Bacteriophage preparation and use

Abstract

The bacteriophage preparation comprises a heterologous bacteriophage preparation, typically obtained from a fermentation process. The bacteriophage preparation may be formulated into a pharmaceutical composition. The bacteriophage preparation may be used to treat dysbiosis by administering the bacteriophage preparation to a subject suffering from or at high risk of suffering from dysbiosis. The bacteriophage preparation may be used to prime or precondition the intestinal environment of a healthy subject prior to administering a bacterial source to the subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 255,105, filed October 13, 2021, and is incorporated by reference herein in its entirety. Summary of the Invention

[0002] overview The disclosure, in one aspect, describes a pharmaceutical composition comprising a heterologous bacteriophage preparation obtained from a fermentation process and a pharma- ceutical acceptable carrier. In one or more embodiments, the pharmaceutical composition may further comprise an adjuvant.

[0003] In one or more embodiments, the pharmaceutical composition includes a bacteriophage that is a member of the Myoviridae family, a member of the Podoviridae family, a member of the Siphoviridae family, a member of the Inoviridae family, a member of the Microviridae family, a member of the Corticoviridae family, a member of the Tectiviridae family, a member of the Leviviridae family, a member of the Cystoviridae family, a member of the Rudiviridae family, a member of the Fuselloviridae family, a member of the Liposliviridae family, a member of the Plasmaviridae family, or a bacteriophage from a combination of the above families.

[0004] In one or more embodiments, the fermentation process produces a food product, hi other embodiments, the fermentation process produces a fertilizer or livestock feed. In one or more embodiments, the fermentation process includes fermentation of plant material, plant juice, feedstock, biofuel, or biological waste. In one or more embodiments, the pharmaceutical composition is formulated for delivery to epidermal tissue.

[0005] In another aspect, the disclosure describes a method for preparing a bacteriophage preparation. Generally, the method includes isolating a bacteriophage from a fermentation process and combining the isolated bacteriophage with a pharma- ceutical acceptable carrier.

[0006] In one or more embodiments, the method further comprises concentrating the bacteriophage preparation such that the concentration of the bacteriophage in the preparation is increased, the biological activity of the bacteriophage in the preparation is increased, or both. In one or more embodiments, the method further comprises culturing the bacteriophage preparation with a suitable bacterial species in a bioreactor.

[0007] In one or more embodiments, the fermentation process produces a food product, hi other embodiments, the fermentation process produces a fertilizer or livestock feed. In one or more embodiments, the fermentation process includes the fermentation of plant material, plant juice, feedstock, biofuel, or biological waste.

[0008] In another aspect, this disclosure describes a method of treating dysbiosis in a subject suffering from or at risk of suffering from dysbiosis. Generally, the method includes administering to the subject a bacteriophage preparation in an amount effective to ameliorate at least one symptom or clinical sign of the dysbiosis.

[0009] In one or more embodiments, the method further comprises administering to the subject a second pharmaceutical composition for treating the dysbiosis. In some of these embodiments, the second pharmaceutical composition for treating the dysbiosis may comprise an antibiotic formulation, a prebiotic formulation, a probiotic formulation, a synbiotic formulation, a fecal microbiota transplant, a membrane vesicle, or an autophagy inducer.

[0010] In one or more embodiments, the dysbiosis is localized to epidermal tissue, in some of these embodiments, epidermal tissue includes the epidermis, a mucosal surface, at least a portion of the skin or scalp, at least a portion of the oral cavity, at least a portion of the gastrointestinal tract, at least a portion of the nasal cavity, at least a portion of the respiratory tract, at least a portion of the genitourinary tract, or a body cavity.

[0011] In one or more embodiments, the bacteriophage preparation is incorporated into a medical device. In one or more embodiments, the dysbiosis causes bacterial overgrowth.

[0012] In one or more embodiments, the method further comprises fasting the subject for an extended period of time prior to administering the bacteriophage preparation to the subject. In some of these embodiments, the extended fast is from about 1 to 3 days.

[0013] In one or more embodiments, the method further comprises feeding the subject one or more foods prior to, concomitantly with, or after administration of the bacteriophage preparation to the subject. In one or more embodiments, the bacteriophage preparation is administered orally, topically, by inhalation, or parenterally.

[0014] In yet another aspect, the disclosure describes a method of preparing or prepping the intestinal environment of a healthy subject for administration of a bacterial source. Generally, the method comprises administering to the subject a bacteriophage preparation in an amount effective to improve susceptibility of the subject's intestine to the bacterial source.

[0015] In one or more embodiments, the bacterial source comprises one or more (eg, a cocktail) of bacteria grown in culture, a probiotic preparation, or a fecal microbiota explant. In one or more embodiments, the bacteriophage preparation is administered orally, delivered directly to the intestine, or encapsulated as a microbial implant.

[0016] In one or more embodiments, the method further comprises fasting the subject for an extended period of time prior to administering the bacteriophage preparation to the subject. In some of these embodiments, the extended fast is from about 1 to 3 days.

[0017] In one or more embodiments, the method further comprises fasting the subject for an extended period of time prior to administering the bacteriophage preparation to the subject. In some of these embodiments, the extended fast is from about 1 to 3 days.

[0018] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided by lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief description of the drawings]

[0019] [Figure 1] Water avoidance stress (WAS) test animals receiving PBS placebo (WAS-PBS) had a 3.3-fold increase in bacterial density compared to control animals (p<0.005), demonstrating psychological stress-induced bacterial overgrowth. However, bacterial density in WAS test mice receiving bacteriophage preparations (WAS-phage) was not significantly different from control animals, demonstrating the efficacy of the bacteriophage preparation in inhibiting stress-induced bacterial overgrowth. All results were expressed as fold change in 16S rRNA gene copy number relative to that of the control (set as 1.0). [Diagram 2] WAS test animals receiving either PBS placebo (WAS-PBS) or bacteriophage preparation (WAS-phage) had significantly different β-diversity compared to control animals as measured by the Bray-Curtis dissimilarity index (p<0.01). These data demonstrated that testing with WAS is associated with changes in the overall composition of the gut microbiota. [Diagram 3] Only WAS test animals that received the bacteriophage preparation (WAS-phage) had enhanced α-diversity versus controls as measured using the Chao1 α-diversity index (p<0.05), whereas WAS test animals that received the PBS placebo (WAS-phage) did not have significantly enhanced α-diversity versus controls (p=0.2). These data on α-diversity interpreted with data from Figures 1 and 2 demonstrate the efficacy of the bacteriophage preparation in enhancing overall gut microbiota diversity in the context of stress-induced gut microbiota dysbiosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS This disclosure describes pharmaceutical compositions and methods involving the use of bacteriophages to alleviate dysbiosis. In the context of the gut microbiota, dysbiosis is an abnormal state in which the microbiota is associated with numerous health disorders and adverse conditions. A dysbiotic microbiota is typically characterized by a reduced overall diversity of bacterial membership and an increased density of selected bacterial members compared to a healthy microbiota. Similarly, a dysbiotic gut viral biota is typically characterized by a reduced overall diversity of viral membership and an increased density of selected viral members compared to a healthy gut viral biota. Conversely, greater bacterial and / or viral diversity is associated with a healthy gut microbiota. In contrast, many bacterial infections typically involve a single bacterial pathogen, and in the case of methicillin-resistant Staphylococcus aureus infections, dysbiosis can involve many more members or even the entire community of >500-1000 bacterial species (many of which are resident, mutualistic, and non-pathogenic) that make up the gut microbiota. Gut microbiota interact closely with their human host, and this interaction directly impacts human health. In addition, other relevant components of the gut ecosystem that show low diversity in the context of dysbiosis are the archaebacteria (archaea), phagebiota (bacteriophages), viribiota (viruses), mycobiota (fungi), metagenomics (total catalog of genes), metatranscriptome (total catalog of expressed genes), and metaproteome (total catalog of proteins).

