylococcus aureus mutant confers broad immunity against staphylococcal infection in mice. PLoS One 5, 1-13 (2010). Chan, B. K. et al. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa. Sci. Rep. 6, 26717 (2016). Conly, J. M. & Johnston, B. L. Where are all the new antibiotics? The new antibiotic paradox. Can. J. Infect. Dis. Med. Microbiol. 16, 159-160 (2005). Fish, F. Phage therapy for the treatment of human intestinal bacterial infections: soon to be a reality? Expert Rev. Gastroenterol. Hepatol. 00, 1-4 (2013). Fish, R, Kutter, E, Wheat, G, Blasdel, B, Kutateladze, M, Kuhl, S. Journal of wound care: Bacteriophage treatment of intransigent diabetic toe ulcers: a case series. 7, 27-33. Gaba, S. & Ebert, D. Time-shift experiments as a tool to study antagonistic coevolution. Trends Ecol. Evol. 24, 226-232 (2009). Gandon, S., Buckling, A., Decaestecker, E. & Day, T. Host-parasite coevolution and patterns of adaptation across time and space. J. Evol. Biol. 21, 1861-1866 (2008). Green, S. I. et al. Bacteriophages from ExPEC Reservoirs Kill Pandemic Multidrug-Resistant Strains of Clonal Group ST131 in Animal Models of Bacteremia. Sci. Rep.7, 46151 (2017). Green, S. I. et al. Bacteriophages from ExPEC Reservoirs Kill Pandemic Multidrug-Resistant Strains of Clonal Group ST131 in Animal Models of Bacteremia. Sci. Rep.7, 46151 (2017). Green, S. I. et al. Murine model of chemotherapy-induced extraintestinal pathogenic Escherichia coli translocation. Infect. Immun. 83, 3243-3256 (2015). Johnson, J. R. & Russo, T. A. Extraintestinal pathogenic Escherichia coli: ‘The other bad E coli’. J. Lab. Clin. Med. 139, 155-162 (2002). Johnson, J. R., Johnston, B., Clabots, C., Kuskowski, M. A. & Castanheira, M. Escherichia coli Sequence Type ST131 as the Major Cause of Serious Multidrug-Resistant E . coli Infections in the United States. 55417, 286-294 (2010). Leon, M. & Bastias, R. Virulence reduction in bacteriophage resistant bacteria. Front. Microbiol. 6, 1-7 (2015). Levin, B. R. Nasty viruses, costly plasmids, population dynamics, and the conditions for establishing and maintaining CRISPR-mediated adaptive immunity in bacteria. PLoS Genet. 6, 1-12 (2010). Ma, L., Green, S. I., Trautner, B. W., Ramig, R. F. & Maresso, A. W. Metals Enhance the Killing of Bacteria by Bacteriophage in Human Blood. Sci. Rep. 8, 1-11 (2018). Poullain, V., Gandon, S., Brockhurst, M. A., Buckling, A. & Hochberg, M. E. The evolution of specificity in evolving and coevolving antagonistic interactions between a bacteria and its phage. Evolution (N. Y). 62, 1-11 (2008). Rhoads, D. D. et al. Bacteriophage therapy of venous leg ulcers in humans: results of a phase I safety trial. J. Wound Care 18, 237-243 (2009). Schooley, R. T. et al. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob. Agents Chemother. 61, 1-14 (2017). Seed, K. D. et al. Evolutionary consequences of intra-patient phage predation on microbial populations. Elife 3, e03497 (2014). Shabbir, M. A. B. et al. Bacteria vs. bacteriophages: Parallel evolution of immune arsenals. Front. Microbiol. 7, 1-8 (2016). Wright A, Hawkins CH, Anggard EE, Harper, D. A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic resistant Pseudomonas aeruginosa: a preliminary report of efficacy. Clin Otolaryngol34, 349-357 (2009). Yen, M., Cairns, L. S. & Camilli, A. A cocktail of three virulent bacteriophages prevents Vibrio cholerae infection in animal models. Nat. Commun. 8, 14187 (2017).

Claims

1. A composition comprising: (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.

1.

2. The composition of claim 1 , wherein the composition comprises bacteriophage ES17.

3. The composition of claim 1 , wherein the composition comprises bacteriophage ES19.

4. The composition according to any one of claims 1 to 3, further comprising bacteriophage HP3.

2.

