Phage Compositions and Kits and Related Methods

Abstract

Compositions for influencing the gut microbiome of a mammalian subject can comprise a pharmaceutically acceptable carrier and phages at a concentration greater than or equal to 1x10 4 PFU / mL or PFU / mg, wherein the phages have tropisms that include obesity-causing bacteria and / or inflammatory bacteria associated with the gut microbiome of a mammal. Processes for preparing the disclosed compositions can include isolating the phages from environmental sources, characterizing the phages, and combining the phages with a pharmaceutically acceptable carrier in an amount greater than or equal to 1x10 4 PFU / mL or PFU / mg. Methods can include administering the disclosed compositions to a mammalian subject with or without co-administering the disclosed probiotics. Administering the disclosed compositions can reduce the concentration of obesity-causing bacteria and / or inflammatory bacteria in the gut microbiome of a mammalian subject, promote or induce weight loss, reduce inflammation, support metabolic health, and / or support a healthy balance / diversity within the gut microbiome of a mammalian subject.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority and the benefit of U.S. Provisional Patent Application Serial No. 62 / 675,652, filed on May 23, 2018, entitled "Methods and Compositions for Treating Chronic Diseases with Bacteriophages", and claims priority of U.S. Provisional Patent Application Serial No. 62 / 815,844, filed on March 8, 2019, entitled "Bacteriophage Compositions and Methods of Use". Each of the foregoing is incorporated herein by reference in its entirety. Technical field

[0003] The present invention generally relates to compositions comprising one or more bacteriophages, kits comprising such compositions, and methods of preparing such compositions and methods of using or administering such compositions to affect the number or concentration of bacteria within the gut microbiome of a mammalian subject, preferably for promoting or inducing weight loss, reducing inflammation, supporting metabolic health, and / or supporting a healthy balance / diversity within the gastrointestinal microbiome. Background art

[0004] Obesity has become a worldwide epidemic. Over the past few decades, the rates of overweight and obesity in the United States have risen sharply, with up to 37% of Americans (i.e., 120 million people) meeting the criteria for obesity. In the United States, from 2013 to 2014, the prevalence of obesity was 35% in men and 40% in women. Another 34% of American adults are overweight. 6% of American adults are morbidly obese, which has increased by 400% since 1986. Among children and adolescents, the prevalence of obesity is 17%. Obesity is typically associated with chronic low - level inflammation and is a major cause of many other chronic diseases, such as type 2 diabetes, insulin resistance, prediabetes, heart disease, stroke, fatty liver disease, kidney disease, osteoarthritis, depression, anxiety, certain cancers, and / or other conditions, which generally lead to a decline in quality of life and soaring healthcare costs.

[0005] To improve their quality of life, some overweight or obese individuals have adopted calorie - restricted diets and / or increased their physical activity, such as by participating in cardiovascular fitness regimens. However, certain physical limitations can impede an individual's weight - loss efforts. For example, some overweight or obese individuals have difficulty increasing their level or intensity of physical activity due to complications or secondary effects of their body size and weight. For some overweight or obese individuals, even with strict adherence to a diet and exercise program, their weight - loss progress is unexpectedly slow or sometimes non - existent.

[0006] Recent evidence suggests that factors other than diet and exercise may play a role in an individual's ability to promote or induce weight loss. For example, an individual may be genetically predisposed to have a higher body mass index and / or a poorer metabolism. Environmental stress may exacerbate the genetic predisposition and play a role in an individual's ability to promote or induce weight loss. Recently, it has been hypothesized that an individual's ability to absorb and digest nutrients affects their health and wellness and may impact the effectiveness of dieting or other weight loss programs.

[0007] Recently, scientists have been able to establish a correlation between the metabolic potential of mammals and the diversity and composition of their respective gut microbiomes. For example, groundbreaking studies conducted in mice have linked pregnancy-induced insulin resistance to the composition and diversity of the mouse gut microbiome. Fecal transplants between pregnant mice exhibiting pregnancy-induced insulin resistance and non-pregnant mice with normal or baseline insulin sensitivity resulted in the non-pregnant mice exhibiting signs and symptoms of similar pregnancy-induced insulin resistance as their corresponding pregnant mice. In another study, fecal transplants from obese mice to healthy, average-weight mice resulted in the average-weight mice gaining weight, even when the mice had the same diet.

[0008] These and other studies suggest that the gut microbiome plays a role in our digestive and metabolic health. Unfortunately, it has proven difficult to tease out the complex interactions between the state of an individual's gut microbiome and the various effects it may have on their physiology and metabolism. The gut microbiome is a complex ecosystem composed of bacteria, fungi, and viruses that exist in different physiological states and structures. For example, bacteria in the gut microbiome can exist as planktonic cells or as structured communities of monoclonal (or even polyclonal) biofilms. Additionally, some bacterial species may be located in different regions of the digestive tract, and the practical significance of the spatial organization of the microbes associated with the digestive tract is poorly understood. Adding to the complexity, the composition and diversity of the microbes associated with the digestive tract vary among individuals, and the commonalities or predictive features of a "healthy" gut microbiome are poorly known.

[0009] Most research in this area has relied on whole fecal transplants between hosts to determine the overall correlation between physiological states and the abundance and diversity of the associated gut microbiota. This is due in part to the wide diversity and proportional concentrations of the microorganisms that make up the mammalian gut microbiome. Many gut microbiota cannot be cultured using known laboratory conditions and media, which complicates their identification and tractable contributions to host physiology or metabolism. Thus, while research on the gut microbiome has provided some illuminating insights, there remains a lack of tools to specifically increase the members or concentrations of select microbial cohorts in the mammalian microbiome. It is clear that there is a lack of available tools and methods for transitioning the mammalian microbiome from an undesirable state to a desirable state.

[0010] For example, aspects of the microbiome have been shown to be associated with body weight (e.g., body mass index, BMI) and obesity. Bacterial cohorts associated with the gut microbiome have been correlated with "healthy" individuals with an average or ideal BMI, while other bacteria associated with the gut microbiome have been correlated with "unhealthy" overweight or obese individuals. However, there is a lack of compositions, methods, and / or kits for reducing or eliminating the prevalence of microbes associated with obesity and / or for increasing the prevalence of "beneficial" microbes associated with non-obese individuals.

[0011] Accordingly, many of the drawbacks associated with currently available compositions, methods, and kits can be addressed, and there is a need for targeted methods to meaningfully affect microbiome alterations to improve the health and well-being of an individual, particularly with regard to weight gain, obesity, and the detrimental consequences associated therewith. SUMMARY OF THE INVENTION

[0012] Embodiments of the present disclosure address one or more of the foregoing or other problems in the art through compositions, methods, and kits for effecting changes in the gut microbiome. In particular, aspects (or embodiments) of the present disclosure relate to methods, systems, and compositions for reducing the amount or concentration of one or more bacteria in the gut microbiome of a mammalian (preferably human) subject and, in certain aspects, increasing the amount or concentration of one or more additional bacteria. Specifically, aspects of the present disclosure relate to methods, systems, and compositions for treating one or more conditions that are caused, at least in part, by an excess of one or more bacteria and / or a deficiency of one or more additional bacteria in the digestive tract of the subject. Such conditions may include, for example, (i) obesity (or overweight), (ii) one or more obesity (or overweight)-related conditions (e.g., (metabolic, chronic, and / or acute) diseases, disorders, and / or illnesses), (iii) inflammation, particularly low levels of systemic inflammation, and / or (iv) one or more inflammation-related conditions (e.g., (metabolic, chronic, and / or acute) diseases, disorders, and / or illnesses). Embodiments include one or more bacteriophages, as well as compositions or kits containing the bacteriophage, and methods for preparing and using the compositions or kits.

[0013] Aspects of the present disclosure include a composition comprising an amount of one or more bacteriophages and a pharmaceutically acceptable carrier or excipient.

[0014] The compositions or their bacteriophages of various aspects of the present disclosure can be used for and / or are effective in altering or changing the gut microbiome or the composition of the gut microbiome of a mammalian subject when the composition or one or more of its bacteriophages are administered to the subject in a manner that delivers or disposes the composition or one or more of its bacteriophages to the digestive tract or gut microbiome of the subject. For example, the composition or its bacteriophages can be used for and / or are effective in reducing the concentration of one or more bacteria in the gut microbiome of a subject when administered to the subject in a manner that delivers or disposes the composition or one or more of its bacteriophages to the digestive tract or gut microbiome of the subject. In certain aspects, the one or more bacteria may include at least one species or strain of obesity-causing, inflammatory, and / or other bacteria. The one or more bacteriophages may have tropism for (or include) one or more (obesity-causing, inflammatory, and / or other) bacteria or their species or strains.

[0015] In at least one aspect, reducing the concentration of one or more obesity-causing, inflammatory, and / or other bacteria in the gut microbiome of a subject can be effective in: inducing, promoting, and / or supporting weight loss; treating obesity (or overweight) and / or one or more obesity-related conditions; promoting or supporting weight management; supporting metabolic health; supporting a healthy balance and / or diversity within the digestive tract microbiome, treating and / or reducing (low-level, systemic) inflammation, reducing the presence and / or concentration of inflammatory markers; and / or treating one or more conditions in a subject associated with (low-level systemic) inflammation. In some aspects, conditions related to obesity and / or related to (low-level systemic) inflammation can include one or more of type 2 diabetes, insulin resistance, prediabetes, heart disease, stroke, fatty liver disease, kidney disease, osteoarthritis, depression, anxiety, certain cancers, and / or other disorders, illnesses, diseases, and / or conditions (including metabolic and / or acute disorders, illnesses, and / or diseases). Accordingly, aspects of the present disclosure relate to compositions for treating (i) obesity and / or one or more obesity-related conditions in a subject, (ii) inflammation and / or one or more conditions related to (low-level systemic) inflammation in a subject, (iii) an excess of one or more bacteria in the gut microbiome of a subject, and (iv) combinations thereof.

[0016] In at least one aspect, reducing the concentration of one or more obesity-causing, inflammatory, and / or other bacteria in the gut microbiome of a subject can be effective in: increasing energy and / or improving stamina, improving the regularity and ease of bowel movement (softer stools and / or reduced / less frequent constipation), reducing appetite / food cravings, improving muscle tone, reducing arm and / or waist circumference, improving skin color and / or reducing acne, improving mood, reducing feelings of depression, improving concentration, mental awareness, and / or focus, alleviating and / or relieving chronic stomach pain, and / or improving overall health. Accordingly, the compositions of the present disclosure can be used to treat a variety of conditions ranging from obesity (or overweight), stomach pain, constipation, and food cravings to low energy or stamina, impaired mental concentration, depression / mood disorders, and pain / skin conditions.

[0017] One or more aspects (or embodiments) of the present disclosure include a composition having a pharmaceutically acceptable carrier and one or more bacteriophages, wherein the one or more bacteriophages are included in the composition in any suitable amount and / or concentration. For example, the one or more bacteriophages may be included in the composition in an amount and / or concentration that is effective in reducing the concentration of one or more (target or host) bacteria of the one or more bacteriophages when the composition or one or more bacteriophages thereof are delivered or configured to the digestive tract or intestinal microbiome of a mammalian (preferably human) subject. Illustratively, the composition may have or include (i) greater than or equal to 1x10 4 One or more phages, (ii) greater than or equal to 1x10 4 Plaque forming units of one or more phages, (iii) greater than or equal to 1x10 4 PFU / mL of one or more bacteriophages, and / or (iv) greater than or equal to 1x10 4 PFU / mg (PFU / mg) of one or more bacteriophages. As used herein, an "effective" amount can be or include any of the foregoing or other specific amounts or concentrations.

[0018] In certain aspects, the one or more phages (of the composition) have a tropism against or include the one or more bacterial species or strains. Illustratively, the one or more phages may have a tropism against or include a single bacterial species or strain or a plurality of (e.g., closely related) bacteria. In other aspects, the one or more phages may have a tropism against or include a plurality of bacteria that are not closely related.

[0019] In some aspects, the composition includes one or a single phage (strain). The single phage (strain) may have a tropism for or include a single bacterial species or strain or multiple bacteria. In other aspects, the composition comprises multiple phages (strains). In at least one aspect, the multiple phages (strains) may include a first phage (strain), a second phage (strain), and optionally a third, fourth, fifth, sixth, etc. phage (strain). The multiple phages (strains) may each have a tropism for or include a single bacterium (species or strain) or multiple bacteria (species or strains). The single bacteria (species or strain) may be the same bacteria or different bacteria. The multiple bacteria (species or strains) may be the same multiple bacteria or different bacteria. Illustratively, the first phage (strain) may have a tropism for or include a single bacteria (species or strain) or multiple bacteria (species or strains), and the second phage (strain) may have a tropism for or include the same or different single bacteria (species or strains), or the same or different multiple bacteria (species or strains).

[0020] In at least one aspect, a mixture (or cocktail) of bacteriophages can be selected to be included in the compositions of the present disclosure. The bacteriophage cocktail can be configured for, adapted to, or otherwise capable of targeting, infecting, and killing (e.g., lysing) a single bacterium (species or strain) or multiple bacteria (species or strains). In certain aspects of the present disclosure, redundancy or overlap in tropism may be desirable. In certain aspects of the present disclosure, variability and diversity in tropism may be desirable. For example, some subjects may have an unconventional single bacterium species or strain in their gut microbiome, while other subjects may have multiple unconventional bacterium species or strains in their gut microbiome. Different combinations or two or more bacteriophages can be selected to target a single or multiple bacteria. Additionally, the bacteria can be adapted to or capable of mutating to evade the targeting, infection, or destruction by one or more bacteriophage strains. Thus, the compositions according to certain aspects of the present disclosure include at least two bacteriophages having tropisms for the same bacterium or at least some of the same bacteria, but preferably by different (genetic or tropic) mechanisms of action.