[0021] Dysbiosis has been linked to many disorders including, but not limited to: chronic non-communicable diseases in developed countries (such as atopy, metabolic syndrome, inflammatory diseases, cancer, some behavioral disorders), irritable bowel syndrome (IBS, including IBS-associated gastrointestinal, neurological, and psychiatric conditions, and IBS-associated neurodegenerative and psychiatric comorbidities), chronic fatigue syndrome, fibromyalgia, gastroesophageal reflux disease, functional abdominal pain and other functional disorders, Crohn's disease, ulcerative colitis, inflammatory bowel disease (IBD), eating disorders, idiopathic pulmonary fibrosis (IPF), autoimmune diseases, COVID-19, Post COVID-19 Long Syndrome, Interstitial Cystitis, Chronic Pelvic Pain Syndrome, Chronic Prostatitis, Chronic Epididymitis, Liver Cirrhosis, Hypercontractile Circulation, Ascites, Varicose Veins, Encephalopathy, Renal Insufficiency, Hepatorenal Syndrome, Microbiome Translocation, End Stage Renal Disease, Celiac Disease, Allergy, Asthma, Gluten Sensitivity or Intolerance, Cardiovascular Disease, Gulf War Syndrome or Illness, Attention Deficit Hyperactivity Disorder (ADHD), Post Traumatic Stress Disorder, Anxiety Disorder, Diabetes, Metabolic Syndrome, Impaired Glucose Tolerance, Leaky Gut Syndrome, Intestinal Barrier Dysfunction, Hypersensitivity Disorder, Cystic Fibrosis Fibrosis, Multi-symptom disorders, Environmental illnesses, Food intolerances and allergies, Food sensitivities, Multiple food or chemical sensitivities, Autism, Post-traumatic depression, Neurodegeneration, Multiple sclerosis, Alzheimer's disease, Mild cognitive impairment, Parkinson's disease, Peripheral neuropathy, Dementia with Lewy bodies (DLB), Idiopathic REM Sleep Behavior Disorder (iRBD), Restless legs syndrome, Sleep disorders, Obstructive sleep apnea, Orthostatic hypotension, Postural correction syndrome, Systemic lupus erythematosus, Scleroderma, Rheumatoid arthritis (RA), Osteoarthritis (OA), Skin disorders (e.g., acne, atopic dermatitis, psoriasis, rosacea, hives, Acne vulgaris, bullous pemphigus, etc.), Raynaud's syndrome, osteoarthritis, Sjögren's syndrome and other autoimmune diseases, atherosclerosis and related cardiovascular diseases, costochondritis, hyperhomocysteinemia, Hashimoto's thyroiditis, atopic dermatitis, eczema, vaginal diseases, gingivitis, periodontal disease, dental pulp chamber disease, dental caries, uveitis, iritis, hyperalgesia, opioid-induced hyperalgesia, allodynia, opioid bowel syndrome, anesthetic hypersensitivity syndrome, hematopoietic cell transplantation, impaired protective antibacterial mechanisms (e.g., achlorhydria, exocrine pancreatic insufficiency, immune deficiency syndrome, etc.), anatomical abnormalities (e.g.,Small bowel obstruction, diverticulum, fistula, surgical blind loop, previous ileocecal resection, etc.), motility disorders (e.g., scleroderma, autonomic neuropathy in diabetes, post-irradiation enteropathy, small bowel pseudo-obstruction, etc.), mastitis, nutritional deficiencies, critical illness, chronic illness, oral mucositis, intestinal mucositis, Candida albicans (Candida albicans infection, diabetes, atopic constitution, allergic rhinitis conjunctivitis, food allergy, eosinophilic esophagitis, pancreatitis, butyrate deficiency, infertility, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), liver cancer, liver disease, liver transplantation, bronchiectasis, chronic obstructive pulmonary disease (COPD), gastrointestinal cancer, esophageal cancer, gastric cancer, colorectal carcinoma, pancreatic cancer, hepatocellular carcinoma, surgical site infection, enteritis, colorectal cancer, fatty liver, graft-versus-host disease (GVHD), oral squamous cell carcinoma, sarcopenia, urinary tract stone disease, kidney disease, Clostridium difficile difficile infection (CDI), allogeneic stem cell transplantation, osteopathogenic disorders, promotion of fungal metastasis and invasive disease, fungal bloodstream infections (e.g., Candida bloodstream infections), chronic rhinosinusitis (CRS), neuroinflammation, autism spectrum disorders, attenuated morphine analgesic intolerance, retinal degenerative diseases (e.g., retinitis pigmentosa), necrotizing enterocolitis (NEC), hepatic encephalopathy, cirrhosis-related cognitive abilities, addictive disorders, aging, chronic otitis media, immunosenescence, urgency incontinence, chronic kidney disease, vitiligo, insulin resistance, vaginal microbiota imbalance, nasopharyngeal carcinoma, hypertension, dyslipidemia, idiopathic intracranial hypertension syndrome (IIH), peri-implant mucositis (PM), Peri-implantitis (PI), cardiometabolic health during menopause, senile pruritus (SP), primary biliary cholangitis, irritable scalp, seborrheic dermatitis, autoimmune thyroid disease, Grave-Basedow disease, bacterial vaginosis, sexually transmitted diseases, glaucoma, recurrent miscarriage, recurrent implantation failure, atherogenic dyslipidemia, vertical sleeve gastrectomy, axial spondyloarthritis, chronic physiological stress, age-related decline in microbiota diversity, urinary tract infection, visceral hypersensitivity, intestinal membrane permeability, fibromyalgia, schizophrenia, attention-deficit / hyperactivity disorder, leaky gut syndrome, polycystic ovary syndrome, ischemic stroke, atherosclerotic plaque development, atherosclerotic thrombosis, microscopic colitis, and substance use disorders.

[0022] Dysbiosis may also be associated with chemotherapy, hemodialysis, the use of antibiotics (e.g., during childhood), bariatric surgery, and being on a ventilator. Symptoms and clinical signs of disorders associated with dysbiosis include, but are not limited to, fatigue, low energy, flu-like illness, poor sleep, insomnia, panic, depression, bone loss, borborygmi, abdominal bloating, flatulence, bloating, heartburn, indigestion, picky eating, constipation, diarrhea (e.g., refractory diarrhea, bile acid diarrhea, antibiotic diarrhea, etc.), vagal gut-brain communication, abdominal pain, paresthesias, tingling, neuropathy, brain fog, bad breath, sugar cravings, depression, anxiety, poor psychiatric outcome, intellectual disability, difficulty concentrating, dementia, mild cognitive impairment, memory impairment, synaptic plasticity, irritability, headaches, migraines, dizziness, aches and pains, chronic widespread musculoskeletal pain, shortness of breath, nausea or vomiting, loss of appetite, increased appetite, acne, rash, cold hands and feet, unwanted weight loss, or obesity.