5. The composition according to any one of claims 1 to 4, further comprising a pharmaceutically acceptable excipient.

6. The composition of any one of claims 1 to 5, further comprising one or more metals.

7. 7. The composition of claim 6, wherein the one or more metals comprise calcium, magnesium, iron, sodium and / or potassium.

8. The composition of any one of claims 1 to 7, wherein the amounts of said bacteriophages in said compositions are substantially equivalent.

9. The composition of any one of claims 1 to 7, wherein the amounts of said bacteriophage in said composition are not substantially equal.

10. The composition according to any one of claims 1 to 9, wherein the composition is a liquid formulation.

11. The composition according to any one of claims 1 to 9, wherein the composition is a solid formulation.

12. The composition of any one of claims 1 to 11, wherein the composition is contained in a delivery device.

13. A method of treating or preventing an E. coli infection in an individual, the method comprising administering to said individual a composition according to any one of claims 1 to 12.

14. A device comprising (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1 on, within, and / or around the device.

15. 15. The device of claim 14, wherein the device comprises bacteriophage ES17.

16. 15. The device of claim 14, wherein the device comprises bacteriophage ES19.

17. The device of any one of claims 14 to 16, further comprising bacteriophage HP3.

2.

18. 18. The device of any one of claims 14 to 17, wherein the device is a catheter, a drive line, a syringe, a tube, an implant, a defibrillator, a prosthetic joint, a pacemaker, a screw, a rod, a disc, an intrauterine device, a pin, a plate, a stent, a dental device, an intraocular lens, a shunt, a valve, a neurological or neurosurgical device, a gastrointestinal device, a genitourinary device, a catheter cuff, a vascular access device, or a wound drain.

19. The device according to any one of claims 14 to 17, further characterized as a device having a coating comprising the bacteriophage.

20. A kit comprising (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.1 housed in a suitable container.

21. 21. The kit of claim 20, wherein the kit comprises bacteriophage ES17.

22. 21. The kit of claim 20, wherein the kit comprises bacteriophage ES19.

23. The kit according to any one of claims 20 to 22, further comprising bacteriophage HP3.

2.

24. The kit of any one of claims 20 to 23, further comprising a device.

25. 24. The kit of claim 23, wherein the device comprises a respective bacteriophage.

26. 24. The kit of claim 23, wherein each of the bacteriophages is separate from the device.

27. A method of treating or preventing an E. coli infection in an individual, the method comprising administering to the individual (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.

1.

28. 28. The method of claim 27, wherein the method comprises administering bacteriophage ES17 to the individual.

29. 28. The method of claim 27, wherein the method comprises administering bacteriophage ES19 to the individual.

30. 30. The method of any one of claims 27 to 29, further comprising administering to the individual bacteriophage HP3.

2.

31. The method of any one of claims 27 to 30, wherein the bacteriophages are in the same formulation.

32. The method of any one of claims 27 to 30, wherein the bacteriophages are not in the same formulation.

33. 33. The method of claim 32, wherein each of the bacteriophages is administered to the individual simultaneously.

34. 33. The method of claim 32, wherein each of the bacteriophages is administered to the individual at a different time.

35. 35. The method of any one of claims 27 to 34, wherein the bacteriophage is administered to the individual intravenously, orally, and / or on a device.

36. 36. The method of any one of claims 27 to 35, wherein the bacteriophage is administered to the individual multiple times.

37. 37. The method of claim 36, wherein the bacteriophage is administered daily, twice daily, weekly, twice weekly, monthly, or twice monthly.

38. 37. The method of claim 36, wherein the bacteriophage is administered twice a week for 6 to 12 weeks.

39. 39. The method of any one of claims 27-38, wherein the individual has an infection in the urinary tract, blood, intestines, abdomen, stomach, lungs, skin, kidney, prostate, bladder, brain, vaginal tract, heart, liver, spleen, or a combination thereof.

40. 40. The method of claim 39, wherein the individual has a catheter-associated urinary tract infection.

41. The method of any one of claims 27 to 40, wherein the E. coli is multidrug resistant.

42. The method according to any one of claims 27 to 41, wherein the E. coli is extraintestinal pathogenic E. coli.

43. 43. The method of any one of claims 27 to 42, wherein the E. coli is ST69, ST73, ST96, or ST131.

44. 44. The method of claim 43, wherein the E. coli is ST131.

45. 45. The method of any one of claims 27-44, wherein the individual has a urinary tract infection, neonatal meningitis, a bloodstream infection, pneumonia, sepsis, a surgical wound infection, a skin infection, a prostate infection, meningitis, a vaginal infection, or a combination thereof.