[0021] In at least one aspect, the composition comprises a pharmaceutically acceptable carrier and an amount of one or more bacteriophages. The amount of the one or more bacteriophages can be (i) an amount effective to reduce the concentration of one or more target bacteria of the one or more bacteriophages when the composition or one or more of its bacteriophages are administered to a subject in a manner that delivers or configures the composition or one or more of its bacteriophages to the digestive tract or gut microbiome of a mammalian (preferably human) subject, (ii) one or more bacteriophages greater than or equal to 1x10 4 of one or more bacteriophages, (iii) one or more bacteriophages greater than or equal to 1x10 4 plaque forming units, (iv) one or more bacteriophages greater than or equal to 1x10 4 PFU / mL (plaque forming units per milliliter), (v) and / or one or more bacteriophages greater than or equal to 1x10 4 PFU / mg (plaque forming units per milligram). The one or more bacteriophages can have tropisms that include one or more target bacteria associated with the gut microbiome of a subject or tropisms that exhibit specific infectivity for the target bacteria. The one or more target bacteria can be or include one or more obesity- and / or inflammation-causing bacteria associated with the gut microbiome of a mammal.

[0022] In at least one aspect, the composition comprises a pharmaceutically acceptable carrier and an amount of two or more phages. The amount of each of the two or more phages can be (i) an amount effective to reduce the concentration of one or more target bacteria of each of the two or more phages when the composition or two or more of its phages are administered to a subject in a manner that delivers or disposes the composition or its two or more phages to the digestive tract or gut microbiome of a mammalian (preferably human) subject, (ii) each of the two or more phages greater than or equal to 1x10 4 , (iii) each of the two or more phages greater than or equal to 1x10 4 plaque forming units, (iv) each of the two or more phages greater than or equal to 1x10 4 PFU per milliliter (PFU / mL), (v) and / or each of the two or more phages greater than or equal to 1x10 4 PFU per milligram (PFU / mg). Each of the two or more phages can have a tropism that includes one or more target bacteria associated with the gut microbiome of the subject or exhibits specific infectivity for the target bacteria. The one or more target bacteria can be or include one or more obesity- and / or inflammation-causing bacteria associated with the gut microbiome of a mammal. The tropism of each of the two or more phages can be a similar or identical tropism, or a different tropism, compared to any one or more other phages of the composition.

[0023] In one aspect, the composition comprises a pharmaceutically acceptable carrier and greater than or equal to 1x10 6 or 1x10 6 PFU etc. of a plurality of phages (each), the plurality of phages comprising two or more of the following, preferably each: Optium-12, Optium-18, Optium-38, Optium-79, Optium-125, Optium-126, Optium-148, Optium-155, Optium-162, Optium-169, Optium-212, Optium-712, Optium-719, Optium-712, and Optium-819, which correspond to SEQ ID NO 1–15 of the present disclosure. In one aspect, the composition comprises a pharmaceutically acceptable carrier and greater than or equal to 1x10 8 or 1x10 8Each of a plurality of bacteriophages such as PFU, said plurality of bacteriophages comprising, consisting of, or consisting essentially of: Optium-12, Optium-18, Optium-38, Optium-79, Optium-125, Optium-126, Optium-148, Optium-155, Optium-162, Optium-169, Optium-212, Optium-712, Optium-719, Optium-712, and Optium-819, which correspond to SEQ ID NO 1–15 of the present disclosure.

[0024] One or more embodiments of the present disclosure include a composition having a pharmaceutically acceptable carrier and one or more bacteriophages in an amount greater than or equal to 1x10 4 plaque forming units per milliliter (PFU / ml), plaque forming units per milligram (PFU / mg), or other suitable unit of measurement, said bacteriophages having tropism for one or more obesity-causing bacteria associated with the mammalian gut microbiome. In at least one aspect of the composition, the tropism of the one or more bacteriophages includes Enterobacter cloacae, preferably Enterobacter cloacae strain B29. In at least one aspect of the composition, the one or more bacteriophages include Optium-18, Optium-38, Optium-125, Optium-126, and / or Optium-712.

[0025] In at least one aspect of the composition, the one or more bacteriophages include Optium-12, Optium-18, Optium-38, Optium-79, Optium-125, Optium-126, Optium-148, Optium-155, Optium-162, Optium-169, Optium-212, Optium-712, Optium-719, Optium-712, and / or Optium-819.

[0026] In another aspect, the one or more bacteriophages include Optium-17, Optium-34, Optium-86, Optium-88, Optium-113, Optium-118, Optium-125, and / or Optium-417.

[0027] In at least one aspect, the one or more bacteriophage of the composition has a tropism comprising one or more of: Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Enterobacter aerogenes (also classified as Klebsiella aerogenes), Citrobacter freundii, Kluyvera ascorbata, and Yokenella regensburgei.

[0028] In at least one aspect, the one or more phages are classified or typed as or have a classification selected from the following: Myoviridae, preferably (i) T4-like, more preferably subcluster G, I or a new or unknown subcluster type, or (ii) RV5-like, more preferably subcluster C or a new or unknown subcluster type; Brachyviridae, preferably T7-like, more preferably subcluster B or a new or unknown subcluster type; Longicurus, preferably (i) SO1-like, more preferably subcluster A or a new or unknown subcluster type, (ii) 9g-like, more preferably subcluster B or a new or unknown subcluster type, (iii) T1-like, more preferably subcluster B, D or a new or unknown subcluster type; Jello group, preferably subcluster A, B or a new or unknown subcluster type; FaintSaint cluster; and T1-like phages.

[0029] In at least one aspect, the one or more bacteriophage are lytic.

[0030] In at least one aspect, the pharmaceutically acceptable carrier and the one or more bacteriophage form an aqueous solution further comprising a salt, a peptide and / or a yeast extract.

[0031] In at least one aspect, the aqueous solution comprises greater than or equal to 0.5 mg / mL of salt, greater than 0.5 mg / mL of peptide, and greater than 0.25 mg / mL of yeast extract.

[0032] In at least one aspect, the one or more phage are present in an amount greater than or equal to 1 x 10 5 , 1x10 6 , 1x10 7 , 1x10 8 , 1x10 9 or 1x10 10 The concentration of PFU / mL or PFU / mg is present (in the composition).

[0033] In at least one aspect, the composition further comprises one or more additional bacteriophages at a concentration of greater than or equal to 1x10 4 (or 1x10 5 etc.) PFU / mL or PFU / mg, each of the bacteriophages having a respective tropism, the tropism including one or more additional bacteria associated with the mammalian gut microbiome. In at least one aspect, the one or more additional bacteria include one or more of the following: Enterobacter cloacae, Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Enterobacter aerogenes, Kluyvera ascorbata, Citrobacter freundii, or Yokenella regensburgei. In at least one aspect of the composition, the one or more additional bacteria include one or more of Clostridium ramosum or Bilophila wadsworthia.

[0034] In at least one aspect, the one or more bacteriophages include one or more of the following: a first bacteriophage having a tropism including at least Enterobacter cloacae, a second bacteriophage having a tropism including at least Klebsiella pneumoniae, a third bacteriophage having a tropism including at least Escherichia coli, or a fourth bacteriophage having a tropism including at least Serratia marcescens. In at least one aspect of the foregoing composition, the first bacteriophage may include one or more of Optimum-18, Optium-38, Optimum-125, Optimum-126, and / or Optimum-712; the second bacteriophage may include one or more of Optium-12, Optium-79, and / or Optium-817; wherein the third bacteriophage may include one or more of Optium-212, Optium-719, and / or Optium-819; and / or the fourth bacteriophage may include one or more of Optium-148, Optium-155, Optium-162, and / or Optium-169.

[0035] In at least one aspect, the one or more phages include one or more of the following: a fifth phage having tropism including at least Enterobacter aerogenes (also classified as Klebsiella aerogenes), a sixth phage having tropism including at least Kluyvera ascorbata, a seventh phage having tropism including at least Citrobacter freundii, and / or an eighth phage having tropism including at least Yokenella regensburgei.

[0036] The methods of the present disclosure may also include methods for preparing the disclosed compositions. Aspects of the disclosed methods for preparing the compositions may include aspects of the compositions disclosed herein. In at least one aspect, the method for preparing the disclosed compositions includes isolating the one or more phages from an environmental source, characterizing the one or more phages, and combining the one or more phages at greater than or equal to 1x10 4 (or 1x10 5 etc.) PFU / mL or PFU / mg with a pharmaceutically acceptable carrier.

[0037] In at least one aspect, the one or more phages have a genome with greater than or equal to 80% sequence identity to one of SEQ ID NOs 1-19 (preferably one of SEQ ID NOs 1-15). In at least one aspect, the one or more phages have a genome with greater than or equal to 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to one of SEQ ID NOs 1-19 (preferably one of SEQ ID NOs 1-15). Alternatively, the one or more phages may have a genome having a tropism element with greater than or equal to 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a tropism element of one of SEQ ID NOs 1-19 (preferably one of SEQ ID NOs 1-15).

[0038] In at least one aspect of the method, characterizing the one or more bacteriophages can include measuring the lytic activity of the one or more bacteriophages to determine that the one or more bacteriophages are not lysogenic, and determining the tropism of the one or more bacteriophages. In at least one aspect, the tropism includes a narrow host range that includes one or more obesity-causing, inflammatory, and / or other bacteria.

[0039] In at least one aspect of the method, characterizing the one or more bacteriophages can additionally or alternatively include sequencing the genomes of the one or more bacteriophages and excluding from the composition any bacteriophages having genomes that contain toxin or virulence factor genes or integrase genes. One aspect of the foregoing method can include determining the relatedness of the one or more bacteriophages to a second bacteriophage having a related tropism based on the sequenced genomes.

[0040] In at least one aspect of the method, combining the one or more bacteriophages with a pharmaceutically acceptable carrier includes combining a plurality of bacteriophages with a pharmaceutically acceptable carrier, wherein the plurality of bacteriophages includes at least a first group of bacteriophages having a tropism that includes one or more obesity-causing, inflammatory, and / or other bacteria. In at least one aspect of the foregoing method, the first group of bacteriophages includes two bacteriophages having different host cell receptor attachment specificities and / or having a genomic sequence identity of less than or equal to 97%, preferably less than or equal to 90%, or more preferably less than or equal to 80%.

[0041] The kit of the present disclosure can include a composition and a probiotic and / or a prebiotic. In at least one aspect of the kit, the composition contains a pharmaceutically acceptable carrier and greater than or equal to 1x10 4 (or 1x10 5 etc.) PFU / mL or PFU / mg of one or more bacteriophages having a tropism that includes one or more obesity-causing, inflammatory, and / or other bacteria related to the mammalian gut microbiome.

[0042] In at least one aspect of the kit, the probiotic includes a mixture of bacteria that includes species of Bifidobacterium and species of Lactobacillus.

[0043] In at least one aspect of the kit, the probiotics include one or more of the following: Lactobacillus rhamnosus, Lactobacillus reuteri, Bacteroides fragilis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium pseudocatenulatum. In at least one aspect, the probiotics of the kit may additionally or alternatively include one or more of the following: Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus lactis, Bifidobacterium bifidum or Bifidobacterium lactis. In at least one aspect, the probiotics of the kit may additionally or alternatively include one or more of the following: Bifidobacterium infantis, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactococcus cremoris or Enterococcus faecium. In at least one aspect, the probiotics of the kit may additionally or alternatively include one or more of Roseburia hominis, Akkermansia muciniphila or Faecalibacterium prausnitzii. In at least one aspect, the probiotics of the kit may additionally or alternatively include one or more bacteria of the family Lachnospiraceae.

[0044] In at least one aspect, the kit may include any composition disclosed herein and any probiotics disclosed herein.

[0045] The method of the present disclosure may further include administering the composition to a mammalian subject, preferably in a manner that delivers or disposes the composition or one or more of its bacteriophages into the digestive tract or gut microbiome of the subject. In at least one aspect of the method, the composition comprises a pharmaceutically acceptable carrier and greater than or equal to 1x10 4 (or 1x10 5one or more bacteriophages at a titer of plaque forming units per milliliter (PFU / mL) or PFU per milligram (PFU / mg), said bacteriophages preferably having tropisms that include one or more obesity-causing, inflammatory, and / or other bacteria associated with the mammalian gut microbiome. In at least one aspect, the foregoing method may additionally include administering a probiotic to a mammalian subject, preferably in a manner that delivers or configures the probiotic to the subject's digestive tract or gut microbiome. In at least one aspect, the administered probiotic comprises a mixture of bacteria, said bacteria including species of the genus Bifidobacterium and species of the genus Lactobacillus. In at least one aspect, the administered probiotic comprises one or more of the following: Lactobacillus rhamnosus, Lactobacillus reuteri, Bacteroides fragilis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium pseudocatenulatum. In at least one aspect, the administered probiotic may additionally or alternatively comprise one or more of the following: Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus lactis, Bifidobacterium bifidum, or Bifidobacterium lactis. In at least one aspect, the administered probiotic may additionally or alternatively comprise one or more of the following: Bifidobacterium infantis, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactococcus cremoris, or Enterococcus faecium.