[0023] One type of dysbiosis associated with reduced bacterial density is small intestinal bacterial overgrowth (SIBO), which is present in up to 78% of patients suffering from irritable bowel syndrome. SIBO is characterized by a reduced diversity of the intestinal microbial community and / or excessive bacterial colonization in the proximal end of the small intestine (which may be independent of excessive bacterial density in the more distal end of the gastrointestinal tract). Intestinal bacterial overgrowth represents an excessive bacterial density in the proximal and / or distal parts of the gastrointestinal tract. In the case of SIBO, this overgrowth is not limited to one or a few bacterial species. Rather, like many dysbiosis, SIBO can encompass many or all species of the gut microbiota and is generally caused by a high density of resident non-pathogenic bacterial species that become pathogenic commensals. In a healthy gastrointestinal tract, the proximal end of the small intestine is substantially sterile, with a progressively higher bacterial density towards the distal end. High colonization of the small intestine with Enterobacteriaceae, especially in the more proximal end of the small intestine, is associated with induction of host immunity, which can lead to high proinflammatory / immune responses, leaky gut, impaired intestinal barrier function, and / or bacterial translocation across the intestinal barrier into the bloodstream.

[0024] Thus, SIBO and intestinal bacterial overgrowth are associated with a range of conditions including, but not limited to, irritable bowel syndrome (IBS), chronic fatigue syndrome, fibromyalgia, gastroesophageal reflux disease, functional abdominal pain and other functional disorders, Crohn's disease, ulcerative colitis and other inflammatory bowel disease post-COVID-19 long-term syndrome, interstitial cystitis, chronic pelvic pain syndrome, chronic prostatitis, chronic epididymitis, liver cirrhosis, hypercontractile circulation, ascites, varicose veins, encephalopathy, renal insufficiency, hepatorenal syndrome, microbiome translocation, end stage renal disease, celiac disease, allergies, asthma, gluten sensitivity or intolerance, cardiovascular disease, Gulf War syndrome or illness, attention deficit hyperactivity disorder (ADHD), post traumatic stress disorder, anxiety disorders, diabetes, metabolic syndrome, impaired glucose tolerance, leaky gut syndrome, intestinal barrier dysfunction, hypersensitivity disorders, cystic fibrosis, multi-symptom disorders , Environmental Illnesses, Food Intolerances and Allergies, Food Sensitivities, Multiple Food or Chemical Sensitivities, Autism, Post-Traumatic Depression, Multiple Sclerosis, Alzheimer's Disease, Mild Cognitive Impairment, Parkinson's Disease, Peripheral Neuropathy, Restless Legs Syndrome, Sleep Disorders, Orthostatic Hypotension, Postural Correction Syndrome, Systemic Lupus Erythematosus, Multiple Sclerosis, Scleroderma, Rheumatoid Arthritis, Psoriasis, Rosacea, Hives, Raynaud's Syndrome, Osteoarthritis, Shea In some embodiments, the inflammatory bowel syndrome is associated with a number of conditions, symptoms, and / or clinical signs, including, but not limited to, rheumatoid arthritis ...

[0025] Available treatments for SIBO and intestinal bacterial overgrowth are limited to antibiotic therapy, including, for example, rifaximin, doxycycline, augmentin, ciprofloxacin, metronidazole, norfloxacin, or neomycin, which have varying degrees of clinical effectiveness and therefore are not successful in all patients.

[0026] This disclosure describes compositions and methods involving the use of bacteriophages for the treatment of dysbiosis exemplified by, but not limited to, SIBO, intestinal bacterial overgrowth, and dysbiosis conditions characterized by reduced or lost bacterial diversity. Bacteriophages are ubiquitous viruses that infect bacterial hosts. Each bacteriophage is dependent on a bacterial host for reproduction and has a limited range of susceptible bacterial hosts. One method of replicating a bacteriophage using its bacterial host involves the bacteriophage attaching to a receptor on the exterior of the bacterial cell and injecting its bacteriophage DNA into the bacterial chromosome, replication of the bacteriophage particle by the bacterial host, and lysis of the bacterial host, which ultimately results in the release of new bacteriophage progeny that are found in a new host to repeat the cycle. In microbial communities such as the gut microbiota, community-wide infection of bacteria by bacteriophages is a mechanism that helps regulate bacterial community composition by downregulating the abundance of bacterial species with high population densities, a process that helps drive and maintain bacterial species diversity. Bacteriophages are abundant in the gut microbiota, accounting for approximately 10 of the total population in the gut, a population roughly the same size as gut bacteria. 13 Bacteriophage interactions are the basis of healthy microbial communities, including a healthy gut microbiome, and dysfunction in bacteriophage-bacteria dynamics is associated with reduced bacterial diversity and high densities of specific bacterial members (i.e., dysbiosis).

[0027] The co-evolutionary correlation between bacteriophages and bacteria (where bacteriophages regulate the growth and density of bacterial species) coupled with the ability of bacteriophages to replicate and grow gives bacteriophage-based gut microbiome interventions the potential to address SIBO, gut bacterial overgrowth, and dysbiosis conditions characterized by reduced and lost bacterial diversity, or other forms of dysbiosis. Moreover, bacteriophage-based interventions can produce more sustained effects by establishing a healthy and diverse gut microbiome. In contrast, many existing antibiotic therapies reduce gut microbiome diversity and disrupt the gut microbial community, leading to dysbiosis.

[0028] Conventional bacteriophage therapy involves the use of bacteriophages that infect specific bacterial pathogens involved in infectious diseases. A typical strategy for bacteriophage therapy involves culturing a single bacteriophage or a "cocktail" of multiple bacteriophages that show efficacy in vitro against a specific disease-causing pathogen that is harvested from the patient and cultured at the site of infection. Since each bacteriophage has a limited number of susceptible hosts, this strategy relies on the careful selection of bacteriophages that can infect and lyse the bacterial pathogen of interest. This "one bacteriophage per bacterium" approach can lead to a loss of clinical efficacy if the bacterial pathogen develops resistance to infection by the bacteriophage.

[0029] In contrast to the established use of bacteriophages, this disclosure describes an alternative approach that uses an entire bacteriophage community to treat any dysbiotic condition associated with a disturbed microbiota or reduced or lost bacterial diversity. The approach described herein uses a community-level solution (a community of bacteriophages) to address a community-level problem. In one or more embodiments, the bacteriophage community can be isolated from a source, including fermented plant material.Exemplary fermented plant materials include, but are not limited to, appam, atchara, bagoong, balao-balao, banh cuon, brem, burong isda, cheonggukjang, cincalok, curtido, dhokla, doenjang, doubanjiang, douzhi, fermented coconut water, fermented pineapple juice, fermented tofu, fermented miso, fish sauce, galapong, ganjang, gochujang, gundruk, hakarl, jeotgal, kenkey, khanomjin, and the like. chin, kimchi, kombucha, kusaya, lufu, miso, mixian, mohnyin tjin, murri, nata de coco, nata de pina, naem, natto, nem chua, ngapi, ogi, ogiri, oncom, palappam, peuyeum, pickles, pickle brine, poi, pon yegi Some examples of sauces that are considered to be good for cooking include gyi, sauerkraut, salted fish, shrimp paste, sinki, soweans, soy sauce, sumbala, tarhana, tempeh, tianmianjiang, tungtoh, and Worcestershire sauce.

[0030] In one or more embodiments, a bacteriophage preparation may be prepared by isolating the bacteriophage from a source, hi other cases, a bacteriophage preparation may be prepared by a method that includes growing a bacteriophage population in a suitable bacterial culture (e.g., using a bioreactor) and then isolating the bacteriophage from the grown bacterial culture.

