Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/76—Viruses; Subviral particles; Bacteriophages
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present disclosure generally relates to compositions that include one or more bacteriophages, kits including the same, and methods for preparing such compositions and for using or administering such compositions to impact the number or concentration of bacteria within the intestinal microbiome of a mammalian subject, preferably to promote or induce weight loss, reduce inflammation, support metabolic health, and/or support a healthy balance/diversity within the gut microbiome.
• Obesity is often accompanied by chronic low-level inflammation and is the major contributor to many other chronic illnesses such as type 2 diabetes, insulin resistance, pre-diabetes, heart disease, stroke, fatty liver disease, kidney disease, osteoarthritis, depression, anxiety, certain cancers, and/or other conditions that often result in diminished quality of life and skyrocketing healthcare costs.
• the intestinal microbiome is a complex ecosystem of bacteria, fungi, and viruses existing in different physiological states and structures. Bacteria within the intestinal microbiome, for example, can exist as planktonic cells or as structured communities of monoclonal— or even polyclonal— biofilms. Additionally, some bacterial species may localize to distinct regions within the gut, and the practical implications of the spatial organization of gut-associated microbes is poorly understood. Further complicating matters, the makeup and diversity of gut-associated microbes varies between individuals, and few commonalities or predictive features of a“healthy” intestinal microbiome are known.
• aspects of the microbiome have been shown to correlate with weight (e.g body mass index, BMI) and obesity.
• BMI body mass index
• Cohorts of bacteria associated with the intestinal microbiome have been correlated with“healthy” individuals having an average or ideal BMI, while other bacteria associated with the intestinal microbiome have been correlated with “unhealthy” overweight or obese individuals.
• compositions, methods, and kits that can be addressed, and there is a need for targeted approaches of meaningfully affecting changes to the microbiome to improve the health and well-being of individuals, particularly with respect to weight gain, obesity, and the deleterious consequences associated therewith.
• aspects of the present disclosure solve one or more of the foregoing or other problems in the art with compositions, methods, and kits for affecting changes to the intestinal microbiome.
• aspects (or embodiments) of the present disclosure relate to methods, systems, and compositions for reducing the amount or concentration of one or more bacterium and, in certain aspects, increasing the amount or concentration of one or more additional bacterium, in the intestinal microbiome of a mammalian, preferably human, subject.
• aspects of the present disclosure relate to methods, systems, and compositions for treating one or more conditions resulting, at least in part, from the overpopulation of the one or more bacterium and/or the underpopulation of the one or more additional bacterium in the gut of said subject.
• Such conditions can include, for example, (i) obesity (or over-weight), (ii) one or more obesity- (or over-weight-) associated condition (e.g., (metabolic, chronic, and/or acute) illnesses, disorders, and/or diseases), (iii) inflammation, particularly low-level, systemic inflammation, and/or (iv) one or more inflammation-associated conditions (e.g., (metabolic, chronic, and/or acute) illnesses, disorders, and/or diseases).
• obesity or over-weight
• one or more obesity- (or over-weight-) associated condition e.g., (metabolic, chronic, and/or acute) illnesses, disorders, and/or diseases
• inflammation particularly low-level, systemic inflammation
• inflammation-associated conditions e.g., (metabolic, chronic, and/or acute) illnesses, disorders, and/or diseases.
• Embodiments include one or more bacteriophage, and compositions or kits comprising the same, and processes for preparing and using said compositions or kits.
• compositions comprising an amount of one or more bacteriophage and a pharmaceutically acceptable carrier or excipient.
• the composition, or bacteriophage thereof, of various aspects of the present disclosure can be useful and/or effective for altering or to alter the intestinal microbiome or the composition of the intestinal microbiome of a mammalian subject when the composition is administered to the subject in a manner that delivers or disposes the composition, or the one or more bacteriophage thereof, into the gut or intestinal microbiome of the subject.
• the composition, or bacteriophage thereof can be useful and/or effective for reducing the concentration of one or more bacterium in the intestinal microbiome of the subject, when administered to the subject in a manner that delivers or disposes the composition, or the one or more bacteriophage thereof, into the gut or intestinal microbiome of the subject.
• the one or more bacterium can include at least one species or strain of obesogenic, inflammatory, and/or other bacteria.
• the one or more bacteriophage can have tropism for (or that includes) the one or more (obesogenic, inflammatory, and/or other) bacterium, or species or strain thereof.
• reducing the concentration of the one or more obesogenic, inflammatory, and/or other bacteria in the intestinal microbiome of the subject can be effective to: induce, promote, and/or support weight loss; treat obesity (or over-weight) and/or one or more obesity-associated condition; promote or support weight management; support metabolic health; support a healthy balance and/or diversity within the gut microbiome, treat and/or reduce (low-level, systemic) inflammation, reduce the presence and/or concentration of inflammatory markers; and/or treat one or more condition associated with (low-level systemic) inflammation, in the subject.
• a condition associated with obesity and/or associated with (low- level systemic) inflammation can include one or more of type 2 diabetes, insulin resistance, pre- diabetes, heart disease, stroke, fatty liver disease, kidney disease, osteoarthritis, depression, anxiety, certain cancers, and/or other disorder, illness, disease, and/or condition, including metabolic and/or acute disorders, illnesses, and/or diseases.
• aspects of the present disclosure relate to compositions for use in treating (i) obesity and/or one or more obesity- associated condition in the subject, (ii) inflammation and/or one or more condition associated with (low-level systemic) inflammation in the subject, (iii) over-population of one or more bacterium in the intestinal microbiome of the subject, and (iv) combinations thereof.