[0046] In at least one aspect of the disclosed method, administering the composition to a mammalian subject reduces the concentration of one or more obesity-causing bacteria in the gut microbiome of the mammalian subject. In at least one aspect, administering the composition to a mammalian subject additionally or alternatively promotes or induces weight loss in the mammalian subject. In at least one aspect, administering the composition to a mammalian subject can reduce inflammation, support metabolic health, and / or support a healthy balance / diversity within the digestive tract microbiome of the mammalian subject.

[0047] In at least one aspect of the disclosed method, the composition is co-administered with any of the disclosed probiotics.

[0048] This summary is provided to introduce a selection of concepts in a simplified form that will be further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an indication of the scope of the claimed subject matter.

[0049] Additional features and advantages of the present disclosure will be set forth in the description below, and in part will be obvious from the description, or may be learned by practice of the present disclosure. The features and advantages of the present disclosure may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims. These and other features of the present disclosure will become more fully apparent from the following description and the appended claims, or may be learned by the practice of the present disclosure as hereinafter described. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] To describe the manner in which the above and other advantages and features of the present disclosure can be obtained, a more particular description of the present disclosure briefly described above will be given by reference to specific embodiments of the invention shown in the drawings. It should be understood that these drawings only depict typical embodiments of the present disclosure and should not be considered as limiting its scope. The present disclosure will be described and explained with additional characteristics and details by using the drawings, in which:

[0051] Figure 1 is a diagram showing improved glucose tolerance in mice with a gut microbiota colonized with Enterobacter cloacae strain B29 and treated with phage for 8 weeks compared to control mice with a gut microbiota colonized with Enterobacter cloacae strain B29 but not treated with phage. Throughout the experiment, all mice were on a high-fat diet.

[0052] Figures 2A - 2I Shows the results of a four-week trial in 17 individuals who took a daily single dose of a composition comprising a phage cocktail co-administered with a probiotic. Figure 2AShows a diagram depicting the hsCRP results of a cohort of participants whose hsCRP levels were above the healthy range at the start of the study. Figure 2B Shows a diagram of the hsCRP results of all participants. Figure 2C Shows a diagram of the weight loss of the participants. Figure 2D Shows a diagram of the decrease in the body mass index (BMI) of the participants. Figure 2E Shows a diagram of the increase in high-density lipoprotein (HDL) cholesterol of the participants. Figure 2F Shows a diagram of the fasting blood glucose levels of the participants. Figure 2G and 2H Show diagrams of the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of the participants, respectively. Figure 2I Shows a diagram of the triglyceride levels of the participants.

[0053] Figure 3 Shows a table illustrating the tropism of selected phages of the present disclosure.

[0054] Figure 4A –4I shows a diagram demonstrating the lytic activity and tropism of different phage strains against Enterobacter cloacae strain B29.

[0055] Figure 5 Shows a diagram demonstrating that mice with a gut microbiome colonized with Enterobacter cloacae strain B29 and treated with an Enterobacter cloacae - tropic phage have impaired weight gain compared to untreated control mice with a gut microbiome colonized with Enterobacter cloacae strain B29. Detailed Description

[0056] Before detailing various embodiments of the present disclosure, it should be understood that, unless otherwise indicated, the numbers expressing quantities, ingredients, or other metrics used in the specification and claims are to be understood as being modified by the term "about" as defined herein. Thus, unless there is a contrary indication, the numerical parameters set forth in the specification and the appended claims are approximations that can vary depending upon the desired characteristics sought to be obtained by the subject matter presented herein. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Although the numerical ranges and parameters setting forth the broad scope of the subject matter presented herein are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0057] It should also be understood that any headings and subheadings used herein are for organizational purposes only and are not meant to limit the scope of the specification or claims.

[0058] The PCT application No. PCT / US18 / 22419, filed on March 14, 2018, is incorporated herein by reference in its entirety.

[0059] Overview of the disclosed embodiments

[0060] It has only recently been realized that dysregulation of the gut microbiota promotes obesity. Efforts are currently underway to identify pathogenic bacterial strains within the microbiota of obese individuals. There is evidence that these “obesogenic bacteria” are not only associated with obesity but may also be associated with increased low-grade inflammation throughout the body, which can impair insulin responsiveness and / or contribute to a variety of inflammation-related conditions. In fact, the obesogenic effects of certain bacteria may at least in part stem from the inflammation-inducing activity of these bacteria. Obesogenic bacteria may also increase the host's ability to collect and store energy from food.

[0061] The Gram-negative opportunistic pathogen Enterobacter cloacae strain B29, isolated from the gut of an obese human, is the first human gut bacterium to cause obesity when transplanted into germ-free mice. Studies conducted according to Koch's postulates have shown that Enterobacter cloacae strain B29 causes obesity and chronic inflammation in its host. Another potential obesogenic bacterium, Clostridium ramosum, is increased in the gut microbiota of obese humans and also has an increased prevalence in the gut microbiota of women with type 2 diabetes. When transplanted into germ-free mice, Clostridium ramosum has been shown to induce obesity.

[0062] It is hypothesized that altering the composition or concentration of certain bacteria in the gut microbiome can support weight management, weight loss, and / or contribute to the prevention and / or treatment of metabolic disorders. For example, US Publication 2011 / 0123501 proposes a strategy for modulating, in particular reducing, the number of Proteobacteria in the gut with a composition containing an agent that reduces the number of Proteobacteria in the gut. The disclosure ‘501 focuses primarily on antibiotics for reducing the Proteobacteria population in the gut and food-grade bacteria for further influencing the gut microbiome, in particular probiotics. In addition to probiotics, the disclosure ‘501 also broadly lists prebiotics, yeast, phytochemicals, and bacteriophages as potential components in the composition.

[0063] However, the disclosure '501 does not disclose any actual phages, or even which of the nearly 50 species of Enterobacter listed in the specification are or should be the targets of the phages to reduce their prevalence in the digestive tract and thereby have a certain beneficial effect on their host. In fact, there is no evidence in the disclosure that the group has ever had, tested, classified, identified, formulated, and / or administered a single phage. Instead, with respect to phage-based compositions, the present disclosure is similar to a wish list, admitting that there is still a need in the art for compositions comprising phages that can target and kill obesity-causing bacteria in the digestive tract.

[0064] Moreover, although adding or introducing specific obesity-causing bacteria to the host microbiome may cause or contribute to obesity, the converse is not necessarily true. It is entirely unclear from the disclosure '501 whether simply reducing the concentration of obesity-causing bacteria in the host microbiome is sufficient to cause weight loss, particularly a weight loss comparable to the weight gain caused by the introduction or presence of obesity-causing bacteria in the host digestive tract microbiome. While weight gain has been associated with the introduction of Enterobacter cloacae strain B29, for example, into the digestive tract microbiome of a mammal, to the inventors' knowledge, there is no objective data indicating that targeting the removal of Enterobacter cloacae strain B29 or any other single obesity-causing bacteria from the host microbiome can cause or induce weight loss and / or promote sustained weight loss.

[0065] Studies have also shown that the rapid weight regain after weight loss caused by dieting is attributed to the persistence of obesity-causing bacteria in the host digestive tract microbiota. Thus, it is reasonable to believe that the composition of the digestive tract microbiota has the ability to resist change, and although dieting may result in temporary weight loss, this is likely to occur without restoring the digestive tract microbiota to a balanced "healthy" composition or diversity. In this way, the presence or elevated concentration of obesity-causing bacteria in the host digestive tract microbiome may cause changes in the membership or concentration of other microorganisms, thereby producing an altered microbiota or altered digestive tract environment that is conducive to weight gain or other adverse effects.

[0066] The adverse or negative effects of so-called "bad" bacteria in the gut microbiome are generally due to their production of endotoxins. Without being bound by any particular theory, it is generally known that Gram-negative bacteria possess or produce such endotoxins (e.g., as part of their cell wall). Bad bacteria in the gut microbiome may be Gram-negative, endotoxin-producing bacteria. Many of the so-called "good" bacteria in the gut microbiome are also Gram-negative. In a healthy gut, endotoxin-rich good bacteria are generally harmless; they are mostly symbiotic in nature. However, in the case of so-called "leaky gut" (increased gut permeability where bacteria and toxins can "leak" through the intestinal wall) or other similar pathologies, even good bacteria (or more specifically, the endotoxins of said good bacteria or endotoxins produced by said good bacteria) may cause (additional) inflammation or otherwise exacerbate certain diseases.

[0067] To address these and other problems in the art, the compositions, kits, and methods of the present disclosure target and reduce the amount or concentration of obesity-causing, inflammatory, and / or other bacteria within the gut microbiome. By reducing or eliminating these bacteria, an ecological space is created for the proliferation of beneficial (or at least non-obesity-causing) bacteria, which can improve the balance of the gut microbiota. The inventors of the present disclosure have found that individuals experienced weight loss after administration of a phage cocktail targeting obesity-causing bacteria. By targeting obesity-causing bacteria using the disclosed compositions, individuals may also obtain other unexpected physiological benefits beyond weight loss, such as reduced inflammation, support for metabolic health (e.g., increased high-density lipoprotein (HDL), decreased triglyceride levels, decreased fasting blood glucose levels, decreased alanine aminotransferase (ALT) levels, and decreased aspartate aminotransferase (AST) levels), and / or support for a healthy balance / diversity within the gut microbiome. Accordingly, the compositions of the present invention can be used to and / or are effective in reducing inflammation, supporting metabolic health (e.g., increased high-density lipoprotein (HDL), decreased triglyceride levels, decreased fasting blood glucose levels, decreased alanine aminotransferase (ALT) levels, decreased aspartate aminotransferase (AST) levels), and / or supporting a healthy balance / diversity within the gut microbiome.

[0068] The compositions of the present disclosure can provide a targeted therapy for conditions caused or contributed to, at least in part, by specific bacteria found in the mammalian gastrointestinal microbiome. For example, the compositions disclosed herein include one or more bacteriophages that specifically target and kill obesity-causing, inflammatory, and / or other bacteria without infecting human cells or killing non-target bacteria. Unlike broad-spectrum antibiotics that kill without selectivity, each of the one or more bacteriophages in the disclosed compositions has a narrow tropism that only kills a specific subset of bacterial genera and / or bacterial species. This advantageously allows for the reduction and / or eradication of the targeted subset of bacteria (obesity-causing bacteria), while leaving other non-obesity-causing bacteria in the microbiome unharmed.

[0069] It has been shown that treatment with the bacteriophage compositions of the present invention results in weight loss, improved glucose tolerance, and a reduction in inflammatory markers in human and / or murine subjects. Although not wishing to be bound by any theory, bacteriophages can be (highly) specific to their target (or tropic) bacterial hosts. Thus, the downstream effects of bacteriophage treatment are (presumably) directly and / or indirectly attributable to the reduction of target bacteria in the subject's (gastrointestinal microbiome).

[0070] Treatment of human subjects with the bacteriophage compositions of the present invention has also shown the following beneficial effects: (i) increased energy and / or better physical strength; (ii) more regular and easier bowel movements, softer stools, and / or reduced / relieved constipation; (iii) decreased appetite / cravings for food; (iv) improved muscle tone and / or reduced arm and / or waist circumference measurements; (v) improved skin color / complexion and / or reduced acne; (vi) improved mood and / or better mental awareness; (vii) improved attention and / or mental concentration; (viii) reduced feelings of depression; (ix) reduced, alleviated, and / or eliminated chronic stomach pain; and / or (x) an overall positive effect. Thus, the compositions of the present disclosure can be used to treat a variety of conditions, ranging from obesity (or overweight), stomach pain, constipation, and food cravings to low energy or physical strength, impaired mental concentration, depression / mood disorders, and pain / skin conditions. The foregoing results are also attributable to the reduction of target bacteria in the subject's (gastrointestinal microbiome).

[0071] Also disclosed are various kits, which include (i) the compositions of the present disclosure, the compositions having one or more bacteriophages for reducing or eliminating one or more obesity-causing, inflammatory, and / or other bacteria, and (ii) probiotics and / or prebiotics. Traditionally, probiotic foods and pills have proven to be impossible and / or unable to produce lasting changes because the bacterial species that have been stably established in the gastrointestinal microbiome ecosystem have a strong advantage over newly introduced species. By combining the disclosed bactericidal compositions with probiotics, the kits disclosed herein provide an improved approach that incorporates beneficial probiotic bacteria into the gastrointestinal microbiome while also beneficially preventing or resisting the return of target bacteria to their previous positions and / or concentrations in the gut microbiome.

[0072] Compositions and kits that affect the mammalian gut microbiome

[0073] Embodiments of the present disclosure include compositions that, when administered to a mammalian subject in a manner that delivers or configures the composition or one or more of its bacteriophages to the digestive tract or gut microbiome of the mammalian subject, affect the number or concentration of the microorganisms that make up the gut microbiome of the subject. In some exemplary embodiments, the subject is a mammal, preferably a human. Mammals include any mammal, such as but not limited to cows, pigs, sheep, goats, horses, camels, buffalo, cats, dogs, rats, mice, and humans.

[0074] Each of the disclosed compositions further comprises a pharmaceutically acceptable carrier in addition to one or more bacteriophages, the bacteriophages having a (narrow) tropism for one or more obesity-causing, inflammatory, and / or other bacteria. As used herein, the term "tropism" should be understood to mean the host range, number, and / or type of prokaryotes (preferably bacteria) that a given bacteriophage can successfully infect. Successful infection includes the ability of the phage to bind to and enter the host cell, replicate its genome, and package the replicated genome into a capsid to form virion particles and preferably leave the host cell through a lysis event.