[0031] Bacteriophage preparations may include members of the Myoviridae, Podoviridae, Siphoviridae, Inoviridae, Microviridae, Corticoviridae, Tectiviridae, Leviviridae, Cystoviridae, Rudiviridae, Fuselloviridae, Liposliviridae, or Plasmaviridae. In preferred embodiments, the bacteriophage preparation includes members of two or more families.

[0032] In one or more embodiments, the bacteriophage preparation may be concentrated to enhance the concentration and / or bioactivity of the bacteriophage. Exemplary methods for concentrating the bacteriophage preparation include, but are not limited to, inducing the bacteriophage to be released from their bacterial host using processes such as UV treatment, mitomycin C, temperature shift, high salt, high base, high acidity or other bacteriophage inducers. In other embodiments, the bacteriophage preparation may be concentrated by centrifugation, ultracentrifugation, filtration, size fractionation, affinity purification, affinity chromatography, CsCl density gradient, polyethylene glycol treatment, chloroform treatment, or entrapment of bacterial cells in a polymer. In one or more embodiments, periodic harvesting may be applied to the extraction of bacteriophages. Periodic harvesting is novel in the field of phage biology and involves using selective extraction techniques to harvest specific bacteriophage types from a microbial community at their peak population density. In addition, the concentration of bacteriophage in a sample may be increased by adding bacteriophage recovered from one or more other sources.

[0033] Thus, this disclosure describes a novel therapeutic approach for treating intestinal dysbiosis (e.g., SIBO or intestinal bacterial overgrowth) using a treatment that includes bacteriophages and / or diverse bacteriophage communities that are not specifically adapted to the target pathogen. Bacteriophage communities targeted to intestinal dysbiosis can be collected, mixed, and / or separated from various sources, including but not limited to environmental samples or fermentation processes (e.g., fermented plant material, fermented plant juice, fermented food sources, fermented biofuels, fermented biological waste, fermented feedstock, etc.), which are then further cultured, grown, and / or concentrated in a bacterial culture bioreactor, representing a mixed bacterial and / or bacteriophage community. Administration of a diverse bacteriophage population to the intestinal microbiota can reduce the density of the overall bacterial community, thereby correcting bacterial overgrowth. This community-level approach can also increase the overall bacterial diversity in the microbiome, potentially correcting numerous dysbiosis, restoring homeostasis to the host's gut microbial associations, and / or treating disorders associated with reduced bacterial diversity, shifts in bacterial community structure, or abnormal over-abundance of pathogenic symbionts.

[0034] In an exemplary embodiment, a diverse, mixed bacteriophage consortium was tested as a treatment in a mouse model of dysbiosis caused by water avoidance stress. Dysbiosis is characterized by bacterial overgrowth in the small intestine (i.e., SIBO). The water avoidance stress challenge caused dysbiosis in placebo-treated mice, as demonstrated by elevated bacterial density (Figure 1, WAS-PBS). Specifically, the ileum of the WAS-PBS group had a 3.3-fold increase in bacterial density (3.3±0.6) versus the control group (1.0±0.3; p<0.005). The group treated with the bacteriophage consortium (WAS-phage) had a lower bacterial density than the WAS-PBS group. The WAS-phage group had a bacterial density in the ileum (2.4±0.5) that was not significantly different (ns) from the control group.

[0035] In addition, sequence analysis of the cecal microbiota revealed that the WAS-phage group had a 1.5-fold increase in α-diversity (mean = 168 ± 13) versus the control (mean = 115 ± 16; p < 0.05), as measured by the Chao1 index (Figure 3). The Chao1 index of the WAS-PBS group (mean = 144 ± 13) was not significantly different from that of the control (ns). These statistics were measured using a Kruskal-Wallis test with Dunn's correction for multiple comparisons. Analysis of β-diversity between the experimental groups demonstrated that the WAS test was sufficient to alter the composition of the microbiota community. Both WAS groups, WAS-PBS and WAS-phage, were significantly different (p < 0.01) from the control group using Bray-Curtis dissimilarity (Figure 2). Interpreted together with the α-diversity analysis, the β-diversity data demonstrated that the WAS-test altered the overall microbiota community composition versus the control (demonstrating dysbiosis). Furthermore, treatment with the bacteriophage preparation was the only treatment associated with an overall increase in the diversity of species present in the microbiota, indicating that it may have utility in correcting dysbiosis.

[0036] These findings provide evidence that treatment with a bacteriophage consortium is effective in restoring ileal bacterial density to the levels of healthy untreated controls in subjects with experimentally induced gut microbiota dysbiosis. These data on resolving SIBO in mice provide the first evidence for the use of a diverse, mixed bacteriophage consortium cultured in an artificial environment using sources including fermented plant material as a treatment for gut microbial dysbiosis.

[0037] Thus, this disclosure describes bacteriophage preparations and uses of the bacteriophage preparations for the treatment of conditions caused by dysbiosis. As used herein, "treat" or variations thereof refers to any degree of reducing, limiting, improving, or resolving a symptom or sign associated with a condition. "Treatment" can be therapeutic or prophylactic. "Therapeutic" and variations thereof refer to treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition. "Prophylactic" and variations thereof refer to treatment that limits, to some extent, the onset and / or appearance of a symptom or clinical sign of a condition. Generally, a "therapeutic" treatment is initiated after the onset of a condition in a subject, whereas a "prophylactic" treatment is initiated prior to the onset of a condition in a subject.

[0038] A treatment that is preventative - for example, treatment that is initiated before the subject displays symptoms or clinical signs of a disease condition, even though, for example, the infection remains asymptomatic - is referred to herein as treatment of a subject at "high risk" of having a disease condition. As used herein, the term "high risk" refers to a subject who may or may not actually have the described risk. Thus, for example, a subject at "high risk" of an infectious condition is one who is likely to have been in an area where other individuals have been identified as being infectious and / or exposed to infectious material, even if the subject has not yet displayed any detectable indicators of infection by a microorganism, and regardless of whether the subject may carry a subclinical amount of the microorganism. As another example, a subject at "high risk" of a non-infectious condition is one who has one or more risk factors associated with the condition, such as, for example, genetic predisposition, ancestry, age, sex, geographic location, lifestyle, or medical history. In addition, a subject may be considered "at high risk" due to exposure or anticipated exposure to factors that disrupt the gut microbiota, such as, for example, antibiotic treatment, an episode of acute gastroenteritis, surgery or general anesthesia, prolonged stress, deployment to a combat zone, travel, illness, etc. As yet another example, a subject who has been treated for dysbiosis is at "high risk" of recurrence or relapse of dysbiosis. Prophylactic treatment may be initiated before the subject exhibits symptoms or clinical signs, or when the subject exhibits prodromal symptoms or clinical signs that are precursors to full recurrence or relapse of dysbiosis. Such prophylactic treatment may be directed at reducing the likelihood or severity of any recurrence or relapse, or at alleviating the dysbiosis. For example, fluctuating energy levels may be a precursor of impending recurrence or relapse of dysbiosis in chronic fatigue syndrome. As another example, a subject suffering from idiopathic REM sleep behavior disorder (iRBD), a precursor to progression to Parkinson's disease, may be treated before the condition develops into full Parkinson's disease.