• reducing the concentration of the one or more obesogenic, inflammatory, and/or other bacteria in the intestinal microbiome of the subject can be effective to: increase energy and/or improve stamina, improve regularity and ease of bowel movements (with softer stools and/or reduced/less-frequent constipation), reduce appetite/food cravings, improve muscle tone, reduce inches around arms and/or waist, improve complexion and/or reduced acne, improve mood, reduce feeling of depression, improve concentration, mental awareness and/or focus, reduce and/or alleviate chronic stomach pain, and/or improve overall well-being.
• compositions of the present disclosure can be used to treat a variety of conditions, ranging from obesity (or over-weight), stomach pain, constipation, and food cravings, to low-energy or stamina, impaired mental focus, depression/mood disorders, and ache/skin conditions.
• One or more aspects (or embodiments) of the present disclosure include a composition having a pharmaceutically-acceptable carrier and one or more bacteriophage, the one or more bacteriophage being included in the composition in any suitable amount and/or concentration.
• one or more bacteriophage can be included in the composition in an amount and/or concentration effective to reduce the concentration of one or more (target or host) bacterium of the one or more bacteriophage when the composition is administered to a mammalian, preferably human, subject in a manner that delivers or disposes the composition, or one or more bacteriophage thereof, into the gut or intestinal microbiome of the subject.
• the composition can have or comprise (i) greater than or equal to lxlO 4 of the one or more bacteriophage, (ii) greater than or equal to lxlO 4 plaque forming units of the one or more bacteriophage, (iii) greater than or equal to lxlO 4 PFU per milliliter (PFU/mL) of the one or more bacteriophage, and/or (iv) greater than or equal to lxlO 4 PFU per milligram (PFU/mg) of the one or more bacteriophage.
• the“effective” amount can be or comprise any of the foregoing or other specific amounts or concentrations.
• the one or more bacteriophage (of the composition) has a tropism for or that includes the one or more species or strain of bacterium.
• the one or more bacteriophage can have a tropism for or that includes a single species or strain of bacterium, or a plurality of (e.g., closely-related) bacterium.
• the one or more bacteriophage can have a tropism for or that includes a plurality of bacterium that are not closely related.
• the composition includes one, or a single (strain of) bacteriophage.
• the single (strain of) bacteriophage can a tropism for or that includes a single species or strain of bacterium, or a plurality of bacterium.
• the composition includes a plurality of (strains of) bacteriophage.
• the plurality of (strains of) bacteriophage can include a first (strain of) bacteriophage, a second (strain of) bacteriophage, an optional third, fourth, fifth, sixth, and so forth, (strains of) bacteriophage.
• the plurality of (strain of) bacteriophage can each have a tropism for or that includes a single (species or strain of) bacterium, or a plurality of (species or strains of) bacteria.
• the single (species or strain of) bacterium can be the same bacterium or different bacterium.
• the plurality of (species or strains of) bacteria can be the same plurality of bacteria or different bacteria.
• the first (strain of) bacteriophage can have a tropism for or that includes a single (species or strain of) bacterium, or a plurality of (species or strains of) bacteria
• the second (strain of) bacteriophage can have a tropism for or that includes the same or a different single (species or strain of) bacterium, or the same or a different plurality of (species or strains of) bacteria.
• a mixture (or cocktail) of bacteriophages can be selected to include in a composition of the present disclosure.
• the cocktail of bacteriophages can be configured for, adapted for, or otherwise capable of targeting, infecting, and killing (e.g. lysing) includes a single (species or strain of) bacterium, or a plurality of (species or strains of) bacteria. Redundancy or overlap in tropism may be desirable in some aspects of the present disclosure. Variety and diversity in tropism may be desirable in some aspects of the present disclosure.
• compositions according to some aspects of the present disclosure include at least two bacteriophage have tropism for the same bacterium or at least some of the same bacteria, but preferably through different (genetic or tropic) mechanisms of action.
• the composition comprises a pharmaceutically-acceptable carrier and an amount of one or more bacteriophage.
• the amount of the one or more bacteriophage can be (i) an amount effective to reduce the concentration of one or more target bacterium of the one or more bacteriophage when the composition is administered to a mammalian, preferably human, subject in a manner that delivers or disposes the composition, or one or more bacteriophage thereof, into the gut or intestinal microbiome of the subject, (ii) greater than or equal to lxlO 4 of the one or more bacteriophage, (iii) greater than or equal to lxlO 4 plaque forming units of the one or more bacteriophage, (iv) greater than or equal to lxlO 4 PFU per milliliter (PFU/mL) of the one or more bacteriophage, (v) and/or greater than or equal to lxlO 4 PFU per milligram (PFU/mg) of
• the one or more bacteriophage can have a tropism that comprises, or exhibits a specific infectivity to, one or more target bacterium associated with the intestinal microbiome of the subject.
• the one or more target bacterium can be or comprise one or more obesogenic and/or inflammatory bacterium associated with an intestinal microbiome of a mammal.
• the composition comprises a pharmaceutically-acceptable carrier and an amount of two or more bacteriophage.
• the amount of each of the two or more bacteriophage can be (i) an amount effective to reduce the concentration of one or more target bacterium of each of the two or more bacteriophage when the composition is administered to a mammalian, preferably human, subject in a manner that delivers or disposes the composition, or two or more bacteriophage thereof, into the gut or intestinal microbiome of the subject, (ii) greater than or equal to lxlO 4 of each of the two or more bacteriophage, (iii) greater than or equal to lxlO 4 plaque forming units of each of the two or more bacteriophage, (iv) greater than or equal to lxlO 4 PFU per milliliter (PFU/mL) of each of the two or more bacteriophage, (v) and/or greater than or equal to lxlO 4 PFU per mill
• Each of the two or more bacteriophage can have a tropism that comprises, or exhibit a specific infectivity to, one or more target bacterium associated with the intestinal microbiome of the subject.