[0075] Some bacteriophages are understood to have a broad microbial tropism and are capable of successfully infecting bacteria from different phylogenetic orders and / or more than 10, preferably more than 8, or more preferably more than 6 different genera and / or species of bacteria within the same phylogenetic order, while other bacteriophages have a narrower tropism and are capable of successfully infecting, preferably, bacteria within the same phylogenetic order, and more preferably, only selected genera and / or species of bacteria. In some cases, a bacteriophage may have a narrow tropism for bacteria derived from a single genus or bacterial species or strain. A bacteriophage with a "narrow tropism" is understood to be capable of successfully infecting less than or equal to 6, preferably less than or equal to 5, or more preferably less than or equal to 4, still more preferably less than or equal to 3, still more preferably less than or equal to 2, and most preferably a single bacterial genus and / or species or strain, particularly within the genera Enterobacter, Escherichia, Serratia, Klebsiella, Citrobacter, Kluyvera, and Yokenella. In some embodiments, the tropism of the bacteriophage is limited to genera within the Enterobacteriaceae family.

[0076] In some embodiments, the composition comprises a single bacteriophage with a narrow tropism. Such a composition can be used to target and reduce the concentration of a single or narrow range of bacterial genera and / or species or strains in the mammalian digestive tract. In at least one aspect, the bacteriophage has a tropism for at least one species of the genus Enterobacter, preferably Enterobacter cloacae, and more preferably Enterobacter cloacae strain B29. The bacteriophage can comprise, for example, any one of Optimum-18, Optimum-38, Optimum-125, Optimum-126, and / or Optium-712.

[0077] In another aspect, the bacteriophage has a tropism for at least one species of the genus Klebsiella, preferably Klebsiella pneumoniae. The bacteriophage can comprise, for example, any one of Optimum-12, Optimum-79, and / or Optium-817.

[0078] In another aspect, the bacteriophage has a tropism for at least one species of the genus Serratia, preferably Serratia marcescens. The bacteriophage can comprise, for example, any one of Optimum-148, Optimum-155, Optimum-162, and / or Optium-169.

[0079] In another aspect, the bacteriophage has a tropism for at least one species of the genus Escherichia, preferably Escherichia coli. The bacteriophage can comprise, for example, any one of Optimum-212, Optimum-719, and / or Optium-819.

[0080] In another aspect, the composition comprises a plurality of bacteriophages. Each of the plurality of bacteriophages may have a narrow tropism and / or the plurality of bacteriophages may have an overall narrow tropism. In at least one aspect, the narrow tropisms may be similar or identical tropisms. For example, each of the plurality of bacteriophages may target the same single or narrow range of bacterial genera and / or species or strains. Illustratively, the plurality of bacteriophages may have tropism for the same genus Enterobacter, preferably Enterobacter cloacae, more preferably Enterobacter cloacae strain B29. The bacteriophages may include, for example, any two or more of Optimum-18, Optimum-38, Optimum-125, Optimum-126, and / Optium-712. Such a composition can be used to target, for example, the same Enterobacter species in the digestive tract of a mammal and reduce its concentration, preferably without showing cross-reactivity with other bacterial genera and / or species or strains in the digestive tract of the mammal. Further, such a composition can be used to target and kill a single or narrow range of bacterial genera and / or species or strains in the digestive tract of a mammal before the bacteria have time to adapt to any one or more of the plurality of bacteriophages and / or develop resistance thereto.

[0081] In another aspect, the plurality of bacteriophages have tropism for the same Klebsiella species (preferably Klebsiella pneumoniae). The bacteriophages may include, for example, any two or more of Optimum-12, Optimum-79, and / Optium-817.

[0082] In another aspect, the plurality of bacteriophages have tropism for the same Serratia species (preferably Serratia marcescens). The bacteriophages may include, for example, any two or more of Optimum-148, Optimum-155, Optimum-162, and / Optium-169.

[0083] In another aspect, the plurality of bacteriophages have tropism for the same Escherichia species (preferably Escherichia coli). The bacteriophages may include, for example, any two or more of Optimum-212, Optimum-719, and / Optium-819.

[0084] On the other hand, the plurality of phages can each have a narrow and distinct tropism. For example, the plurality of phages can each target a different single or narrow range of bacterial genera and / or species or strains. Such a composition can be used to target and reduce the concentration of a plurality of bacterial genera and / or species or strains, for example, in the digestive tract of a mammal, preferably without exhibiting cross-reactivity with other bacterial genera and / or species or strains in the digestive tract of the mammal. Illustratively, the plurality of phages can each have a tropism for a different single or narrow range of bacterial genera and / or species or strains, and have a tropism for at least two bacterial genera and / or species or strains preferably selected from: Enterobacter species, Escherichia species, Serratia species, and / or Klebsiella species, more preferably selected from Enterobacter cloacae (preferably Enterobacter cloacae strain B29), Escherichia coli, Serratia marcescens, and / or Klebsiella pneumoniae.

[0085] Illustratively, the composition can comprise one or more phages selected from each of two or more phage groups, the groups comprising (1) phages having a tropism for the same Enterobacter species, preferably Enterobacter cloacae, more preferably Enterobacter cloacae strain B29, and most preferably the phages are selected from any one or more of Optium-18, Optimum-38, Optimum-125, Optimum-126, and Optium-712, (2) phages having a tropism for the same Klebsiella species, preferably Klebsiella pneumoniae, more preferably the phages are selected from any one or more of Optium-12, Optimum-79, and / or Optium-817, (3) phages having a tropism for the same Serratia species, preferably Serratia marcescens, more preferably the phages are selected from any one or more of Optimum-148, Optimum-155, Optimum-162, and / or Optium-169, (4) phages having a tropism for the same Escherichia species, preferably Escherichia coli, more preferably the phages are selected from any one or more of Optium-212, Optimum-719, and / or Optium-819. In some embodiments, the composition can comprise two or more phages selected from each of two or more of the above phage groups. In some embodiments, the composition can comprise three or more phages selected from each of two or more of the above phage groups. Such a composition can be used to target and kill the target bacterial genera and / or species or strains in each of two or more groups in the digestive tract of a mammal before the bacteria have time to adapt to any one or more of the plurality of phages and / or develop resistance to them.

[0086] Illustratively, the composition can comprise one or more phages selected from each of three or more of the above phage groups. In some embodiments, the composition can comprise two or more phages selected from each of three or more of the above phage groups. In some embodiments, the composition can comprise three or more phages selected from each of three or more of the above phage groups. This composition can be used to target and kill the target bacterial genera and / or species or strains in each of three or more groups in the mammalian digestive tract before the bacteria have had time to adapt to any one or more of the multiple phages and / or develop resistance to them.

[0087] Illustratively, the composition can comprise one or more phages selected from each of four of the above phage groups. In some embodiments, the composition can comprise two or more phages selected from each of four of the above phage groups. In some embodiments, the composition can comprise three or more phages selected from each of four of the above phage groups. This composition can be used to target and kill the target bacterial genera and / or species or strains in each of four of the above groups in the mammalian digestive tract before the bacteria have had time to adapt to any one or more of the multiple phages and / or develop resistance to them.

[0088] In some embodiments, the phages are one or more of Optium-12, Optium-18, Optium-38, Optium-79, Optium-125, Optium-126, Optium-148, Optium-155, Optium-162, Optium-169, Optium-212, Optium-712, Optium-719, Optium-712, and / or Optium-819. In an exemplary embodiment, the composition comprises each of the following phages: Optium-12, Optium-18, Optium-38, Optium-79, Optium-125, Optium-126, Optium-148, Optium-155, Optium-162, Optium-169, Optium-212, Optium-712, Optium-719, Optium-712, and / or Optium-819, and the phages have a generally narrow tropism for Enterobacter cloacae, Escherichia coli, Serratia marcescens, and / or Klebsiella pneumoniae.

[0089] On the other hand, the phage is tropic to at least one of the species of Enterobacter, Escherichia, Serratia, Klebsiella, Citrobacter, Kluyvera and / or Yokenella, preferably tropic to at least one of Enterobacter cloacae, Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Enterobacter aerogenes, Kluyvera ascorbata, Citrobacter freundii and / or Yokenella regensburgei.

[0090] It should be understood that the bacterium Enterobacter aerogenes has recently been reclassified as Klebsiella aerogenes, and for the purposes of the present disclosure, the bacterial species Enterobacter aerogenes is synonymous with Klebsiella aerogenes.

[0091] In some embodiments, the phage is one or more of the following: Optium-12, Optium-17, Optium-18, Optium-24, Optium-27, Optium-34, Optium-35, Optium-38, Optium-46, Optium-67, Optium-79, Optium-82, Optium-86, Optium-88, Optium-113, Optium-116, Optium-117, Optium-118, Optium-121, Optium-125, Optium-126, Optium-148, Optium-155, Optium-162, Optium-169, Optium-212, Optium-219, Optium-225, Optium-304, Optium-356, Optium-417, Optium-502, Optium-531, Optium-574, Optium-688, Optium-712, Optium-716, Optium-719, Optium-747, Optium-812, Optium-817, Optium-819, Optium-835 and / or Optium-971, and the phage has a generally narrow tropism to Enterobacter cloacae, Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Enterobacter aerogenes, Kluyvera ascorbata, Citrobacter freundii and / or Yokenella regensburgei.

[0092] For example, the compositions of the present disclosure can include multiple bacteriophages that generally have a narrow tropism for two or more obesity-causing, inflammatory, and / or other bacteria. The first bacteriophage or the first group of bacteriophages can have a tropism for Enterobacter species, preferably Enterobacter cloacae, more preferably Enterobacter cloacae strain B29. The first bacteriophage can be selected from the group consisting of Optimum-18, Optimum-38, Optimum-125, Optimum-126, and Optium-712. In some embodiments, the first bacteriophage is included in a first group of bacteriophages that includes at least one other bacteriophage selected from the group consisting of Optimum-18, Optimum-38, Optimum-125, Optimum-126, and Optium-712. In some embodiments, the first group of bacteriophages includes each of Optimum-18, Optimum-38, Optium-125, Optimum-126, and Optimum-712.

[0093] Alternatively, the first bacteriophage and / or the first group of bacteriophages can have a tropism for Escherichia species, preferably Escherichia coli. The first bacteriophage can be selected from the group consisting of Optimum-212, Optimum-719, and Optium-819. In some embodiments, the first bacteriophage is included in a first group of bacteriophages that includes at least one other bacteriophage selected from the group consisting of Optimum-212, Optimum-719, and Optium-819. In some embodiments, the first group of bacteriophages includes each of Optium-212, Optimum-719, and Optimum-819.

[0094] Alternatively, the first bacteriophage and / or the first group of bacteriophages can have a tropism for Serratia species, preferably Serratia marcescens. The first bacteriophage can be selected from the group consisting of Optimum-148, Optimum-155, Optimum-162, and Optium-169. In some embodiments, the first bacteriophage is included in a first group of bacteriophages that includes at least one other bacteriophage selected from the group consisting of Optimum-148, Optimum-155, Optimum-162, and Optium-169. In some embodiments, the first group of bacteriophages includes each of Optimum-148, Optimum-155, Optimum-162, and Optium-169.

[0095] Alternatively, the first phage and / or the first group of phages may be tropic to Klebsiella species, preferably Klebsiella pneumoniae. The first phage may be selected from the group consisting of Optimum-12, Optimum-79, and Optium-817. In some embodiments, the first phage is included in the first group of phages, and the first group of phages includes at least one other phage selected from the group consisting of Optimum-12, Optimum-79, and Optium-817. In some embodiments, the first group of phages includes each of Optium-12, Optimum-79, and Optimum-817.

[0096] The first phage and / or the first group of phages may be included in a composition having a second phage and / or a second group of phages, and the second phage and / or the second group of phages are tropic to at least one of Enterobacter species, Escherichia species, Serratia species, and Klebsiella species, preferably tropic to at least one of Enterobacter cloacae, Escherichia coli, Serratia marcescens, and Klebsiella pneumoniae.

[0097] In embodiments where the second phage and / or the second group of phages are tropic to Enterobacter species, preferably Enterobacter cloacae, the second phage may be any one of Optimum-18, Optimum-38, Optimum-125, Optimum-126, and Optimum-712. The second phage may also be included in the second group of phages, and the second group of phages includes at least one other phage selected from Optimum-18, Optimum-38, Optimum-125, Optimum-126, and Optium-712. In some embodiments, the second group of phages includes each of Optimum-18, Optimum-38, Optium-125, Optimum-126, and Optimum-712.

[0098] In embodiments where the second phage and / or the second group of phages are tropic to Escherichia species, preferably Escherichia coli, the second phage may be any one of Optimum-212, Optimum-719, and Optimum-819. The second phage may also be included in the second group of phages, and the second group of phages includes at least one other phage selected from Optimum-212, Optimum-719, and Optium-819. In some embodiments, the second group of phages includes each of Optium-212, Optimum-719, and Optimum-819.

[0099] In embodiments where the second phage and / or the second group of phages are tropic to species of the genus Serratia, preferably Serratia marcescens, the second phage can be any one of Optimum-148, Optimum-155, Optimum-162, and Optimum-169. The second phage can also be included in the second group of phages, and the second group of phages includes at least one other phage selected from Optimum-148, Optimum-155, Optimum-162, and Optimum-169. In some embodiments, the second group of phages includes each of Optimum-148, Optimum-155, Optimum-162, and Optium-169.