[0039] Thus, the bacteriophage preparation may be administered to a subject before, during, or after the subject first exhibits a symptom or clinical sign of a condition caused by dysbiosis. Treatment initiated before the subject first exhibits a symptom or clinical sign associated with the condition may result in a reduced likelihood that the subject will experience clinical evidence of the condition, a reduced severity of the symptoms and / or clinical signs of the condition, and / or complete resolution of the condition, compared to a subject to whom the bacteriophage preparation is not administered. Treatment initiated after the subject first exhibits a symptom or clinical sign associated with the condition may result in a reduced severity of the symptoms and / or clinical signs of the condition, and / or complete resolution of the condition, compared to a subject to whom the bacteriophage preparation is not administered.

[0040] Thus, the method comprises administering to a subject suffering from or at risk of suffering from a condition caused by dysbiosis an effective amount of the composition, In this embodiment, an "effective amount" is an amount effective to alleviate, limit the progression of, ameliorate, or resolve, to any extent, a symptom or clinical sign associated with the condition.

[0041] The bacteriophage preparations described herein may be formulated with a pharma- ceutically acceptable carrier. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and / or agents for pharma- ceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic bacteriophage preparations is contemplated. Any conventional media components may also be incorporated into the bacteriophage preparation. As used herein, "pharma-ceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual together with the bacteriophage preparation without causing any undesirable biological effects or interacting in a deleterious manner with any other components of the pharmaceutical bacteriophage preparation in which it is included.

[0042] Thus, the bacteriophage preparations may be formulated into pharmaceutical compositions. Pharmaceutical compositions may be formulated in a variety of forms adapted to the preferred route of administration. Thus, the compositions may be administered via known routes, including, for example, orally, parenterally (e.g., intradermally, transdermally, subcutaneously, intramuscularly, intravenously, intraperitoneally, etc.), via a ring located in an organ such as the gastrointestinal tract or a body cavity, or topically (e.g., intranasally, intrapulmonary, intramammary, intravaginally, intrauterine, intradermally, transdermally, rectally, intravesically, etc.). Pharmaceutical compositions may also be administered to mucosal surfaces, such as, for example, to the nasal mucosa, respiratory mucosa, vaginal mucosa, rectal mucosa, etc. (e.g., by spray or aerosol), or applied to the skin or scalp (e.g., cream, gel, solution, ointment, spray, or patch). The compositions may also be administered via sustained or delayed release.

[0043] Thus, the bacteriophage preparation may be provided in any suitable form, including, but not limited to, a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The bacteriophage preparation may be delivered in a formulation comprising any pharma- ceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in, for example, a conventional topical dosage form, such as a cream, an ointment, a skin patch, an aerosol formulation, a non-aerosol spray, a gel, a lotion, a hand wash, a body wash, a shampoo, a surgical or dental cleanser or rinse, a dentrifice, an eye drop, an inhaler, and the like.

[0044] Formulations designed for oral delivery include, but are not limited to, microspheres (coated, uncoated, or combinations of coated and uncoated), particles (coated, uncoated, or combinations of coated and uncoated), powders, granular formulations, suspensions, emulsions, solutions, syrups, elixirs, tablets (coated, uncoated, or combinations of coated and uncoated), troches, capsules, caplets, lozenges, dentrifices, chewing gum, or sprays.

[0045] Formulations designed for delivery to the skin include, but are not limited to, microspheres (coated, uncoated, or combinations of coated and uncoated), particles (coated, uncoated, or combinations of coated and uncoated), powders, granular formulations, suspensions, emulsions, solutions, hand or body washes, shampoos, patches, or sprays.

[0046] Formulations designed for delivery to the eye or conjunctiva include, but are not limited to, microspheres (coated, uncoated, or combinations of coated and uncoated), particles (coated, uncoated, or combinations of coated and uncoated), powders, granular formulations, suspensions, emulsions, or solutions, and may be delivered as eye drops or as a spray.

[0047] Formulations designed for delivery to the nasal or respiratory passages include, but are not limited to, microspheres (coated, uncoated, or combinations of coated and uncoated), particles (coated, uncoated, or combinations of coated and uncoated), powders, granular formulations, suspensions, emulsions, or solutions, and may be delivered as an aerosol or spray.

[0048] The formulation may further include one or more additives including, for example, adjuvants, skin penetration enhancers, dyes, fragrances, flavorings, moisturizers, thickeners, and the like.

[0049] In some cases, the method of treatment may involve treating dysbiosis localized to epidermal tissues, such as, for example, the epidermis, mucosal surfaces, at least a portion of the skin or scalp, at least a portion of the oral cavity including the teeth or gums, at least a portion of the gastrointestinal tract, at least a portion of the nasal cavity, at least a portion of the respiratory tract, at least a portion of the genitourinary tract, or a body cavity. Formulations designed for delivery to the genitourinary tract (e.g., the vagina or rectum) include, but are not limited to, microspheres (coated, uncoated, or combinations of coated and uncoated), particles (coated, uncoated, or combinations of coated and uncoated), powders, granular formulations, suspensions, emulsions, solutions, syrups, elixirs, tablets (coated, uncoated, or combinations of coated and uncoated), troches, bidets, enemas, capsules, caplets, lozenges, suppositories, or sprays. Thus, the bacteriophage preparation may be provided in a formulation suitable for delivery to epidermal tissues by a route of administration suitable for delivery to epidermal tissues.

[0050] In one or more embodiments, the bacteriophage preparation is incorporated into a medical device, such as, for example, a pacemaker, a catheter, or a stent. As used herein, "incorporated into a medical device" refers to any manner of associating a bacteriophage preparation with a medical device. Exemplary ways in which a bacteriophage preparation may be incorporated into a medical device include, but are not limited to, a bacteriophage preparation incorporated into one or more components of a medical device, a bacteriophage preparation contained in a coating applied to a portion of a medical device, or a bacteriophage preparation contained within a patch or reservoir of a medical device.

[0051] The formulations may be conveniently provided in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods for preparing compositions with pharmaceutically acceptable carriers include the step of bringing the bacteriophage preparation into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations involve uniformly and / or intimately bringing into association an active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0052] The dose of the bacteriophage preparation administered can vary depending on a variety of factors, including, but not limited to, the weight, physical condition, and / or age of the subject, and / or the route of administration. Thus, the concentration of the bacteriophage in a given formulation volume can vary widely and depends on factors such as the species, age, weight and physical condition of the subject, and / or the method of administration. Thus, it is not practical to generally indicate what constitutes an amount of bacteriophage preparation effective for all possible applications. However, one skilled in the art can easily determine the appropriate amount taking such factors into account.

[0053] According to one or more embodiments, the method comprises administering to the subject about 10 4 Bacteriophage / ml ~ approx. 10 12The method may include administering sufficient bacteriophage preparation to provide a dose of 100 mg / ml of bacteriophage, although in one or more embodiments the method may be practiced by administering the bacteriophage preparation at a dose outside this range. In some of these embodiments, the method may include administering sufficient bacteriophage preparation to provide a dose of 100 mg / ml of bacteriophage, although in one or more embodiments the method may be practiced by administering the bacteriophage preparation at a dose outside this range. 5 Bacteriophage / ml ~ approx. 10 10 Dose of bacteriophage / ml, from about 4 x 10 bacteriophage / ml to about 3 x 10 11 A dose of bacteriophage / ml, for example, or about 5×10 10 Bacteriophage / ml ~ approx. 1.2 x 10 11 In one exemplary embodiment, the method comprises administering a sufficient bacteriophage preparation to provide a dose of about 10 7 A dose of bacteriophage / ml is administered to the subject.