• the one or more target bacterium can be or comprise one or more obesogenic and/or inflammatory bacterium associated with an intestinal microbiome of a mammal.
• the tropism of each of the two or more bacteriophage can be a similar or same tropism, or a different tropism, than any one or more other bacteriophage of the composition.
• the composition comprises a pharmaceutically-acceptable carrier and greater than or equal to lxlO 6 or lxlO 6 PFU, etc. of (each of) a plurality of bacteriophages, the plurality of bacteriophages comprising two or more of, preferably each of, Optium-l2, Optium- 18, Optium-38, Optium-79, Optium-l25, Optium-l26, Optium-l48, Optium-l55, Optium-l62, Optium-l69, Optium-2l2, Optium-7l2, Optium-7l9, Optium-7l2, and Optium-8l9, corresponding to corresponding to SEQ ID NOs 1-15 of the present disclosure.
• the composition comprises a pharmaceutically-acceptable carrier and greater than or equal to lxlO 8 or lxlO 8 PFU, etc. of each of a plurality of bacteriophages, the plurality of bacteriophages comprising, consisting of, or consisting essentially of Optium-l2, Optium-l8, Optium-38, Optium-79, Optium-l25, Optium-l26, Optium-l48, Optium-l55, Optium-l62, Optium-l69, Optium-2l2, Optium-7l2, Optium-7l9, Optium-7l2, and Optium-8l9, corresponding to corresponding to SEQ ID NOs 1-15 of the present disclosure.
• One or more embodiments of the present disclosure include a composition having a pharmaceutically-acceptable carrier and greater than or equal to lxlO 4 plaque forming units per milliliter (PFU/mL), per milligram (PFU/mg), or other suitable unit of measurement, of one or more bacteriophage that has a tropism that includes one or more obesogenic bacterium associated with an intestinal microbiome of a mammal.
• the tropism of the one or more bacteriophage comprises Enterobacter cloacae , preferably Enterobacter cloacae strain B29.
• the one or more bacteriophage comprises Optium-l8, Optium-38, Optium-l25, Optium-l26, and/or Optium-7l2.
• the one or more bacteriophage comprises Optium-l2, Optium-l8, Optium-38, Optium-79, Optium-l25, Optium-l26, Optium-l48, Optium- 155, Optium-l62, Optium-l69, Optium-2l2, Optium-7l2, Optium-7l9, Optium-7l2 and/or Optium-8l9.
• the one or more bacteriophage comprises Optium-l7, Optium- 34, Optium-86, Optium-88, Optium-l l3, Optium-l l8, Optium-l25, and/or Optium-4l7.
• the one or more bacteriophage of the composition has a tropism that comprises one or more of Escherichia coli , Serratia marcescens, Klebsiella pneumoniae , Enterobacter aerogenes (also classified as Klebsiella aerogenes ), Citrobacter freundii , Kluyvera ascorbata, and Yokenella regensburgei.
• the one or more bacteriophage are classified or typed as, or have a classification selected from the group consisting of: Myoviridae, preferably (i) T44ike, 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) SOl- 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) Tl-like, more preferably subcluster B, D, or a new or unknown subcluster type; Jello cluster, preferably subcluster A, B, or a new or unknown subcluster type; Faint Saint
• the one or more bacteriophage is lytic.
• the pharmaceutically-acceptable carrier and the one or more bacteriophage form an aqueous solution further comprising salt, peptides, and/or yeast extract.
• the aqueous solution comprises greater than or equal to 0.5 mg/mL salts, greater than 0.5 mg/mL peptides, and greater than 0.25 mg/mL yeast extract.
• the one or more bacteriophage is present (in the composition) at a concentration greater than or equal to lxlO 5 , lxlO 6 , lxlO 7 , lxlO 8 , lxlO 9 , or lxlO 10 PFU/mL or PFU/mg.
• the composition further comprises greater than or equal to lxlO 4 (or lxlO 5 , etc.) PFU/mL or PFU/mg of one or more additional bacteriophage having a respective tropism that comprises one or more additional bacterium associated with the intestinal microbiome of a mammal.
• the one or more additional bacterium comprises one or more of: Enterobacter cloacae, Escherichia coli , Serratia marcescens , Klebsiella pneumoniae , Enterobacter aerogenes , Kluyvera ascorbata , Citrobacter freundii , or Yokenella regensburgei.
• the one or more additional bacterium comprises one or more of Clostridium ramosum or Bilophila wadsworthia.
• the one or more bacteriophage comprises one or more of: a first bacteriophage having a tropism that comprises at least Enterobacter cloacae , a second bacteriophage having a tropism that comprises at least Klebsiella pneumoniae , a third bacteriophage having a tropism that comprises at least Escherichia coli , or a fourth bacteriophage having a tropism that comprises at least Serratia marcescens.
• the first bacteriophage can include one or more of Optium-l8, Optium-38, Optium- 125, Optium-l26, and/or Optium-7l2;
• the second bacteriophage can include one or more of Optium-l2, Optium-79, and/or Optium-8l7;
• the third bacteriophage can include one or more of Optium-2l2, Optium-7l9, and/or Optium-8l9;
• the fourth bacteriophage can include one or more of Optium-l48, Optium-l55, Optium-l62, and/or Optium-l69.