[0100] In embodiments where the second phage and / or the second group of phages are tropic to species of the genus Klebsiella, preferably Klebsiella pneumoniae, the second phage can be any one of Optimum-12, Optimum-79, and Optimum-817. The second phage can also be included in the second group of phages, and the second group of phages includes at least one other phage selected from Optimum-12, Optimum-79, and Optium-817. In some embodiments, the second group of phages includes each of Optium-12, Optimum-79, and Optimum-817.

[0101] It should be understood that when selecting the second phage and / or the second group of phages, the composition can additionally include a third and / or fourth phage or a third and / or fourth group of phages. Each or each group of the third and / or fourth phage or the third and / or fourth group of phages can be selected from the remaining phage groups that are tropic to species of the genus Enterobacter, the genus Escherichia, the genus Serratia, or the genus Klebsiella, preferably tropic to Enterobacter cloacae, Escherichia coli, Serratia marcescens, or Klebsiella pneumoniae.

[0102] In addition to the foregoing, the composition can include additional bacteriophage populations that are tropic to additional obesity-causing, inflammatory, and / or other bacteria. For example, the composition can include one or more bacteriophages that are tropic to Enterobacter aerogenes, the bacteriophages including any one (or all) of Optium-27, Optium-35, Optium-82, Optium-688, Optium-747, and Optium-971. Additionally or alternatively, the composition can include one or more bacteriophages that are tropic to Kluyvera species, preferably Kluyvera ascorbata, the bacteriophages including any one (or all) of Optium-67, Optium-121, Optium-502, and Optium-531. Additionally or alternatively, the composition can include one or more bacteriophages that are tropic to Citrobacter species, preferably Citrobacter freundii, the bacteriophages including any one (or all) of Optium-24, Optium-117, Optium-219, Optium-225, Optium-574, and Optium-716. Additionally or alternatively, the composition can include one or more bacteriophages that are tropic to Yokenella species, preferably Yokenella regensburgei, the bacteriophages including any one (or all) of Optium-46, Optium-116, Optium-304, Optium-356, Optium-812, and Optium-835.

[0103] In some embodiments, the composition comprises only one or multiple bacteriophages selected from: Optium-24, Optium-27, Optium-35, Optium-46, Optium-67, Optium-82, Optium-116, Optium-117, Optium-121, Optium-219, Optium-225, Optium-304, Optium-356, Optium-502, Optium-531, Optium-574, Optium-688, Optium-716, Optium-747, Optium-812, Optium-835, and Optium-971.

[0104] The number and type of phages included in the composition can vary depending on the application and the obesogenic, inflammatory, and / or other bacteria to be targeted. In some embodiments, the composition comprises at least two, preferably 3-5, phages that are tropic to the same target bacterium. Having more than one phage specific to a given bacterium can beneficially increase the potency of the composition. Figure 3 Illustrates the tropism of at least some of the phages disclosed herein. In some cases, for example Figure 3 The illustrated examples of tropism assist in the selection of the number and type of phages to include in a composition with a single phage or phage cocktail. For example, the minimum (or lesser number) of phages can be selected based on the overlapping tropism of the phages such that at least two, preferably 3-5, phages are tropic to each of the target bacteria.

[0105] In a preferred embodiment, phages are additionally selected for greater genetic diversity from each other and / or the manner of attachment or infection. Targeting the same bacterium with multiple genetically distinct phages and / or phages with different attachment or infection patterns can beneficially reduce the likelihood of escape mutants within the bacterial population.

[0106] It should be understood that in addition to and / or as a supplement to the foregoing, the phages included in the composition can be selected de novo and can be selected for narrow tropism against any target obesogenic, inflammatory, and / or other bacteria. Such identification and selection can be performed, for example, using the methods disclosed for preparing the disclosed compositions.

[0107] Alternatively or additionally, the phages can be classified or typed as follows, or the phages have a classification (or type) selected from the group consisting of: Myoviridae, preferably (i) T4-like, more preferably subcluster G, I, or a new or unknown subcluster type, or (ii) RV5-like, more preferably subcluster C or a new or unknown subcluster type; Podoviridae, preferably T7-like, more preferably subcluster B or a new or unknown subcluster type; Siphoviridae, preferably (i) SO1-like, more preferably subcluster A or a new or unknown subcluster type, (ii) 9g-like, more preferably subcluster B or a new or unknown subcluster type, (iii) T1-like, more preferably subcluster B, D, or a new or unknown subcluster type; Jellovirus group, preferably subcluster A, B, or a new or unknown subcluster type; FaintSaint cluster; and T1-like phages. Table 1 (below) shows the various classifications of 15 individual phages of the present disclosure. Other phages of the present disclosure can be classified similarly.

[0108] Table 1. Possible classifications of the various phages disclosed

[0109]

[0110]

[0111] Tests have shown that each of the bacteriophages listed in Table 1 above (see Column 2 - Bacteriophage Name), and in addition, Figure 3 each of the bacteriophages listed in [see Column 2 - Bacteriophage Name] has, exhibits, and / or possesses (i) the ability to successfully and specifically target, infect, and kill (e.g., lyse) its corresponding bacterial host target (see Column 1 - Bacterial Target), and in some cases (ii) a narrow tropism for said target, with little or no significant cross-reactivity with the targets listed in Table 1, Figure 3 and / or other host targets shared by the mammalian and / or human digestive tract (microbiome). See Figure 3 . When a first bacteriophage exhibits a narrow tropism for a single host bacterium, the first bacteriophage can be used to highly specifically target and kill the single bacterium, either alone (e.g., administered) or in combination with one or more additional bacteriophages. The one or more additional bacteriophages can have (i) the same or (ii) a different narrow tropism (e.g., specific infectivity for the same single bacterial host or a different single bacterial host). Alternatively, the one or more additional bacteriophages can have a broader tropism than the tropism of the first bacteriophage (e.g., specific infectivity for a range of bacterial hosts, which may or may not include the bacterial host of the first bacteriophage).

[0112] When a first bacteriophage exhibits a broad tropism for multiple host bacterial hosts, the first bacteriophage can be used to broadly target and kill the multiple host bacterial hosts, either alone (e.g., administered) or in combination with one or more additional bacteriophages. The one or more additional bacteriophages can have (i) the same or (ii) a different broad tropism (e.g., specific infectivity for the same multiple bacterial hosts or bacterial hosts other than the multiple bacterial hosts). Alternatively, the one or more additional bacteriophages can have a narrow tropism for a single or narrower range of bacterial hosts. In each case, for example, Figure 3 each of the bacteriophages listed in [see Column 2 - Bacteriophage Name] can be used alone (e.g., as a component of a composition administered to a mammalian (preferably human) subject), or for example, in combination with Figure 3 any one or more of the other bacteriophages listed in [see Column 2 - Bacteriophage Name].

[0113] In some embodiments, targeted therapeutic agents can be developed, prescribed, and / or administered to treat a specific combination of (potentially harmful or adverse) bacteria in the gastrointestinal microbiome of a subject. Alternatively, a composition comprising a standard or general phage cocktail (or mixture) can be developed, prescribed, and / or administered, where the phages can generally target and kill any of a variety of (potentially harmful or adverse) bacteria that may be present in the gastrointestinal microbiome of the subject.

[0114] The tropism of each phage listed in Table 3 was determined using a lysis activity assay as described in Example 4 below, where each phage was combined with a culture of a specific target microorganism. Aliquots of the culture over time were plated to determine the presence or absence (to what extent) of the bacteria over time. A reduced bacterial titer corresponds to a phage that is tropic for and kills the bacteria. For example, as Figure 4A shown in –4I, each of Optimum-125, Optimum-417, Optimum-18, Optimum-118, Optimum-86, Optimum-712, Optimum-113, Optimum-34, and Optium-38 was analyzed using a lysis activity assay, and as Figures 4A - 4I shown, the phage was able to infect and lyse Enterobacter cloacae, particularly strain B29. This demonstrates the lytic ability and tropism of the phage for a given target bacterium. It should be understood that the lysis activity assay is one method of characterizing the lytic activity and / or tropism of an identified phage, but other methods and assays exist and are known in the art and can be used as an alternative or supplement to the plaque-based assay. For example, a plaque assay or a spot assay can be used.

[0115] The phages incorporated into the compositions of the present disclosure preferably do not have genes encoding bacterial toxins that may have a negative impact on humans, such as cholera toxin, botulinum toxin, and diphtheria toxin. Additionally, the phages preferably do not contain any genes encoding bacterial virulence factors or phage integrase genes. This can advantageously prevent the phage from delivering virulence factors to its host bacteria, which can then cause non-pathogens or opportunistic pathogens to become virulent.

[0116] The phage of various embodiments may include mutants or recombinant strains of isolated phages capable of infecting and lysing obesogenic, inflammatory and / or other bacteria. The mutants of various embodiments can be prepared, for example, by exposing the isolated phage to a mutagen (e.g., a chemical mutagen) or electromagnetic radiation. The recombinant phage strains of various embodiments can be prepared by inserting a polynucleotide into the genome of the isolated phage strain, wherein the polynucleotide encodes an antimicrobial protein or a therapeutic protein. Examples of antimicrobial proteins and therapeutic proteins are disclosed in PCT Application Publication Nos. WO 2018 / 174810 and WO2018 / 030323, the disclosures of which are incorporated herein by reference in their entirety.

[0117] As described above, the compositions of the present disclosure include bacteriophages, which, when applied to a mammal (preferably a human), reduce the number or concentration of obesogenic, inflammatory and / or other bacteria in the intestinal microbiome of a mammal. The compositions disclosed herein may be included in a kit that additionally includes probiotics and / or prebiotics. When the bacteria in the probiotics must compete with the natural bacteria in the digestive tract, the use of probiotics alone may not be effective or have as many beneficial effects. However, when combined with the compositions disclosed herein and co-administered, the disclosed probiotics can beneficially improve and / or stabilize the digestive tract microbiome and provide a healthier and generally more diverse microbiome. This may be due at least in part to the bacteriophages in the composition, which remove the niches in the intestine previously maintained by the target bacteria. Then, the probiotic microorganisms can colonize the newly cleared niches throughout the digestive tract and thereby provide a dual beneficial effect: preventing the recolonization of obesogenic, inflammatory and / or other bacteria by occupying the newly cleared niches, and providing beneficial nutrients and / or metabolites to the host.

[0118] In one embodiment, the probiotics include a variety of bacteria selected from Bifidobacterium species and Lactobacillus species. For example, the probiotics of the present disclosure may include any (or all) of Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus lactis, Bifidobacterium bifidum and / or Bifidobacterium lactis.

[0119] As a supplement or replacement of the aforementioned bacterial strains, the probiotics may also include any (or all) of Lactobacillus rhamnosus, Lactobacillus reuteri, Bacteroides fragilis, Bifidobacterium breve, Bifidobacterium longum and / or Bifidobacterium pseudocatechu. In some embodiments, the probiotics may additionally include one or more or all of the following: Bifidobacterium infantis, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactococcus cremoris and / or Enterococcus faecalis.

[0120] As a supplement or alternative to the aforementioned bacterial strains, the probiotic may further include any (or all) of Roseburia hominis, Akkermansia muciniphila, or Faecalibacterium prausnitzii. Additionally or alternatively, the probiotic may include one or more bacteria of the family Lachnospiraceae.

[0121] Formulations and dosages of the composition

[0122] As described above, each of the disclosed compositions further comprises a pharmaceutically acceptable carrier in addition to one or more bacteriophages having a narrow tropism for one or more obesity-causing, inflammatory, and / or other bacteria. As used herein, the term "pharmaceutically acceptable" refers to a biocompatible formulation suitable for one or more routes of administration, in vivo delivery, or contact, in gaseous, liquid, or solid form, or mixtures thereof. The formulation is compatible in that it does not destroy the activity of the active ingredient therein (e.g., bacteriophage or bacteriophage cocktail) or cause adverse side effects that outweigh any prophylactic or therapeutic effects or benefits.

[0123] It should be understood that the disclosed compositions may include one or more (pharmaceutically acceptable) carriers or excipients. The pharmaceutically acceptable carriers and excipients (or their pharmaceutical acceptability) depend in part on the particular composition being administered and the particular method used to administer the composition. Accordingly, there are a variety of suitable pharmaceutical composition formulations (see, e.g., Remington's Pharmaceutical Sciences, which is incorporated herein by reference). Suitable excipients may be or include carrier molecules and may include antioxidants such as ascorbic acid; chelating agents such as EDTA; carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, and stearic acid; liquids such as oils, water, saline, glycerol, and ethanol; wetting or emulsifying agents; pH buffering substances; and the like. Liposomes are also included within the definition of pharmaceutically acceptable excipients. Additional or alternative examples of carriers include silica (silica, silica gel), carbohydrates or carbohydrate polymers (polysaccharides), cyclodextrins, starches, degraded starches (starch hydrolysis products), chemically or physically modified starches, modified celluloses, gum arabic, Indian gum, tragacanth gum, karaya gum, carrageenan, guar gum, locust bean gum, alginates, pectins, inulin, or hydrolysates of xanthan gum or maltodextrin. In various embodiments, the bacteriophage may be dispersed throughout the carrier. In various embodiments, the pharmaceutically acceptable excipient or carrier may be suitable for oral administration.