[0054] A single dose may be administered all at once, continuously over a defined period of time, or in multiple separate doses. When multiple doses are used, the amount of each dose may be the same or different. For example, 10 doses per day may be administered. 7 The dose of bacteriophage was 10 7 When administered as a single dose of bacteriophage, it caused a sustained, 5 × 10 6 Either administered as two doses of bacteriophage or as a total of 10 7 The bacteriophage may be administered as unequal doses within a 24 hour period. When multiple doses are used to deliver a single dose, the intervals between doses may be the same or different.

[0055] In certain embodiments, the bacteriophage preparation may be administered as a single dose or multiple doses once per week, however, in one or more embodiments, the method may involve a course of treatment that includes administering doses of bacteriophage at frequencies exceeding this range. When a course of treatment involves administering multiple doses within a particular time period, the amount of each dose may be the same or different. For example, a course of treatment may include a loading dose, followed by a maintenance dose that is less than the loading dose. Also, when multiple doses are used within a particular time period, the interval between doses may be the same or different.

[0056] In one or more embodiments, the bacteriophage preparation may be administered after an extended period of fasting. As used herein, "extended fasting" refers to a period of time that is longer than the time between normal meals. As used herein, "fasting" refers to any reduction in access to calories or nutrients. Thus, in one or more embodiments, an extended period of fasting includes fasting for a minimum period of at least 8 hours, such as, for example, at least 12 hours, a minimum of 18 hours, at least 24 hours, at least 30 hours, or at least 36 hours. An extended period of fasting includes fasting for a maximum period of 72 hours or less, such as, for example, 60 hours or less, 54 hours or less, 48 ​​hours or less, 42 hours or less, 36 hours or less, 30 hours or less, 24 hours or less, or 18 hours or less. In some cases, an extended period of fasting includes fasting for a period of time within a range having an endpoint defined by any of the minimum fasting periods described above and any of the maximum fasting periods described above that is longer than the minimum fasting period. In certain embodiments, the prolonged fasting includes fasting for 12 to 36 hours, hi one particular exemplary embodiment, the prolonged fasting includes fasting for 24 hours.

[0057] Fasting reduces the food sources available to resident gut bacteria, thereby inhibiting the growth of the gut bacteria population. When used in conjunction with bacteriophage therapy, fasting can reduce resident gut bacteria, thereby making the administration of bacteriophage therapy more effective. In one or more alternative embodiments, the bacteriophage therapy can be administered before, during, or after one or more meals.

[0058] In one or more embodiments, the bacteriophage preparation may be administered for a period ranging from, for example, a single dose to the remaining lifespan of the subject. In certain embodiments, the bacteriophage preparation may be administered as a one-time, single dose. In other embodiments, the bacteriophage preparation may be administered for a period ranging from one day to about one year, for example, from about three days to about seven days. Treatment with the bacteriophage preparation may also occur periodically as a re-treatment to reduce the likelihood and / or severity of recurrence of the dysbiosis.

[0059] In one or more embodiments, the method may include administering to the subject a second pharmaceutical composition for treating dysbiosis. Exemplary pharmaceutical compositions for treating dysbiosis include, but are not limited to, an antibiotic preparation, a prebiotic preparation, a probiotic preparation, a synbiotic preparation, a fecal microbiota transplant, a membrane vesicle, an autophagy inducer, or a combination of any two or more pharmaceutical compositions for treating dysbiosis.

[0060] Exemplary antibiotic agents include, but are not limited to, rifaximin, metronidazole, cephalexin, trimethoprim-sulfomethoxazole, doxycycline, colistin, tetracycline, clindamycin, ciprofloxacin, levofloxacin, moxifloxacin, ofloxacin, norfloxacin, ampicillin, amoxicillin, erythromycin, tinidazole, nitazoxanide, albendazole, paromomycin, quinacrine, lactulose, bismuth disalicylate, gentamicin, neomycin, or any combination of two or more antibiotics.

[0061] Exemplary prebiotic preparations include, but are not limited to, fructo-oligosaccharides, disaccharides, monosaccharides, polyols, galacto-oligosaccharides, inulin, short chain carbohydrates, sugar alcohols, oligofructose, or a combination of two or more prebiotic preparations.

[0062] Exemplary probiotic formulations include, but are not limited to, members of the lactic acid bacteria group, such as, for example, Pediococcus, Streptococcus, Lactococcus, Lactobacillus, Oenococcus, Weissella, or Leuconostoc spp., such as, for example, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus brevis, Lactobacillus spp. brevis, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus sakei, Lactobacillus bulgarius, Lactobacillus reuteri, Lactobacillus rhamnosus GG, Lactobacillus lactis, Streptococcus thermophilus, Saccharomyces boulardii boulardii, Saccharomyces bayanus, Saccharomyces cerevisiaecerevisiae, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium animalis, Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Pediococcus acidilactici acdilactici, Leuconostoc mesenteroides, Escherichia coli Nissle, Enterococcus durans, Weissella cibaria, Enterococcus faecium, or a combination of any two or more probiotic preparations.

[0063] Exemplary synbiotic formulations include, but are not limited to, combinations of prebiotic and probiotic formulations. Exemplary fecal microbiota transplants include, but are not limited to, whole fecal matter, partial fecal matter, altered fecal matter, whole microbiota, partial microbiota, fecal filtrate, or altered microbiota.

[0064] In one or more embodiments, the dysbiosis to be treated may include dysbiosis localized to superficial tissues, such as, for example, the epidermis, a mucosal surface, at least a portion of the skin or scalp, at least a portion of the oral cavity including the teeth or gums, at least a portion of the gastrointestinal tract, at least a portion of the nasal cavity, at least a portion of the respiratory tract, at least a portion of the genitourinary tract, or a body cavity.

[0065] In the foregoing description and in the claims that follow, the term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements; the terms "comprise", "comprising" and variations thereof should be interpreted as open-ended, i.e., additional elements and steps are optional and may or may not be present: unless otherwise stated, "a", "an", "the" and "at least one" are used interchangeably and mean one or more than one; and the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0066] In the above description, certain embodiments may be described separately for clarity. Throughout this specification, reference to "one embodiment," "an embodiment," "particular embodiment," or "one or more embodiments" means that the particular feature, form, composition, or characteristic described in connection with that embodiment is included in at least one embodiment of the disclosure. Thus, the appearance of such phrases in various places throughout this specification does not necessarily refer to the same embodiment of the disclosure. Moreover, certain features, forms, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, certain features, forms, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, a feature described in connection with one embodiment may be combined with a feature described in connection with a different embodiment, except where the features are mutually exclusive.

[0067] For any method disclosed herein that includes discrete steps, the steps may be performed in any practicable order and, where appropriate, any combination of two or more steps may be carried out simultaneously.

[0068] As used herein, the terms "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0069] The present invention is illustrated by the following examples, it being understood that the particular examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein. EXAMPLES

[0070] A diverse and mixed community preparation of bacteriophages was generated in a spontaneous lactobacillus culture of fresh cabbage in a 3% NaCl w / v solution. The culture was incubated at 18°C ​​for 21 days and then subjected to centrifugation, filtration, and concentration to isolate a pure preparation of bacteriophages. The culture was pelleted of bacterial cells and particulate matter by centrifugation of the sample at 5000 x g for 30 minutes. The remaining supernatant (containing the bacteriophages) was filtered at 450 nm to filter-sterilize the solution and remove any remaining bacteria. The bacteriophages were concentrated and isolated using a centrifugal filter with a pore size of 3 nm, a pore size known to be small enough to capture all known bacteriophages. Subsequent filling and centrifugation of the centrifugal filter concentrated the bacteriophages to 10x the level of the original culture. The bacteriophage was clarified by centrifugal filter using phosphate buffered saline (PBS) solution. The presence of bacteriophage in the solution was confirmed using electron microscopy.