• the one or more bacteriophage comprises one or more of a fifth bacteriophage having a tropism that comprises at least Enterobacter aerogenes (also classified as Klebsiella aerogenes ), a sixth bacteriophage having a tropism that comprises at least Kluyvera ascorbata, a seventh bacteriophage having a tropism that comprises at least Citrobacter freundii , and/or an eighth bacteriophage having a tropism that comprises at least Yokenella regensburgei .
• Methods of the present disclosure can also include a process for preparing the disclosed compositions. Aspects of the disclosed methods for preparing the compositions can include aspects of the compositions disclosed herein. In at least one aspect, a method for preparing the disclosed compositions includes isolating the one or more bacteriophage from an environmental source, characterizing the one or more bacteriophage, and combining the one or more bacteriophage with the pharmaceutically acceptable carrier at greater than or equal to lxlO 4 (or lxlO 5 , etc.) PFU/mL or PFU/mg.
• the one or more bacteriophage has a genome with greater than or equal to 80% sequence identity to one of SEQ ID NOs 1-19, preferably one or SEQ ID NOs 1- 15. In at least one aspect, the one or more bacteriophage has 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 or SEQ ID NOs 1-15.
• the one or more bacteriophage can have a genome with a tropic element having greater than or equal to 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a tropic element of one of SEQ ID NOs 1-19, preferably one or SEQ ID NOs 1-15.
• characterizing the one or more bacteriophage can include measuring a lytic activity of the one or more bacteriophage to determine that the one or more bacteriophage is not lysogenic and determining a tropism for the one or more bacteriophage.
• the tropism comprises a narrow host range that includes the one or more obesogenic, inflammatory, and/or other bacterium.
• characterizing the one or more bacteriophage can additionally or alternatively include sequencing the genome of the one or more bacteriophage and excluding from the composition any bacteriophage having a genome that comprises a toxin or virulence factor gene or an integrase gene.
• One aspect of the foregoing methods can include determining, based on the sequenced genome, a relatedness of the one or more bacteriophage to a second bacteriophage having a related tropism.
• combining the one or more bacteriophage with the pharmaceutically acceptable carrier includes combining a plurality of bacteriophages with the pharmaceutically acceptable carrier where the plurality of bacteriophages comprises at least a first set of bacteriophages sharing a tropism that includes the one or more obesogenic, inflammatory, and/or other bacterium.
• the first set of bacteriophages comprises two bacteriophages having a different host cell receptor specificity for attachment and/or that have less than or equal to 97% genomic sequence identity, preferably less than or equal to 90% genomic sequence identity, or more preferably less than or equal to 80% genomic sequence identity.
• Kits of the present disclosure can include a composition and a probiotic and/or prebiotic.
• the composition includes a pharmaceutically-acceptable carrier and greater than or equal to lxlO 4 (or lxlO 5 , etc.) PFU/mL or PFU/mg of one or more bacteriophage having a tropism that comprises one or more obesogenic, inflammatory, and/or other bacterium associated with an intestinal microbiome of a mammal.
• the probiotic comprises a mixture of bacteria comprising Bifidobacterium sp. and Lactobacillus sp.
• the probiotic comprises one or more of: Lactobacillus rhamnosus , Lactobacillus reuteri, Bacteroides fragilis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium pseudocatenulatum.
• the probiotic of the kit can additionally or alternatively include one or more of: Lactobacillus acidophilus , Lactobacillus brevis, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus lactis , Bifidobacterium bifidum, or Bifidobacterium lactis.
• the probiotic of the kit can additionally or alternatively include one or more of: Bifidobacterium infantis, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactococcus cremoris, or Enterococcus faecium.
• the probiotic of the kit can additionally or alternatively include one or more of Roseburia hominis , Akkermansia muciniphila , or Faecalibacterium prausnitzii.
• the probiotic of the kit can additionally or alternatively include one or more bacterium of the Lachnospiraceae family.
• kits can include any of the compositions disclosed herein and any of the probiotics disclosed herein.
• Methods of the present disclosure can also include administering a composition to a mammalian subject, preferably in a manner that delivers or disposes the composition, or the one or more bacteriophage thereof, into the gut or intestinal microbiome of the subject.
• the composition includes a pharmaceutically-acceptable carrier and greater than or equal to lxlO 4 (or lxlO 5 , etc.) plaque forming units per milliliter (PFU/mL) or PFU per milligram (PFU/mg), of one or more bacteriophage, preferably having a tropism that comprises one or more obesogenic, inflammatory, and/or other bacterium associated with an intestinal microbiome of a mammal.
• the foregoing method can additionally include administering a probiotic to the mammalian subject, preferably in a manner that delivers or disposes the probiotic into the gut or intestinal microbiome of the subject.
• the administered probiotic comprises a mixture of bacteria comprising Bifidobacterium sp. and Lactobacillus sp.
• the administered probiotic comprises one or more of: Lactobacillus rhamnosus , Lactobacillus reuteri, Bacteroides fragilis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium pseudocatenulatum.
• the administered probiotic can additionally or alternatively include one or more of: Lactobacillus acidophilus , Lactobacillus brevis, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus lactis , Bifidobacterium bifidum, or Bifidobacterium lactis.
• the administered probiotic can additionally or alternatively include one or more of: Bifidobacterium infantis, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactococcus cremoris, or Enterococcus faecium.
• administering the composition to the mammalian subject reduces a concentration of one or more obesogenic bacterium in the intestinal microbiome of the mammalian subject. In at least one aspect, administering the composition to the mammalian subject additionally or alternatively promotes or induces weight loss in the mammalian subject. In at least one aspect, administering the composition to the mammalian subject reduces inflammation, supports metabolic health, and/or supports a healthy balance/diversity within the gut microbiome of the mammalian subject.
• the composition is co-administered with any one of the disclosed probiotics.