46. 46. ​​The method of any one of claims 27 to 45, wherein the individual has diarrhea, stomach cramps, nausea, and / or vomiting.

47. The method of any one of claims 27 to 46, wherein the individual is immunosuppressed.

48. 48. The method of claim 47, wherein the individual has an immune cell dysregulation, asplenia, splenic dysfunction, nephrotic syndrome, or an autoimmune disease.

49. 49. The method of any one of claims 27 to 48, wherein the individual receives the bacteriophage prior to a medical procedure or therapeutic regimen.

50. 50. The method of claim 49, wherein the individual is to be exposed to an immunosuppressive condition.

51. 51. The method of claim 50, wherein the individual is undergoing or will undergo chemotherapy.

52. 50. The method of claim 49, wherein the individual is taking or will be taking an immunosuppressant.

53. 53. The method of claim 52, wherein the immunosuppressant is a glucocorticoid, a calcineurin inhibitor, an antimetabolite, or an antibody therapy.

54. 50. The method of claim 49, wherein the medical procedure comprises the insertion of a device into the individual.

55. 55. The method of any one of claims 27 to 54, wherein the E. coli originates from a beverage, food, other individual, or the environment.

56. 56. The method of claim 55, wherein the environment is groundwater or surface water, water used to irrigate crops, a public water system, a hospital, a school, a nursing home, a petting zoo, a cruise ship, a train, or an airplane.

57. 57. The method of any one of claims 27 to 56, wherein the individual is at higher risk of E. coli infection than the general population.

58. 58. The method of claim 57, wherein the individual is under 12 years of age or over 65 years of age.

59. 59. The method of any one of claims 27 to 58, wherein the individual has consumed undercooked meat, unpasteurized milk, apple juice or cider, or soft cheese made from raw milk.

60. 60. The method of any one of claims 27 to 59, wherein the individual is taking a medication to reduce stomach acid.

61. A method for reducing the level of drug-resistant and / or pathogenic E. coli strains in an individual, comprising administering to the individual therapeutically effective amounts of (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.

1.

62. 62. The method of claim 61 , wherein the method comprises administering bacteriophage ES17 to the individual.

63. 63. The method of claim 61 or 62, wherein the method comprises administering bacteriophage ES19 to the individual.

64. 64. The method of any one of claims 61 to 63, further comprising administering to the individual a therapeutically effective amount of bacteriophage HP3.

2.

65. 65. The method of any one of claims 61 to 64, wherein the reduction is in the intestine of the individual.

66. 66. The method of any one of claims 61 to 65, wherein the individual has undergone or is to undergo insertion of a device.

67. 67. The method of any one of claims 61 to 66, wherein the individual is to be exposed to an immunosuppressive condition.

68. 68. The method of any one of claims 61 to 67, wherein the E. coli strain is an extraintestinal pathogenic E. coli.

69. 69. The method of any one of claims 61 to 68, wherein the E. coli strain is ST69, ST73, ST96 or ST131.

70. 70. The method of claim 69, wherein the E. coli strain is ST131.

71. A method of manufacturing a device, comprising exposing the device to (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, and (c) bacteriophage HP3.

1.

72. 72. The method of claim 71, wherein the bacteriophage is disposed on, within, and / or around a device.

73. 73. The method of claim 71 or 72, wherein one or more surfaces of the device are coated with the bacteriophage.

74. 52. The method of any one of claims 49 to 51, wherein the device is a catheter, a driveline, a syringe, a tube, an implant, a defibrillator, an artificial joint, a pacemaker, a screw, a rod, a disc, an intrauterine device, a pin, a plate, a stent, a dental device, or an intraocular lens.

75. 75. The method of any one of claims 71 to 74, further comprising the step of delivering said device to an individual in need thereof.

76. A composition comprising bacteriophage HP3.

2.

77. 77. A method of treating or preventing an E. coli infection in an individual, comprising administering to said individual the composition of claim 76.

78. A device comprising bacteriophage HP3.2, wherein the bacteriophage is present on, within, and / or around the device.

79. A kit comprising bacteriophage HP3.2, housed in a suitable container.

80. 1. A method of treating or preventing an E. coli infection in an individual, the method comprising administering to the individual bacteriophage HP3.

2.

81. A composition comprising: (a) bacteriophage ES17 or bacteriophage ES19, (b) bacteriophage HP3, (c) bacteriophage HP3.1, and (d) bacteriophage HP3.2.

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