[0071] Therefore, to establish a typical intestinal dysbiosis model for testing bacteriophage preparations, C57BL / 6 mice (n=6) were challenged with a water avoidance stress (WAS-PBS) test to induce SIBO. The WAS test involved 10 days of 1-h treatments, in which each mouse was placed on a circular platform (2 inches in diameter) in the center of a transparent plastic container filled with water up to 1 cm below the platform surface. In parallel, another group was given the WAS test as well as the bacteriophage preparation (WAS-phage; n=6). The WAS-phage mice were given approximately 10 stools per day for a total of 13 treatments, starting 3 days before the start of the WAS test and continuing until the last day of the WAS test. 7 A single treatment consisting of the bacteriophage preparation was given. The WAS-PBS group was given PBS gavage instead of the bacteriophage preparation. During this period, control mice (n=3) remained in their cages. Mice were euthanized 24 hours after the WAS and WAS-phage groups had received their last treatment. Tissue samples were taken from the ileum of the small intestine and stored in Zymo RNA / DNA Shield until analysis.

[0072] For quantification of bacterial density in the ileum of the small intestine, DNA was extracted from approximately 50 mg of ileal tissue by processing using the DNeasy Blood and Tissue Kit (Qiagen). The copy number of the gene for the 16s rRNA gene subunit, a universal bacterial marker, was counted in the extracted ileal DNA using quantitative polymerase chain reaction (qPCR). Using a primer pair targeting the 16s rRNA gene, one skilled in the art can count the amount of bacterial cells present in a sample relative to the amount of eukaryotic cells in the host tissue (which can be identified using the 18s rRNA gene). Using this approach, an accurate count of mucosa-associated bacteria in different tissues can be achieved by comparing the amount of bacterial cells relative to the amount of eukaryotic host cells, thereby measuring the bacterial density in the host tissue. The data from qPCR can be used to: ΔΔAnalysis was performed using the Ct method, and results are reported as fold change (mean ± SE) relative to the untreated control group. Differences between the means of each group were analyzed using the Kruskal-Wallis test with Dunn's correction for multiple comparisons. All statistical analyses were performed using PRISM (GraphPad Software, Inc., San Diego, CA).

[0073] For comparison of α- and β-diversity between treatment groups, approximately 200 mg of fecal material was collected from the cecum of each animal and sent to the University of Minnesota Genomics Center (UMGC) for bacterial DNA extraction and 16S sequencing. 16S microbiota sequencing allows for characterization of the entire microbial community composition. Bioinformatics analysis of 16S sequencing data was performed on a commission basis by CD Genomics (Shirley, NY). This analysis compares microbial community composition between treatment groups and provides a quantitative measure of both the diversity of the communities (i.e., α-diversity), as well as the dissimilarity between groups (i.e., β-diversity). Indicators of α-diversity, such as the Chao1 index used in this study, quantify the number of species and provide an objective measure of diversity within a microbial community. Conversely, beta diversity dissimilarity, such as the Bray-Curtis dissimilarity used in this study, is a subjective measure that compares differences in microbial community composition between groups; specifically, it compares groups based on which species are present and their abundance.

[0074] For statistical analysis, differences in α-diversity means between groups were analyzed using the Kruskal-Wallis test with Dunn's correction for multiple comparisons. All statistical analyses were performed with PRISM (GraphPad Software, Inc., San Diego, CA). Dissimilarities in β-diversity were analyzed using Permanova pairwise comparisons with commission method by CD Genomics (Shirly, CA).

[0075] The complete disclosures of all patents, patent applications and publications cited herein, as well as electronically available materials (e.g., nucleotide sequence submissions in GenBank and RefSeg, and amino acid sequence submissions in, e.g., SwissProto, PIR, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeg) are incorporated herein by reference in their entirety. In the event of a discrepancy between the disclosure of this application and the disclosure(s) of any document incorporated herein by reference, the disclosure of this application shall govern. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described, as variations obvious to one of ordinary skill in the art will be included within the invention defined by the claims.

[0076] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims are to be understood in all instances as modified by the term "about." Accordingly, unless specifically indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and without any intention to limit the teaching of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0077] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements. Unless otherwise stated, all headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading.

Claims

1. below: A preparation of bacteriophages of heterologous origin obtained from a fermentation process; and a pharmaceutically acceptable carrier; A pharmaceutical composition comprising:

2. The pharmaceutical composition of claim 1 further comprising an adjuvant.

3. 2. The pharmaceutical composition of claim 1, wherein the bacteriophage comprises a member of the Myoviridae family, a member of the Podoviridae family, a member of the Siphoviridae family, a member of the Inoviridae family, a member of the Microviridae family, a member of the Corticoviridae family, a member of the Tectiviridae family, a member of the Leviviridae family, a member of the Cystoviridae family, a member of the Rudiviridae family, a member of the Fuselloviridae family, a member of the Liposliviridae family, a member of the Plasmaviridae family, or a bacteriophage from a combination of the above families.

4. The ingredients of the fermentation process are as follows: Apam, Achara, Bagoong, Balao Balao, Banquong, Bleem, Brong Isda, Cheonggukjang, Chingkarok, Kulchido, Dokla, Doenjang, Dobanjang, Dochi, Fermented coconut water, Fermented grains, Fermented grapes, Fermented pineapple juice, Fermented tofu, Fermented miso, Fish sauce, Garapong, Soy sauce, Gochujang, Gundeul, Hakkalu, Chotgal, Kefir, Kaengkee, Ka 2. The pharmaceutical composition of claim 1, comprising nomchin, kimchi, kombucha, kusaya, ruhu, miso, mixian, mokuninchin, muri, nata de coco, nata de pina, nem, natto, nem chua, ngapii, ogi, ogiri, onkom, parappam, piyum, pickles, pickle brine, poi, bonyegi, sauerkraut, salted fish, shrimp paste, shinki, sowins, soy sauce, sumbala, tarhana, tempeh, tenmenjang, tungtu, or Worcestershire sauce.

5. 10. The pharmaceutical composition of claim 1, wherein the fermentation process produces fertilizer or livestock feed.

6. 10. The pharmaceutical composition of claim 1, wherein the fermentation process comprises fermentation of plant material, plant juice, feedstock, biofuel, or biological waste.

7. 10. The pharmaceutical composition of claim 1, formulated for delivery to epidermal tissue.

8. 8. The pharmaceutical composition of claim 7, wherein the epidermal tissue comprises the epidermis, a mucosal surface, at least a portion of the skin or scalp, at least a portion of the oral cavity, at least a portion of the gastrointestinal tract, at least a portion of the nasal cavity, at least a portion of the respiratory tract, at least a portion of the genitourinary tract, at least a portion of the conjunctiva, or a body cavity.

9. 8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition comprises a bacteriophage preparation formulated into a microsphere, particle, powder, granular formulation, suspension, emulsion, solution, elixir, syrup, tablet, troche, bidet, suppository, capsule, lozenge, gum, spray, body wash, shampoo, patch, strip, dentrifice, eye drops, or aerosol.