• Figure l is a graphical representation showing improved glucose tolerance in mice with intestinal microbiotas colonized with Enterobacter cloacae strain B29 treated for 8 weeks with bacteriophages as compared to control mice with intestinal microbiotas colonized with Enterobacter cloacae strain B29 but not treated with bacteriophages. All mice were on a high fat diet throughout the experiment.
• Figures 2A-2I illustrate results from a four-week trial of 17 individuals taking a single daily dose of a composition comprising a bacteriophage cocktail co-administered with a probiotic.
• Figure 2A illustrates a graphical representation depicting hsCRP results of a cohort of participants whose hsCRP levels were above the healthy range at the start of the study.
• Figure 2B illustrates a graphical representation of the hsCRP results for all participants.
• Figure 2C illustrates a graphical representation of participants’ reduction in body weight.
• Figure 2D illustrates a graphical representation of the participants’ reduction in body mass index (BMI).
• Figure 2E illustrates a graphical representation of the participants’ increase in high-density lipoprotein (HDL) cholesterol.
• HDL high-density lipoprotein
• Figure 2F illustrates a graphical representation of the participants’ fasting blood glucose levels.
• Figure 2G and 2H illustrate graphical representations of participants’ alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, respectively.
• Figure 21 illustrates a graphical representation of the participants’ triglyceride levels.
• Figure 3 illustrates a table illustrating the tropism of select bacteriophages of the present disclosure.
• Figure 4A-4I illustrate graphical representations showing lytic activity and tropism of different bacteriophage strains for Enterobacter cloacae strain B29.
• Figure 5 illustrates a graphical representation showing arrested weight gain in mice with intestinal microbiomes colonized with Enterobacter cloacae strain B29 that are treated with E. cloacae tropic bacteriophages as compared to untreated control mice with intestinal microbiomes colonized with Enterobacter cloacae strain B29.
• the gram-negative opportunistic pathogen Enterobacter cloacae strain B29 isolated from the gut of an obese human, is the first human gut bacterium shown to cause obesity when transplanted into germ-free mice. Research using Koch’s postulates showed that Enterobacter cloacae strain B29 causes obesity and chronic inflammation in its host.
• Another potentially obesogenic bacterium, Clostridium ramosum is elevated in the gut microbiota of obese humans and also has an increased prevalence within the gut microbiota of women with type-2 diabetes. When transplanted into germ-free mice, C. ramosum has been shown to induce obesity.
• U.S. Publication 2011/0123501 proposes strategies for modulating, in particular reducing, the amount of proteobacteria in the gut with compositions comprising an agent that reduces the amount of proteobacteria in the gut.
• the ’501 publication focuses primarily on antibiotics for reducing the proteobacterial population in the gut and food grade bacteria, in particular probiotics, for further affecting the gut microbiome.
• the ’501 publication also broadly lists prebiotics, yeasts, phytochemicals, and bacterial phages as potential components in the composition.
• The’501 publication does not, however, disclose any actual bacteriophage or even which species of the nearly 50 enterobacteria genera listed in the specification are or should be targeted by said bacteriophages to reduce their prevalence in the gut and thereby afford some beneficial effect to its host. Indeed, there is no evidence in the publication that the group ever possessed, tested, classified, confirmed, formulated, and/or administered a single phage. Rather, the disclosure, with respect to phage-based compositions, is akin to a wish-list, acknowledging that there is a continued need in the art for a composition that comprises phage that can target and kill obesogenic bacteria in the gut.
• compositions, kits, and methods of the present disclosure target and reduce the amount or concentration of obesogenic, inflammatory, and/or other bacteria within the intestinal microbiome. By reducing or eliminating these bacteria, ecological space is created for beneficial (or at least non-obesogenic) bacteria to thrive, which can improve the balance of the gut microbiota.
• the inventors of the present disclosure found that individuals experienced weight loss following administration of bacteriophage cocktails targeted to obesogenic bacteria.
• obesogenic bacteria By targeting obesogenic bacteria using the disclosed compositions, individuals receive other unexpected physiological benefits beyond weight loss, such as reduced inflammation, supported 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 of a healthy balance/diversity within the gut microbiome.
• supported 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
• HDL high-density lipoprotein
• ALT alanine aminotransferase
• AST aspartate aminotransferase
• compositions of the present disclosure can be useful and/or effective to reduce inflammation, support metabolic health (e.g, increased high-density lipoprotein (HDL), decrease triglyceride levels, decrease fasting blood glucose levels, decrease alanine aminotransferase (ALT) levels, decrease aspartate aminotransferase (AST) levels), and/or support a healthy balance/diversity within the gut microbiome.
• metabolic health e.g, increased high-density lipoprotein (HDL), decrease triglyceride levels, decrease fasting blood glucose levels, decrease alanine aminotransferase (ALT) levels, decrease aspartate aminotransferase (AST) levels
• ALT alanine aminotransferase
• AST aspartate aminotransferase
• compositions of the present disclosure can provide a targeted therapy for conditions caused by or resulting, at least in part from, particular bacteria found in the mammalian gut microbiome.
• compositions disclosed herein include one or more bacteriophages that specifically target and kill obesogenic, inflammatory, and/or other bacterium while not infecting human cells or killing non-target bacteria.
• broad-spectrum antibiotics which indiscriminately kill
• each of the one or more bacteriophages within the disclosed compositions has a narrow tropism, killing only a specific subset of bacterial genera and/or species. This beneficially allows the reduction and/or eradication of a targeted subset of bacteria— the obesogenic bacteria— while leaving unharmed other non-obesogenic bacteria within the microbiome.