[0124] The compositions described herein can be formulated in any form suitable for the intended method of administration. When intended for oral use, for example, tablets, troches, lozenges, aqueous or oily suspensions, non-aqueous solutions, dispersible powders or granules (including micronized particles or nanoparticles), emulsions, hard or soft gelatin capsules, syrups or elixirs can be prepared. Oral products can also include semi-solid foods, solid foods, spoonable semi-solid or solid foods, confections, beverages, or dairy products. Dairy products of various embodiments are ice cream, milk, milk powder, yogurt, kefir, or quark. Compositions intended for oral use can be prepared by any method known in the art of manufacturing pharmaceutical compositions, and such compositions can contain one or more agents including sweetening agents, flavoring agents, coloring agents, and preservatives to provide a palatable formulation.

[0125] Pharmaceutically acceptable excipients particularly suitable for use with tablets include, for example, inert diluents such as cellulose, calcium carbonate or sodium carbonate, lactose, calcium phosphate or sodium phosphate; disintegrants such as cross-linked povidone, corn starch or alginic acid; binders such as povidone, starch, gelatin or gum arabic; and lubricants such as magnesium stearate, stearic acid or talc.

[0126] Tablets can be uncoated or can be coated by known techniques including microencapsulation to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, delayed release materials such as glyceryl monostearate or glyceryl distearate used alone or in combination with waxes can be used.

[0127] Preparations for oral use can also be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent such as cellulose, lactose, calcium phosphate or kaolin, or in the form of soft gelatin capsules wherein the active ingredient is mixed with a non-aqueous or oily medium such as glycerol, propylene glycol, polyethylene glycol, peanut oil, liquid paraffin or olive oil.

[0128] In another embodiment, the pharmaceutical composition can be formulated as a suspension comprising the compound of the embodiment mixed with at least one pharmaceutically acceptable excipient suitable for the preparation of a suspension.

[0129] In another embodiment, the pharmaceutical composition is formulated as dispersible powders and granules suitable for the preparation of a suspension by addition of a suitable excipient.

[0130] Excipients suitable for suspensions include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth, gum arabic, dispersing agents or wetting agents such as naturally occurring phospholipids (such as lecithin), condensation products of alkylene oxides and fatty acids (such as polyoxyethylene stearate), condensation products of ethylene oxide and long-chain aliphatic alcohols (such as heptadecaethyleneoxycethanol), condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydrides (such as polyoxyethylene sorbitan monooleate); polysaccharides and polysaccharide-like compounds (such as dextran sulfate); glycosaminoglycan and glycosaminoglycan-like compounds (such as hyaluronic acid); and thickening agents such as carbomer, beeswax, hard paraffin or cetyl alcohol. The suspension may also contain one or more preservatives such as acetic acid, methyl paraben and / or propyl paraben; one or more colorants; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin.

[0131] The pharmaceutical composition may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil (such as olive oil or peanut oil), a mineral oil (such as liquid paraffin) or a mixture thereof. Suitable emulsifying agents include naturally occurring gums such as gum arabic and tragacanth; naturally occurring phospholipids (such as soy lecithin), esters or partial esters derived from fatty acids; hexitol anhydrides such as sorbitan monooleate; and condensation products of these partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening agents and flavoring agents. Syrups and elixirs may be formulated with sweetening agents such as glycerol, sorbitol or sucrose. Such formulations may also contain demulcents, preservatives, flavoring agents or colorants.

[0132] Co-solvents and adjuvants may be added to the formulation. Non-limiting examples of co-solvents include hydroxyl or other polar groups such as alcohols such as isopropyl alcohol; diols such as propylene glycol, polyethylene glycol, polypropylene glycol, ethylene glycol ethers; glycerol; polyvinyl alcohol and polyoxyethylene fatty acid esters. Adjuvants include, for example, surfactants such as soy lecithin and oleic acid; sorbitol esters such as sorbitol trioleate; and polyvinylpyrrolidone.

[0133] It should be understood that the compositions and / or formulations disclosed herein contain a total amount of one or more bacteriophages sufficient (collectively or individually) to achieve the desired effect (such as reducing the number or concentration of obesity-causing, inflammatory and / or other bacteria in the gut microbiome of a subject).

[0134] For convenience, the composition can be prepared or provided in a unit dosage form, and can be packaged in a unit dosage form for ease of administration and uniform dosage. As used herein, "unit dosage form" refers to a physically discrete unit suitable as a unit dosage for a subject to be treated. When administered in one or more doses, each unit containing a predetermined amount of one or more phages is calculated to produce a desired effect (e.g., reducing the number or concentration of obesogenic, inflammatory and / or other bacteria in the subject's intestinal microbiome), and the phages are optionally combined with a pharmaceutically acceptable carrier (e.g., excipient, diluent, vehicle or filler). The unit dosage form may include a daily dose or unit, a daily subdose or an appropriate fraction thereof of the administered compound. The unit dosage form also includes, for example, capsules, lozenges, cachets, lozenges, tablets, ampoules and vials, which may include compositions in a freeze-dried or lyophilized state. For example, a sterile liquid carrier may be added before in vivo administration or delivery. The unit dosage form additionally includes, for example, ampoules and vials containing a liquid composition distributed therein. Each unit dosage form may be included in a multi-dose kit or container.

[0135] The administration (or application) of the composition may be one or more doses. According to one or more aspects or embodiments of the present disclosure, the dose of the composition may include, for example, greater than or equal to 1×10 4 PFU / mL or PFU / mg of a composition of one or more phages. Alternative doses may include greater than or equal to 1x10 5 , 1x10 6 , 1x10 7 , 1x10 8 , 1x10 9 or 1x10 10 PFU / mL or PFU / mg. Suitable dosage sizes or amounts can be 1 μL to 500 mL or more (e.g., liquid or semisolid compositions), 1 μg to 5000 mg or more (e.g., solid or semisolid compositions), or other amounts known in the art.

[0136] The disclosed compositions can be administered at any frequency, such as once, twice, three times, four times, five times or more per hour, per day or per week, or as appropriate, in a single bolus or multiple doses according to the methods. Typical exemplary frequencies are 1-3 times, 2 times or once per day; for example, once a day for 30 days or indefinitely. The time of administration can be determined by the desired physiological properties to be affected, such as reducing or inducing weight loss, reducing inflammation, etc. The skilled person will be aware of factors that may affect the dosage, frequency and time required to provide a sufficient or effective amount to provide a desired effect or benefit. Dosing and application can be adjusted to provide (generally or individually) sufficient levels of one or more phages or to maintain a desired effect.

[0137] In at least one embodiment, the composition can be administered or received as part of a treatment regimen. The treatment regimen can include a first treatment phase (or period). The first treatment period can include the first administration of the composition administered or received according to a first dosage schedule. The first dosage schedule can be, for example, a daily dosage schedule or any other suitable dosage schedule (e.g., twice a day, every other day, once a week, twice a week, etc.). The first administration can include any suitable dose (amount) disclosed herein. In at least one embodiment, the first dose (amount) of the first administration in the first treatment period can be or include a (relatively high) initial treatment dose, e.g., greater than or equal to 1x10 6 、1x10 7 、1x10 8 、1x10 9 、or 1x10 10 PFU / mL or PFU / mg. In some embodiments, the first administration period (or its schedule) can be or last for any suitable amount of time (e.g., greater than or equal to 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, etc.).

[0138] In some embodiments, the treatment regimen can include a second treatment phase (or period). The second treatment period can include the second administration of the composition administered or received according to a second dosage schedule. The second dosage schedule can be, for example, a weekly dosage schedule or any other suitable dosage schedule (e.g., once a day, every other day, twice a week, etc.). The second administration can include any suitable dose (amount) disclosed herein. In at least one embodiment, the second treatment period can include a (lower) maintenance treatment dose, e.g., greater than or equal to 1x10 4 、1x10 5 、1x10 6 、1x10 7 、or 1x10 8 PFU / mL or PFU / mg. Illustratively, the second administration can include a lower phage concentration than the first administration. Alternatively or additionally, the second treatment period can include a lower frequency dosage schedule than the first treatment period. In some embodiments, the second administration period (or its schedule) can be or last for any suitable amount of time (e.g., greater than or equal to 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, etc. or indefinitely).

[0139] In some embodiments, a composition containing one or more phages is co-administered with a probiotic and / or prebiotic.

[0140] As used herein, "co - administration" means administering simultaneously, or administering one substance and then starting to administer the second substance within 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, 1 minute, within the range bounded by any two of the foregoing numbers, and / or within approximately any of the foregoing numbers of time. In some embodiments, co - administration is simultaneous administration.

[0141] Processes and methods for preparing and administering the composition

[0142] Embodiments of the present disclosure further include processes for preparing the compositions disclosed herein and methods for administering the compositions to reduce the number or concentration of obesity - causing, inflammatory, and / or other bacteria in the gut microbiome of a mammal (preferably a human).

[0143] Exemplary processes for the phage component of preparing the compositions disclosed herein include isolating the phage from an environmental source and characterizing the phage. An exemplary detailed process is provided in Example 3 below.

[0144] Generally, "characterizing" a phage includes measuring its lytic activity to ensure that the phage is a lytic phage rather than a lysogenic phage; measuring the host range to ensure that the phage has a narrow tropism including the desired bacterial target; passing it through a mouse to ensure that the phage survives in the mammalian gastrointestinal tract; and sequencing the phage genome to ensure that the phage does not carry any bacterial toxins or virulence factors or integrase genes (which may indicate that the phage may be lysogenic). During the characterization process, any phage that does not meet any one of the criteria is not considered.

[0145] In some embodiments, genomic sequencing of the phages is performed to determine the degree to which each of the various phages is closely related, especially when those phages have a tropism for the same bacteria. This additional (sometimes optional) step can advantageously allow the selection and combination of phages that are not related to each other as much as possible and reduce the likelihood that the targeted bacteria will develop resistance to the selected phages. Thus, after identifying and characterizing the first phage, additional phages specific for each additional putative obesity - causing, inflammatory, and / or other bacteria can be included, each of which is verified to be lytic, have a narrow and specific tropism, be able to survive in the mammalian gastrointestinal tract, and be unlikely to promote the escape mutants of bacteria resistant to the phage cocktail.

[0146] After isolation and characterization, the selected bacteriophages are prepared in any manner for incorporation as a component of a food stuff, feed additive, or liquid additive, and can be in various forms, such as liquid or dry (including as a powder). In other embodiments, the bacteriophages are dried by air drying, natural drying, spray drying, freeze drying, etc. The preparation of bacteriophage strains can also be used to enhance the properties of the composition, including stability. The term "food" should be understood to mean any substance or product in a processed, partially processed, or unprocessed state that is intended or reasonably expected to be ingested by humans. "Food" also includes beverages, chewing gums, and any substance (including water) that is intentionally added to food during manufacture, preparation, or treatment. The term "feed" is understood to cover all forms of animal food. Food can also be used as feed.

[0147] In various embodiments, the bacteriophages are prepared in any manner for incorporation as a component of a nutritional supplement to promote or support weight management and control, support metabolic health, and / or support the healthy balance of the gastrointestinal microbiota, and can be in various forms, such as liquid or dry (including powder). The term "nutritional supplement" should be understood to cover substances or compositions of substances intended to be used as agents having properties that support or promote health but are not used for treating, diagnosing, preventing, or curing any disease.

[0148] As described above, the compositions disclosed herein can be administered alone or in combination with probiotics. The dosage and regimen of administration can be determined based on the desired effect and as known in the art.

[0149] List of abbreviations defining terms

[0150] To assist in understanding the scope and content of the foregoing and following written description and the appended claims, several selected terms are defined directly below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0151] As used herein, the terms "phage" or "phagosome" include any prokaryotic virus that infects and kills bacteria, preferably lytic viruses. Unless otherwise stated, "phage" and "phagosome" are used interchangeably and may include naturally occurring phages and recombinant phages. A "naturally occurring" phage is a phagosome isolated from a natural or artificial environment that has not been genetically engineered. A "recombinant phage" is a phagosome that contains a genome that has been genetically modified by inserting a heterologous nucleic acid sequence into the genome or by removing a nucleic acid sequence from the genome. The genome of a naturally occurring phagosome can be modified by recombinant DNA techniques to introduce a heterologous nucleic acid sequence at a defined site in the genome. Additionally or alternatively, the genome of a naturally occurring phagosome can be modified by recombinant DNA techniques to remove, for example, a nucleic acid sequence encoding a bacterial virulence factor (e.g., a toxin). Further description of phages can be found in U.S. Patent No. 9,617,522, the entire contents of which are incorporated herein by reference.

[0152] The terms "co-administer" and like terms refer to the concurrent, sequential, and / or combined administration of two or more components. For example, two components can be co-administered by administering each component in parallel, simultaneously, or sequentially in separate doses (e.g., different administrations separated by a period of time). The period of time can be short (e.g., substantially immediately after the first administration) or longer (e.g., after 10 - 60 seconds, 1 - 60 minutes, 1 - 24 hours, 1 - 7 days, or any value or range of values therebetween). Concurrent or simultaneous administration can include an overlapping administration schedule for two or more components or the administration of a combined product that is a mixture of two or more components.

[0153] As used herein, the terms "cocktail", "phage cocktail", "phagosome cocktail" or like terms are intended to be understood to include a composition of two or more phages. The composition can have a proportional or non-proportional number or concentration of phages, and the phages included in the cocktail can have overlapping or non-overlapping tropisms. The cocktail can be in dry form or suspended in a pharmaceutically acceptable carrier.