10. below: Isolating the bacteriophage from the fermentation process; and combining the isolated bacteriophage with a pharmaceutically acceptable carrier; 1. A method for preparing a bacteriophage preparation, comprising:

11. 10. The method of claim 1, further comprising concentrating the bacteriophage preparation, wherein the concentration of the bacteriophage preparation comprises: Increasing the concentration of bacteriophage in the preparation; or 11. The method of claim 10, wherein the biological activity of bacteriophage in the preparation is increased.

12. 12. The method of claim 11, wherein the bacteriophage preparation is concentrated by using centrifugation, ultracentrifugation, filtration, size fractionation, affinity purification, affinity chromatography, CsCl density gradient, polyethylene glycol treatment, chloroform treatment, or entrapment of bacterial cells in a polymer.

13. 13. The method of claim 11 or claim 12, wherein the bacteriophage preparation is concentrated by treating the bacteriophage preparation with UV light, mitomycin C, a temperature change, high salt, a pH change, or another bacteriophage-inducing treatment.

14. 12. The method of claim 10 or 11, further comprising culturing the bacteriophage preparation with a suitable bacterial species in a bioreactor.

15. The fermentation process includes the following: Apam, Achara, Bagoong, Balao Balao, Banquong, Bleem, Brong Isda, Cheonggukjang, Chinkarok, Kulchido, Dokla, Doenjang, Doebanjang, Dochi, Fermented coconut water, Fermented grains, Fermented grapes, Fermented pineapple juice, Fermented tofu, Fermented miso, Fish sauce, Garapong, Soy sauce, Gochujang, Gundeul, Hakkalu, Chotgal, Kaengkee, Kanomjin, Kefi The method according to claim 10 or 11, wherein the method is for producing kimchi, kombucha, kusaya, luhu, miso, mixian, mokuninchin, muri, nata de coco, nata de pina, nem, natto, nem chua, ngapi, ogi, ogiri, onkom, parappam, piyum, pickles, pickle brine, poi, ponyegi, sauerkraut, salted fish, shrimp paste, shinki, sowins, soy sauce, sumbara, tarhana, tempeh, tenmenjang, tungtu, or Worcestershire sauce.

16. 12. The method of claim 10 or 11, wherein the fermentation process produces fertilizer or livestock feed.

17. 12. The method of claim 10 or 11, wherein the fermentation process comprises fermentation of plant material, plant juice, feedstock, biofuel, or biological waste.

18. 1. A pharmaceutical composition for use in a method for treating dysbiosis in a subject suffering from or at risk of suffering from dysbiosis, comprising: the method comprising administering to the subject a bacteriophage preparation in an amount effective to ameliorate at least one symptom or clinical sign of the dysbiosis. The pharmaceutical composition according to claims 1 to 9.

19. 20. The pharmaceutical composition of claim 18, wherein the method further comprises administering to the subject a second pharmaceutical composition for treating a dysbiosis.

20. 20. The pharmaceutical composition of claim 19, wherein the second pharmaceutical composition for treating dysbiosis comprises an antibiotic preparation, a prebiotic preparation, a probiotic preparation, a synbiotic preparation, a fecal microbiota transplant, a membrane vesicle, or an autophagy inducer.

21. 21. The pharmaceutical composition of claim 20, wherein the antibiotic agent comprises rifaximin, metronidazole, cephalexin, trimethoprim-sulfomethoxazole, doxycycline, colistin, tetracycline, clindamycin, ciprofloxacin, levofloxacin, moxifloxacin, ofloxacin, norfloxacin, ampicillin, amoxicillin, erythromycin, tinidazole, nitazoxanide, albendazole, paromomycin, quinacrine, lactulose, bismuth disalicylate, gentamicin, neomycin, or a combination thereof.

22. 21. The pharmaceutical composition of claim 20, wherein the prebiotic formulation comprises a fructo-oligosaccharide, a disaccharide, a monosaccharide, a polyol, a galacto-oligosaccharide, inulin, a short chain carbohydrate, a sugar alcohol, oligofructose, or a combination of two or more of the foregoing.

23. 21. The pharmaceutical composition of claim 20, wherein the probiotic preparation comprises lactic acid bacteria.

24. 24. The pharmaceutical composition of claim 23, wherein the lactic acid bacteria include a member of the genus Pediococcus, a member of the genus Streptococcus, a member of the genus Lactococcus, a member of the genus Lactobacillus, a member of the genus Oenococcus, a member of the genus Weissella, or a member of the genus Leuconostoc, or a combination of two or more of the above genera.

25. The lactic acid bacteria may be Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus sakei, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactobacillus rhamnosus GG, Lactobacillus lactis, Streptococcus thermophilus, Saccharomyces boulardii, Saccharomyces baianus, Saccharomyces cerevisiae, or Bifidobacterium diffusum.

25. The pharmaceutical composition of claim 24, comprising Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium alesentis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium animalis, Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Pediococcus acidilactici, Leuconostoc mesenteroides, Escherichia coli Nissle, Enterococcus durans, Weissella cibaria, Enterococcus faecalis, or a combination of two or more of the foregoing.

26. 21. The pharmaceutical composition of claim 20, wherein the synbiotic formulation comprises a combination of a prebiotic formulation and a probiotic formulation.

27. 21. The pharmaceutical composition of claim 20, wherein the fecal microbiota transplant comprises whole fecal material, partial fecal material, altered fecal material, whole microbiota, partial microbiota, fecal filtrate, altered microbiota, or a combination of two or more of the foregoing.

28. 19. The pharmaceutical composition of claim 18, wherein the dysbiosis is localized to the epidermal tissue.

29. 29. The pharmaceutical composition of claim 28, wherein the epidermal tissue comprises the epidermis, a mucosal surface, at least a portion of the skin or scalp, at least a portion of the oral cavity including the teeth or gums, at least a portion of the gastrointestinal tract, at least a portion of the nasal cavity, at least a portion of the respiratory tract, at least a portion of the genitourinary tract, at least a portion of the conjunctiva, or a body cavity.

30. 20. The pharmaceutical composition of claim 18, wherein the bacteriophage preparation is incorporated into a medical device.

31. 31. The pharmaceutical composition of claim 30, wherein the medical device comprises a pacemaker, a catheter, or a stent.

32. 19. The pharmaceutical composition of claim 18, wherein the dysbiosis causes bacterial overgrowth.

33. 20. The pharmaceutical composition of claim 18, further comprising fasting the subject for an extended period of time prior to administering the bacteriophage preparation to the subject.

34. 34. The pharmaceutical composition of claim 33, wherein the prolonged fast is about 1 to 3 days.

35. 20. The pharmaceutical composition of claim 18, wherein the bacteriophage preparation is administered before, during, and after one or more meals.

36. 20. The pharmaceutical composition of claim 18, wherein the bacteriophage preparation is administered orally, topically, by inhalation, or parenterally.

37. The method of claim 37, wherein the method comprises administering to a healthy subject a bacterial source, in order to prepare or pre-condition the intestinal environment of the healthy subject for administration of the bacterial source, the method comprising administering to a healthy subject a bacterial source ...

20. The pharmaceutical composition of claim 18, further comprising administering to the subject a bacteriophage preparation in an amount effective to improve the subject's intestinal susceptibility to the bacterial source.

38. 38. The pharmaceutical composition of claim 37, wherein the bacterial source comprises a probiotic preparation or a fecal microbiota transplant.

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