• Treatment with the inventive phage composition to human subjects has also been shown to have the following beneficial effects: (i) increased energy and/or better stamina; (ii) greater regularity and ease of bowel movements with softer stools and/or reduced/less constipation; (iii) reduced appetite/food cravings; (iv) improved muscle tone and/or reduced inches around arms and/or waist; (v) improved complexion/skin and/or reduced acne; (vi) improved mood and/or better mental awareness; (vii) improved concentration and/or mental focus; (viii) reduced feeling of depression; (ix) reduced, alleviated, and/or elimination of chronic stomach aches; and/or (x) an overall positive impact.
• compositions of the present disclosure can be used to treat a variety of conditions, ranging from obesity (or over-weight), stomach pain, constipation, and food cravings, to low-energy or stamina, impaired mental focus, depression/mood disorders, and ache/skin conditions.
• obesity or over-weight
• stomach pain constipation
• food cravings to low-energy or stamina
• impaired mental focus depression/mood disorders
• ache/skin conditions e.g., ache/skin conditions.
• kits are additionally disclosed that include (i) a composition of the present disclosure, having one or more bacteriophage for reducing or eliminating one or more obesogenic, inflammatory, and/or other bacterium and (ii) a probiotic and/or prebiotic.
• a composition of the present disclosure having one or more bacteriophage for reducing or eliminating one or more obesogenic, inflammatory, and/or other bacterium and (ii) a probiotic and/or prebiotic.
• probiotic foods and pills have proven unlikely and/or ineffective to produce lasting changes because bacterial species that are already stably established in the ecosystem of the gut microbiome have a strong advantage over newly introduced species.
• the kits disclosed herein provide an improved way of incorporating beneficial, probiotic bacteria into the gut microbiome while also beneficially preventing or resisting the targeted bacteria from returning to previously held positions and/or concentrations within the intestinal microbiome.
• Embodiments of the present disclosure include compositions that, when administered to a mammalian subject in a manner that delivers or disposes the composition, or the one or more bacteriophage thereof, into the gut or intestinal microbiome of the subject, affect the number or concentration of microbes constituting the subject’s intestinal microbiome.
• the subject is a mammal, preferably a human.
• a mammal includes any mammal, such as by way of non-limiting example, cattle, pigs, sheep, goats, horses, camels, buffalo, cats, dogs, rats, mice, and humans.
• compositions includes a pharmaceutically-acceptable carrier in addition to one or more bacteriophages having a (narrow) tropism for one or more obesogenic, inflammatory, and/or other bacterium.
• the term“tropism” is understood as the host range, number, and/or type of prokaryotes, preferably bacteria, that a given bacteriophage may successfully infect.
• a successful infection comprises the ability of a phage to bind and enter a host cell, replicate its genome and package the replicated genomes into capsids to form virion particles, and exit the host cell, preferably via a lytic event.
• Some bacteriophages are understood to have broad microbial tropism, being capable of successfully infecting bacteria from different phylogenetic orders and/or greater than 10, preferably greater than 8, or more preferably greater than 6 different genera and/or species of bacteria within the same phylogenetic order, while other bacteriophages have a narrower tropism, being capable of successfully infecting, preferably, bacteria from the same phylogenetic order, more preferably only select genera and/or species of bacteria.
• a bacteriophage may have a narrow tropism for bacteria derived from a single genera or bacterial species or strain.
• a bacteriophage having 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, most preferably a single bacterial genera and/or species or strain, particularly within the Enterobacter, Escherichia, Serratia, Klebsiella, Citrobacter, Kluyvera, and Yokenella genera.
• the tropism of the bacteriophage is limited to genera within the Enterobacteriaceae family.
• the composition includes a single bacteriophage having a narrow tropism. Such compositions can be useful to target and reduce the concentration of a single, or narrow range of, bacterial genera and/or species or strain in the mammalian gut.
• the bacteriophage is tropic for at least one Enterobacter sp., preferably Enterobacter cloacae , more preferably Enterobacter cloacae strain B29.
• the bacteriophage can include, for example, any one of Optium-l8, Optium-38, Optium-l25, Optium-l26, and/or Optium-7l2.
• the bacteriophage is tropic for at least one Klebsiella sp., preferably Klebsiella pneumoniae.
• the bacteriophage can include, for example, any one of Optium-l2, Optium-79, and/or Optium-8l7.
• the bacteriophage is tropic for at least one Serratia sp., preferably Serratia marcescens.
• the bacteriophage can include, for example, any one of Optium-l48, Optium-l55, Optium-l62, and/or Optium-l69.
• the bacteriophage is tropic for at least one Escherichia sp ., preferably Escherichia coli.
• the bacteriophage can include, for example, any one of Optium-2l2, Optium- 719, and/or Optium-8l9.
• the composition includes a plurality of bacteriophages.
• the plurality of bacteriophages can each have a narrow tropism and/or the plurality can have a collectively narrow tropism.
• the narrow tropism can be a similar or the same tropism.
• the plurality of bacteriophages can each target the same single, or narrow range of, bacterial genera and/or species or strain.
• the plurality of bacteriophages can be tropic for the same Enterohacter sp ., preferably Enterohacter cloacae , more preferably Enterohacter cloacae strain B29.
• the bacteriophage can include, for example, any two or more of Optium-l8, Optium-38, Optium-l25, Optium-l26, and Optium-7l2.
• Such compositions can be useful to target and reduce the concentration of, for example, the same Enterohacter sp. in the mammalian gut, preferably while not exhibiting cross reactivity with other bacterial genera and/or species or strain in the mammalian gut.
• Such compositions can be useful to target and kill the single, or narrow range of, bacterial genera and/or species or strain in the mammalian gut before the bacterium has time to adapt and/or develop resistance to any one or more of the plurality of bacteriophages.