[0154] As used herein, the term "gut" is synonymous with "digestive tract", "gastrointestinal tract" or like terms and is intended to include the modified epithelial cells, mucus and associated environment, and the system of secretory factors that span between the mammalian mouth and anus (including intervening organs such as the stomach, small intestine, and large intestine).

[0155] The term "microbiome" generally can refer to the collective genomes of the microbiota or the microbiota themselves and can be used synonymously with the term microbiota.

[0156] The term "microbiota" generally refers to the population, collection, and / or totality of microorganisms in a defined environment, habitat, or ecological community, and typically includes multiple genera, species, or strains of symbiotic, commensal, beneficial, and / or opportunistic pathogenic microorganisms (such as bacteria, archaea, fungi, protists, and / or viruses), and typically includes their genetic components (genomes). For example, as used herein, the term "microbiota" or "microbiome" generally refers to the microbial population inhabiting the gut of a mammal (i.e., the gut microbiome).

[0157] As used herein, the term "narrow host range" specifically pertains to the tropism of a given phage.

[0158] The terms "obesogenic bacteria", "obesity-causing bacteria", or similar terms are intended to encompass those bacteria whose presence, accumulation, or imbalance in the gut microbiome of a mammalian (preferably human) subject is associated with, promotes, or induces weight gain in the mammalian subject, and / or whose removal or reduction in the gut microbiome of a mammalian (preferably human) subject is associated with, promotes, or induces weight loss in the mammalian subject. For example, bacteria within the gut microbiome of a mammalian subject can be understood as obesogenic bacteria if, when targeted by a specific phage (e.g., by administering a tropic phage or phage mixture), the accompanying reduction in bacterial concentration is associated with, promotes, or induces weight loss in the mammalian subject (e.g., over time).

[0159] The terms "inflammatory bacteria", "inflammation-causing bacteria", or similar terms are intended to encompass those bacteria whose presence, accumulation, or imbalance in the gut microbiome of a mammalian (preferably human) subject is associated with, promotes, or induces inflammation (preferably low-level systemic inflammation) in the mammalian subject, and / or whose removal or reduction in the gut microbiome of a mammalian (preferably human) subject is associated with, promotes, or induces a reduction in such inflammation and / or inflammatory markers in the mammalian subject. For example, bacteria within the gut microbiome of a mammalian subject can be understood as inflammatory bacteria if, when targeted by a specific phage (e.g., by administering a narrow-tropic phage or phage cocktail), the accompanying reduction in bacterial concentration is associated with, promotes, or induces a reduction in (low-level systemic) inflammation and / or inflammatory markers in the mammalian subject (e.g., over time).

[0160] The term "prebiotic" generally refers to a component (e.g., an energy source or food, food ingredient, dietary supplement, etc.) that, when administered to a mammalian subject, stimulates the growth, diversity, or activity of at least a portion of the microorganisms within the microbiome of said subject. In certain cases, a prebiotic can be selected to include a preferred food source for one or more bacteria in the subject's gut microbiome and / or in co-administered probiotics to promote the growth or activity of that microbial population.

[0161] The term "probiotic" generally refers to one or more live microorganisms associated with a neutral or beneficial effect in the mammalian gastrointestinal tract. Exemplary probiotics can include one or more species of the genus Lactobacillus, Bifidobacterium, Saccharomyces cerevisiae, and / or components thereof (e.g., food, food ingredient, dietary supplement, etc.). Generally, probiotics can help maintain or restore a beneficial level, diversity, or activity of the microbiome when administered.

[0162] The term "sequence identity" or "identity" refers to the specific percentage of residues that are the same in two nucleic acid or amino acid sequences when aligned over a specified comparison window to obtain maximum correspondence as measured by a sequence comparison algorithm or visual inspection. When sequences differ by conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making such adjustments are well known to those skilled in the art. Generally, this involves counting conservative substitutions as partial mismatches rather than total mismatches, thereby increasing the percent sequence identity.

[0163] As used herein, the term "subject" is synonymous with the terms "patient", "individual", and similar terms and generally refers to an individual or organism of any mammalian species, (i) preferably including humans, whether or not under the care of a healthcare provider (e.g., a physician, nurse, healthcare assistant, or volunteer), and (ii) non-human mammals, whether or not under the care of a veterinarian or other veterinary professional, assistant, or volunteer, which include, for example but not limited to, dogs, cats, horses, cows, rodents, or other domesticated or wild mammals.

[0164] Sequence listing

[0165] For convenience, Table 2 (below) lists each sequenced phage and its corresponding SEQ ID NO.

[0166] Table 2. List of SEQ ID NOs

[0167]

[0168]

[0169] Examples

[0170] Example 1

[0171] Figure 1 is a graph showing improved oral glucose tolerance in mice with a gut microbiota colonized with Enterobacter cloacae strain B29 and treated with phages compared to control mice with a gut microbiota colonized with Enterobacter cloacae strain B29 but not treated with phages. As Figure 1 shown, the phage cocktail inhibits B29-induced glucose intolerance. Mice were pretreated with oral antibiotics to deplete their native gastrointestinal microbiota prior to colonization with the human pathogen Enterobacter cloacae strain B29.

[0172] In week 1, all mice received a daily dose of B29 by oral gavage and a high-fat diet (60% of total energy from fat, 20% from carbohydrates, and 20% from protein) to induce weight gain. In week 2, the treatment group (dashed line) received 7 daily doses of the phage cocktail by oral gavage. In weeks 3 - 8, the treatment group (black line) received the phages in drinking water. The phage cocktail contained Optium-125, Optium-417, Optium-18, Optium-118, Optium-86, Optium-712, Optium-113, Optium-34, and Optium-38 at greater than or equal to 1x10 9 PFU.

[0173] An oral glucose tolerance test was performed at the end of week 8 by administering a glucose solution by oral gavage and monitoring blood glucose levels at specified time points over the next 2 hours. The blood glucose levels of the phage-treated group were significantly improved (p < 0.05).

[0174] Example 2

[0175] A four-week test / trial was conducted using 17 individuals aged 25 to 59 years (10 males and 7 females) who took a single daily dose of the phage cocktail. The cocktail included greater than or equal to 1x10 8Optium-12, Optium-18, Optium-38, Optium-79, Optium-125, Optium-126, Optium-212, Optium-712, Optium-719, Optium-712, and Optium-819 of PFU, wherein the phages have tropism for a combination of Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae. Before being included in the composition, each phage was isolated at a titer of greater than or equal to 1.0x10 9 of the phage.

[0176] Blood samples were collected from the participants and tested before and after a four-week period. Blood tests showed statistically significant improvements in the reduction of inflammation, weight and body mass index (BMI), and increase in high-density lipoprotein (HDL) cholesterol.

[0177] Specifically, as Figure 2A shown, before the 4-week trial, the mean hsCRP level decreased by 20.8% (p = 0.031) in 7 participants whose hsCRP levels were above the normal healthy range. As Figure 2B shown, the mean hsCRP level decreased by 14.6% in all participants. On average, the weight decreased by 1.4% (p = 0.049; Figure 2C ), the BMI decreased by 1.8% (p = 0.003, Figure 2D ), and the HDL cholesterol increased by 6.5% (p = 0.013, Figure 2E ).

[0178] In addition, as Figures 2F - 2I shown, the fasting blood glucose level decreased by 2.4%, alanine aminotransferase (ALT) decreased by 5.3%, aspartate aminotransferase (AST) decreased by 6.4%, and triglycerides decreased by 6.7% in the experimental group.

[0179] Daily follow-up data recorded by the participants further pointed out the following benefits and effects associated with the administration of the composition. Overall, 76% of the participants considered the use of the phage cocktail to have a positive effect (24% considered it neutral or adverse), 59% of the participants reported higher energy and better physical strength, 53% of the participants reported more regular and easier bowel movements and softer stools (29% of the participants noticed initial constipation in the first week), 41% of the participants had a decreased appetite, most significantly in the afternoon, 29% of the participants had better muscle tone and / or a reduction in the size of the arm circumference and / or waist circumference, 24% of the participants reported a significant improvement in skin color / skin and a reduction in acne, 24% of the participants reported an improvement in mood and better mental awareness, 18% of the participants were more able to concentrate, 12% of the participants reported an improvement in self-reported depression levels (2 out of 2 participants with depression noticed this positive effect), and 12% of the participants experienced relief / elimination of chronic stomach pain.

[0180] Example 3

[0181] Exemplary methods for identifying and characterizing phages included in a composition to reduce the number or concentration of bacteria associated with the gut microbiome of a mammal include at least the following methods.

[0182] Generally, the method may include: (1) collecting environmental samples; (2) establishing an enrichment culture; (3) isolating phages from the enrichment culture; (4) purifying the phages; (5) testing the phage titer to a high concentration; (6) characterizing the phages (e.g., using electron microscopy and genomic DNA isolation and sequencing); (7) performing a restriction enzyme assay; (8) performing a lysis activity assay; and (9) optionally preparing a phage cryopreservation stock.

[0183] More specifically, the method may include collecting environmental samples from soil, sewage, or other environmental sources and establishing an enrichment culture. The enrichment culture may include, for example, 25 mL of LB broth, 1 mL of the target bacteria, and 1 mL of the environmental sample. The inoculated enrichment culture is then incubated (e.g., incubated for 2 days at 35 °C with aeration). After incubation, the liquid can be transferred from the flask to a cylindrical or conical tube for centrifugation (e.g., 8000 RPM, 20 minutes). The supernatant can then be filtered (e.g., using a 0.45 μm filter) into another sterile conical tube and optionally frozen.

[0184] The phages can then be isolated from the enrichment culture. For example, the filtered enrichment culture can be mixed and serially diluted (e.g., by a factor of 10) to a final concentration of 1x10 -7PFU / mL. Then, an overnight culture of the target bacterium can be inoculated into each tube of the dilution series (e.g., using 500 μL of the bacterial culture). The inoculated tubes are then incubated at 35 °C for 40 minutes. After incubation, each sample is mixed with 5 mL of melted top agar and plated (e.g., onto a petri dish). The plated samples are incubated for 24 hours or until plaque formation is visible.

[0185] The phage is purified by picking a single plaque with a sterile needle and transferring it to a sterile medium (e.g., LB broth). The inoculated medium is mixed, incubated, and the phage picked from the resulting plaque is filtered as before the serial dilution. Then, an overnight culture of the target bacterium can be inoculated into each tube of the dilution series (e.g., using 500 μL of the bacterial culture). The inoculated tubes are then incubated at 35 °C for 40 minutes. After incubation, each sample is mixed with 5 mL of melted top agar and plated (e.g., onto a petri dish). The plated samples are incubated for 24 hours or until plaque formation is visible. This phage purification process can be repeated two or more times.

[0186] After the purification step, the phage titer is determined. This allows the phage to be isolated to a high enough concentration for further analysis. The phage titer assay can be performed as follows. For example, 20 mL of LB broth, 0.5 mL of an overnight culture of the target bacterium, and a plaque picked from the previous round of phage purification that has been suspended in 100 μL of LB broth are mixed. The flask is then incubated at 35 °C for 2 days with medium shaking at medium speed. After this incubation period, the sample is centrifuged (e.g., 8000 rpm, 10 - 15 minutes) and the supernatant is filtered (e.g., through a 0.45 μm filter). The filtered supernatant can then be used to produce plaques as described above to determine the concentration of phage in the filtered lysate.

[0187] Preferably, the final concentration of the phage lysate is greater than or equal to 1x10 8 PFU / mL.

[0188] The phage can then be characterized. As is known in the art, this includes isolating phage DNA using methods and phage DNA isolation kits known in the art. The isolated phage DNA can be sequenced using any method or suitable technique known in the art. If characterization is performed using an electron microscope, as is known in the art, 10 μL of the phage lysate is combined with 10 μL of a tungsten heavy metal solution, placed on an electron microscope grid, and observed by electron microscopy.

[0189] Electron microscopy data can be used to determine phage morphology and structural classification. Sequenced phage DNA can be used to confirm the absence of any bacterial toxins and / or virulence factors and / or integrase genes and to compare its relative genetic similarity to other phage DNA with similar tropisms.

[0190] Example 4

[0191] Exemplary methods for determining the lytic activity and tropism of the identified phages include at least the following methods.

[0192] To identify lytic phages, the target bacterium (e.g., Enterobacter cloacae strain B29) is diluted from an overnight culture to a concentration of 1 x 10 6 colony forming units (CFU / mL) and inoculated with phages titrated to a concentration of 1 x 10 8 PFU / mL at a multiplicity of infection of 100. One milliliter samples are taken from the flask, serially diluted, plated on LB-agar plates, and incubated overnight. The number of surviving bacteria is determined the next day by colony counting. An additional 1 mL of each sample is taken from their respective cultures every 2 hours within the first 12 hours after inoculation, and then once more from the cultures at 24 hours after inoculation to detect whether the phage is lytic and whether the bacteria develop resistance within the first 24 hours.

[0193] The tropism or target specificity of each phage is tested by measuring its ability to lyse a range of bacteria, which encompasses closely related bacterial strains to distantly related bacterial strains. For example, Table 3 shows the host range testing of 9 phages initially isolated based on their ability to lyse Enterobacter cloacae strain B29. Each phage was tested against the following: two additional Enterobacter cloacae strains, namely ATCC13047 and ATCC 23855, and more distantly related Shigella boydii (ATCC9207), Klebsiella pneumoniae (ATCC 10031), Salmonella enterica Serovar Typhimurium LT2, Escherichia coli strain 814, Serratia marcescens, and Pseudomonas aeruginosa.