• the plurality of bacteriophages can be tropic for the same Klebsiella sp ., preferably Klebsiella pneumoniae.
• the bacteriophages can include, for example, any two or more of Optium-l2, Optium-79, and/or Optium-8l7.
• the plurality of bacteriophages can be tropic for the same Serratia sp ., preferably Serratia marcescens.
• the bacteriophages can include, for example, any two or more of Optium-l48, Optium-l55, Optium-l62, and/or Optium-l69.
• the plurality of bacteriophages can be tropic for the same Escherichia sp ., preferably Escherichia coli.
• the bacteriophages can include, for example, any two or more of Optium-2l2, Optium-7l9, and/or Optium-8l9.
• the plurality of bacteriophages can each have narrow and different tropisms.
• the plurality of bacteriophages can each target a different single, or narrow range of, bacterial genera and/or species or strain.
• Such compositions can be useful to target and reduce the concentration of, for example, a plurality of bacterial genera and/or species or strain in the mammalian gut, preferably while not exhibiting cross reactivity with other bacterial genera and/or species or strain in the mammalian gut.
• the plurality of bacteriophages can each be tropic for a different single, or narrow range of, bacterial genera and/or species or strain and, collectively, be tropic for at least two bacterial genera and/or species or strain, preferably selected from Enterobacter sp., Escherichia sp., Serratia sp., and/or Klebsiella sp., more preferably selected from Enterobacter cloacae, preferably Enterobacter cloacae strain B29, Escherichia coli , Serratia marcescens , and/or Klebsiella pneumoniae.
• the composition can include one or more bacteriophage selected from each of two or more groups of bacteriophage, the groups comprising (1) bacteriophage having tropism for the same Enterobacter sp ., preferably Enterobacter cloacae , more preferably Enterobacter cloacae strain B29 and most preferably selected from any one or more of Optium-l8, Optium-38, Optium-l25, Optium-l26, and Optium-7l2, (2) bacteriophage having tropism for the same Klebsiella sp ., preferably Klebsiella pneumoniae , more preferably selected from any one or more of Optium-l2, Optium-79, and/or Optium-8l7, (3) bacteriophage having tropism for the same Serratia sp ., preferably Serratia marcescens , more preferably selected any one or more of Optium- 148, Optium-l55,
• the composition can include two or more bacteriophages selected from each of two or more of the above groups of bacteriophages. In some embodiments, the composition can include three or more bacteriophage selected from each of two or more the above groups of bacteriophages. Such compositions can be useful to target and kill the target bacterial genera and/or species or strain from each of the two or more groups in the mammalian gut before the bacteria have time to adapt and/or develop resistance to any one or more of the plurality of bacteriophages.
• the composition can include one or more bacteriophage selected from each of three or more the above groups of bacteriophages.
• the composition can include two or more bacteriophage selected from each of three or more the above groups of bacteriophages.
• the composition can include three or more bacteriophage selected from each of three or more the above groups of bacteriophages.
• Such compositions can be useful to target and kill the target bacterial genera and/or species or strain from each of the three or more groups in the mammalian gut before the bacteria has time to adapt and/or develop resistance to any one or more of the plurality of bacteriophages.
• the composition can include one or more bacteriophage selected from each of the four above groups of bacteriophages.
• the composition can include two or more bacteriophage selected from each of the four above groups of bacteriophages.
• the composition can include three or more bacteriophage selected from each of the four above groups of bacteriophages.
• Such compositions can be useful to target and kill the target bacterial genera and/or species or strain from each of the four above groups, in the mammalian gut, before the bacteria has time to adapt and/or develop resistance to any one or more of the plurality of bacteriophages.
• the bacteriophage is one or more of Optium-l2, Optium-l8, Optium-38, Optium-79, Optium-l25, Optium-l26, Optium-l48, Optium-l55, Optium-l62, Optium-l69, Optium-2l2, Optium-7l2, Optium-7l9, Optium-7l2 and/or Optium-8l9.
• a composition includes each of bacteriophages Optium-l2, Optium-l8, Optium-38, Optium-79, Optium-l25, Optium-l26, Optium-l48, Optium-l55, Optium-l62, Optium-l69, Optium-2l2, Optium-7l2, Optium-7l9, Optium-7l2 and/or Optium-8l9 that collectively have a narrow tropism for Enterobacter cloacae , Escherichia coli , Serratia marcescens, and/or Klebsiella pneumoniae.
• the bacteriophage is tropic for at least one of Enterobacter sp ., Escherichia sp ., Serratia sp ., Klebsiella sp ., Citrobacter sp ., Kluyvera sp ., and/or Yokenella sp., preferably at least one of Enterobacter cloacae, Escherichia coli , Serratia marcescens , Klebsiella pneumoniae , Enterobacter aerogenes, Kluyvera ascorbata, Citrobacter freundii , and/or Yokenella regensburgei.
• the bacterium Enterobacter aerogenes has recently been re classified as Klebsiella aerogenes , and for the purposes of this disclosure, the bacterial species Enterobacter aerogenes is synonymous with Klebsiella aerogenes.
• the bacteriophages is one or more of Optium-l2, Optium-l7,
• Optium-l8 Optium-24, Optium-27, Optium-34, Optium-35, Optium-38, Optium-46, Optium-67, Optium-79, Optium-82, Optium-86, Optium-88, Optium-l l3, Optium-l l6, Optium-l l7, Optium- 118, Optium-l2l, Optium-l25, Optium-l26, Optium-l48, Optium-l55, Optium-l62, Optium-
• Optium-8l2 Optium-8l7, Optium-8l9, Optium-835, and/or Optium-97l that collectively have a narrow tropism for Enterobacter cloacae, Escherichia coli , Serratia marcescens , Klebsiella pneumoniae , Enterobacter aerogenes , Kluyvera ascorbata , Citrobacter freundii , and/or Yokenella regensburgei .