[0194] By taking 100 μL containing 1 x 10 9For the spot test, 100 μL of each phage solution at 1x10

[0195] Table 3. Results of spot test

[0196]

[0197] Table 3 (continued). Results of spot test

[0198]

[0199] PFU / mL was dropped onto a lawn of a specific target bacterium and incubated overnight at 37 °C. The results are shown in Table 3, where a positive indication corresponds to clearing of the bacteria at the position where the phage was dropped or "spotted" on the plate. Such clearing indicates lysis of the target bacterium and thus its tropism. For example, as shown in Table 3, different phages showed tropism for different bacterial strains while maintaining tropism for many (or all) of the tested Enterobacter cloacae strains. 9 PFU / mL phage was mixed with 500 μL of 1x10 9 CFU / mL bacteria and incubated overnight with shaking at 37 °C to further quantify the lytic activity of the phages disclosed in Table 3 against each susceptible bacterial strain. The overnight cultures were serially diluted, plated, and the number of plaques was counted on the incubated plates to assess the ability of each phage to replicate in the host bacterium. Table 4 shows the results of the host range test for the 9 phages in Table 3. As shown in Table 4, different phages showed tropism specific for different strains of Enterobacter cloacae (and in one case Pseudomonas aeruginosa) and were able to replicate and lyse different Enterobacter cloacae strains.

[0200] Table 4. Results of infection efficiency

[0201]

[0202] Table 4 (continued). Results of infection efficiency

[0203]

[0204] Example 5

[0205] To consider the identified phages for inclusion in the disclosed compositions, we demonstrated that each phage could survive in the mammalian gastrointestinal tract. Phage cocktails were administered to mice by oral gavage, and then fecal samples were collected, from which the phage concentration could be measured. Phages shown to be able to survive in the digestive tract were considered for subsequent development of therapeutic cocktails.

[0206] Specifically, for each phage to be tested, male C57 mice (6 weeks old) were obtained and divided into a negative control group (n = 3) and a test group (n = 7). The control group was given 0.2 mL of sterile Luria broth (LB) once by oral gavage. The test group was given 0.2 mL of 1x10 10 PFU / mL phage in LB broth once by oral gavage. The phage was grown in and purified from the host bacteria, and the titer was determined by the standard plaque assay, and then the phage was diluted in LB to the said concentration. Immediately before the oral gavage, fecal samples were collected from all mice. Additionally, fecal samples were collected from all mice every 6 hours until 24 hours after gavage. After collection, each fecal sample was immediately resuspended in phosphate-buffered saline and filtered through a 0.45 μm filter to isolate the phage. The filtered solution was grown with the host bacteria in a standard plaque-forming assay to calculate the phage concentration in the fecal sample, thereby determining the survival of the phage in the digestive tract. Table 5 shows the survival ability of the 9 phages disclosed in Tables 3 and 4 above after passing through the mouse gastrointestinal tract. As shown in Table 1, different phages were able to survive in the digestive tract for a longer time.

[0207] Table 5. Test results of survival ability in the digestive tract

[0208]

[0209] Example 6

[0210] In vivo proof-of-concept tests were conducted to determine whether phage treatment of the gut-associated Enterobacter cloacae strain B29 could alleviate obesity, inflammation, or other related metabolic disorders in an animal model. Germ-free mice were orally inoculated with Enterobacter cloacae strain B29, which was shown to cause inflammation and obesity in the mice. The mice were treated by orally administering phages that were tropic for Enterobacter cloacae strain B29. The phages were tested alone and in combination with two or more phages to determine whether inflammation or obesity was reduced and to guide the formulation of the cocktail, for which at least in part due to the fact that the multiple phages are not closely related to each other, the obesity-causing bacteria are less likely to develop resistance.

[0211] First, the natural mouse gut microbiota was eliminated using broad-spectrum antibiotics, so that Enterobacter cloacae strain B29, which normally does not colonize in mice, could be implanted. Nine phages with narrow tropisms were isolated, the tropisms including Enterobacter cloacae strain B29, which were verified as lytic and shown to be able to survive in the mammalian gastrointestinal (GI) tract. Multiple phages that attack their targets through different surface receptors were used to reduce the likelihood of the target bacteria developing resistance to infection.

[0212] Figure 5This is a diagram showing that mice colonized with Enterobacter cloacae strain B29 and subsequently treated with phages had impaired weight gain compared to control mice with a gut microbiota colonized with Enterobacter cloacae strain B29 but not treated with phages. As Figure 5 shown, the administered phage cocktail inhibited weight gain induced by Enterobacter cloacae strain B29.

[0213] During the first week of the in vivo study, all mice received a daily dose of Enterobacter cloacae strain B29 by oral gavage and were fed a high-fat diet to induce weight gain. In the third week, the treatment group (solid line with triangles) received a daily dose of the phage cocktail by oral gavage for 7 days. The phage treatment was repeated in the fifth week. Body weight was monitored weekly throughout the study, and it was shown that in the fifth and sixth weeks, there were significant differences in body weight between the phage treatment group and the control group (p < 0.05).

[0214] The study included 6 groups of 8 mice each. The study groups included:

[0215] Group 1: Obese control: Mice with a normal gastrointestinal microbiota; developed obesity.

[0216] Group 2: Lean and healthy control: Mice with an antibiotic-depleted gastrointestinal microbiota; did not develop obesity.

[0217] Group 3: Obese control #2: Mice with an antibiotic-depleted gastrointestinal microbiota were inoculated with Enterobacter cloacae strain B29 to reconstitute the gastrointestinal microbiota; developed obesity.

[0218] Group 4: Experimental group #1: Mice were administered the phage cocktail one week before the first inoculation with Enterobacter cloacae strain B29 at the start of the third week during antibiotic depletion; administration of the phage cocktail was associated with preventing the implantation of Enterobacter cloacae strain B29 and preventing the development of obesity.

[0219] Group 5: Experimental group #2: Mice were administered the phage cocktail at the start of the seventh week after the implantation of Enterobacter cloacae strain B29 but before the development of obesity; phage treatment was associated with preventing the development of obesity, reducing the level of Enterobacter cloacae strain B29, and reducing inflammation.

[0220] Group 6: Experimental group #3: Mice were administered the phage cocktail at the start of the seventeenth week after the development of obesity; administration of the phage was associated with a reduction in low-level systemic inflammation, weight loss, and a decrease in the level of Enterobacter cloacae strain B29 in the mouse gastrointestinal microbiome.

[0221] The study aimed to measure the efficacy of phage treatment when administered before exposure to Enterobacter cloacae strain B29, after exposure to Enterobacter cloacae strain B29 but before obesity occurred, and after obesity had occurred due to the colonization of Enterobacter cloacae strain B29 within the murine gastrointestinal microbiota. During weeks 1 - 3, broad-spectrum antibiotics were administered to groups 2 - 6 (see group descriptions above) to deplete their native gastrointestinal microbiota, and then during week 4, their microbiota was reconstituted (except for group 2) by oral gavage twice daily with 1x10 10 CFU / mL of Enterobacter cloacae strain B29. At the start of week 5, all mice were fed a high-fat diet and maintained on this diet throughout the study.

[0222] The methods for antibiotic depletion of the gastrointestinal microbiota and reconstitution with Enterobacter cloacae strain B29 were tested and proven effective. During the study, when the levels of Enterobacter cloacae strain B29 in the control of group 3 decreased, "boost" doses of Enterobacter cloacae strain B29 were administered biweekly to groups 3 - 6. Body weight was measured weekly throughout the study. Phage treatment was initiated at the designated time points and administered by daily oral gavage during the first week of treatment and then by inclusion in the drinking water throughout the study.

[0223] Starting at the end of week 4, fecal samples were collected biweekly and cultured to track the levels of Enterobacter cloacae strain B29. Glucose tolerance and insulin sensitivity (as described below) were measured every 4 weeks to detect type 2 diabetes. At week 12, obesity was demonstrated in the control group of mice. At week 24, conclusions were established regarding the effects of the Enterobacter cloacae strain B29 phage cocktail on body weight and glucose tolerance when administered before exposure to Enterobacter cloacae strain B29, after implantation of Enterobacter cloacae strain B29 but before obesity occurred, or after implantation of Enterobacter cloacae strain B29 and after obesity had occurred.

[0224] Molecular-level analysis of murine tissues for markers of inflammation and insulin resistance

[0225] Each mouse was dissected at the end of the study and various tissues were preserved. Molecular markers of inflammation in the liver, intestine, and adipose tissue, such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin (IL-6), toll-like receptor 4 (TLR-4), and I-κB kinase ε (IKKε), were examined using qRT-PCR. ELISA assays were used to measure the serum levels of proteins that affect blood glucose, appetite (e.g., insulin, leptin, and adiponectin), and inflammation (e.g., serum amyloid A and LPS-binding protein).

[0226] In response to the administration of a phage cocktail to mice with Enterobacter cloacae strain B29, some or all of the molecular markers of inflammation and glucose dysregulation were improved. Other markers of insulin activity in tissues (e.g., AccI, Fas, Fiaf, Srebp1, Pparq) and other markers of intestinal permeability (ZO-1, Occludin, Claudin) were examined at the RNA and protein levels. The underlying mechanisms of body weight change were inferred by quantifying the molecular-level changes that occurred in mice in response to the administration of the phage cocktail.

[0227] Conclusion

[0228] It should be understood that the systems, devices, products, kits, methods, and / or processes according to certain embodiments of the present disclosure may include, incorporate, or otherwise include the features, characteristics (e.g., components, elements, parts, and / or portions) described in other embodiments disclosed and / or described herein. Thus, the various features of certain embodiments may be compatible with, combined with, included in, and / or incorporated into other embodiments of the present disclosure. Accordingly, the disclosure of certain features related to a particular embodiment of the present disclosure should not be construed as limiting the application or inclusion of such features to that particular embodiment. Instead, it should be understood that other embodiments may also include such features, elements, parts, and / or portions without necessarily departing from the scope of the present disclosure.

[0229] Similarly, any step described in any method or process described herein and / or in the claims may be performed in any suitable order and is not necessarily limited to the order described and / or recited, unless otherwise stated (explicitly or implicitly). However, in certain embodiments of the present disclosure, these steps may also be performed in a specific order or any suitable order.

[0230] Without departing from the spirit or essential characteristics of the present disclosure, the present disclosure may be embodied in other specific forms. The described embodiments are to be considered in all respects only as illustrative and not restrictive. Thus, the scope of the present invention is indicated by the appended claims rather than by the foregoing description. Although certain embodiments and details have been included herein and in the appended disclosure for purposes of illustration of embodiments of the present disclosure, it will be apparent to those skilled in the art that various changes may be made to the compositions and kits disclosed herein without departing from the scope of the present disclosure or the invention defined in the appended claims. All changes that come within the meaning and range of the equivalents of the claims are embraced within their scope.

[0231] The sequence listing that accompanies the present disclosure and is subsequently filed forms a part of the present disclosure and is incorporated herein by specific reference.

Claims

1. A composition comprising: a pharmaceutically acceptable carrier; and Each of a plurality of bacteriophages, present at greater than or equal to 1x10 4 PFU, said plurality of bacteriophages comprising: a first phage having a genomic sequence according to one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:6; a second phage having a genomic sequence according to another of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:6; a third phage having a genomic sequence according to one of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:14; a fourth phage having a genomic sequence according to another of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:14; a fifth phage having a genomic sequence according to one of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; a sixth phage having a genomic sequence according to another of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; a seventh phage having a genomic sequence according to one of SEQ ID NO:12 or SEQ ID NO:15; and an eighth phage having a genomic sequence according to another of SEQ ID NO:12 or SEQ ID NO:

15.

2. The composition according to claim 1, wherein one or more of the plurality of bacteriophages are present at greater than or equal to 1x10 8 PFU.

3. The composition according to claim 1 or 2, wherein the plurality of phages comprises different phages, each having a respective genomic sequence according to the following: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12 and SEQ ID NO:

15.

4. A composition comprising a pharmaceutically acceptable carrier; and Each of a plurality of bacteriophages, present at greater than or equal to 1x10 4 PFU, wherein the plurality of bacteriophages comprises: a first phage having a genomic sequence according to one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:6; a second phage having a genomic sequence according to another of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:6; a third phage having a genomic sequence according to one of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:14; A fourth bacteriophage having a genomic sequence according to another one of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:14; and At least one pair of bacteriophages selected from the group consisting of the following (i) and (ii): (i) A fifth bacteriophage having a genomic sequence according to one of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; and A sixth bacteriophage having a genomic sequence according to another one of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; and (ii) A seventh bacteriophage having a genomic sequence according to one of SEQ ID NO:12 or SEQ ID NO:15; and An eighth bacteriophage having a genomic sequence according to another one of SEQ ID NO:12 or SEQ ID NO:

15.

5. The composition according to claim 4, wherein the at least one pair of bacteriophages comprises the fifth bacteriophage and the sixth bacteriophage.

6. The composition according to claim 4, wherein the at least one pair of bacteriophages comprises the seventh bacteriophage and the eighth bacteriophage.

7. The composition according to any one of claims 4 - 6, wherein one or more of the plurality of phages are present at greater than or equal to 1x10 8 PFU.

Images