• a composition of the present disclosure can include a plurality of bacteriophages that collectively have a narrow tropism for two or more obesogenic, inflammatory, and/or other bacteria.
• a first bacteriophage or set of bacteriophages can have a tropism for Enterobacter sp., preferably Enterobacter cloacae, more preferably Enterobacter cloacae strain B29.
• the first bacteriophage can be selected from the group consisting of Optium-l8, Optium-38, Optium-l25, Optium-l26, and Optium-7l2.
• the first bacteriophage is included within a first set of bacteriophages that includes at least one other bacteriophage selected from the group consisting of Optium-l8, Optium-38, Optium-l25, Optium-l26, and Optium-7l2.
• the first set of bacteriophages includes each of Optium-l8, Optium-38, Optium-l25, Optium-l26, and Optium-7l2.
• the first bacteriophage and/or first set of bacteriophages can have a tropism for Escherichia sp., preferably Escherichia coli.
• the first bacteriophage can be selected from the group consisting of Optium-2l2, Optium-7l9, and Optium-8l9.
• the first bacteriophage is included within a first set of bacteriophages that includes at least one other bacteriophage selected from the group consisting of Optium-2l2, Optium-7l9, and Optium-8l9.
• the first set of bacteriophages includes each of Optium-2l2, Optium-7l9, and Optium-8l9.
• the first bacteriophage and/or first set of bacteriophages can have a tropism for Serratia sp., Serratia marcescens.
• the first bacteriophage can be selected from the group consisting of Optium-l48, Optium-l55, Optium-l62, and Optium-l69.
• the first bacteriophage is included within a first set of bacteriophages that includes at least one other bacteriophage selected from the group consisting of Optium-l48, Optium-l55, Optium-l62, and Optium-l69.
• the first set of bacteriophages includes each of Optium-l48, Optium-l55, Optium-l62, and Optium-l69.
• the first bacteriophage and/or first set of bacteriophages can have a tropism for Klebsiella sp., preferably Klebsiella pneumoniae.
• the first bacteriophage can be selected from the group consisting of Optium-l2, Optium-79, and Optium-8l7.
• the first bacteriophage is included within a first set of bacteriophages that includes at least one other bacteriophage selected from the group consisting of Optium-l2, Optium-79, and Optium-8l7.
• the first set of bacteriophages includes each of Optium-l2, Optium-79, and Optium-8l7.
• the first bacteriophage and/or first set of bacteriophages can be included in a composition having a second bacteriophage and/or second set of bacteriophages having a tropism for at least one of Enterobacter sp., Escherichia sp., Serratia sp., and Klebsiella sp., preferably at least one of Enterobacter cloacae, Escherichia coli, Serratia marcescens, and Klebsiella pneumoniae.
• the second bacteriophage and/or second set of bacteriophages is tropic for Enterobacter sp., preferably Enterobacter cloacae
• the second bacteriophage can be any of Optium-l8, Optium-38, Optium-l25, Optium-l26, and Optium-7l2.
• the second bacteriophage can additionally be included with a second set of bacteriophages that includes at least one other bacteriophage selected from Optium-l8, Optium-38, Optium-l25, Optium-l26, and Optium-7l2.
• the second set of bacteriophages includes each of Optium-l8, Optium-38, Optium-l25, Optium-l26, and Optium-7l2.
• the second bacteriophage and/or second set of bacteriophages is tropic for Escherichia sp., preferably Escherichia coli
• the second bacteriophage can be any of Optium-2l2, Optium-7l9, and Optium-8l9.
• the second bacteriophage can additionally be included with a second set of bacteriophages that includes at least one other bacteriophage selected from Optium-2l2, Optium-7l9, and Optium-8l9.
• the second set of bacteriophages includes each of Optium-2l2, Optium-7l9, and Optium-8l9.
• the second bacteriophage and/or second set of bacteriophages is tropic for Serratia sp., preferably Serratia marcescens
• the second bacteriophage can be any of Optium-l48, Optium-l55, Optium-l62, and Optium-l69.
• the second bacteriophage can additionally be included with a second set of bacteriophages that includes at least one other bacteriophage selected from Optium-l48, Optium-l55, Optium-l62, and Optium-l69.
• the second set of bacteriophages includes each of Optium-l48, Optium-l55, Optium-l62, and Optium-l69.
• the second bacteriophage and/or second set of bacteriophages is tropic for Klebsiella sp., preferably Klebsiella pneumoniae
• the second bacteriophage can be any of Optium-l2, Optium-79, and Optium-8l7.
• the second bacteriophage can additionally be included with a second set of bacteriophages that includes at least one other bacteriophage selected from Optium-l2, Optium-79, and Optium-8l7.
• the second set of bacteriophages includes each of Optium-l2, Optium-79, and Optium-8l7.
• the composition can additionally include a third and/or fourth bacteriophage or sets of bacteriophages.
• Each of the third and fourth bacteriophages or sets of bacteriophages can be selected from the remaining sets of bacteriophages tropic for Enterobacter sp., Escherichia, sp., Serratia sp., or Klebsiella sp., preferably if cloacae, E. coli, S. marcescens, or K. pneumoniae.
• the composition can include additional sets of bacteriophages having a tropism for additional obesogenic, inflammatory, and/or other bacteria.
• the composition can include one or more bacteriophages tropic for Enterobacter aerogenes, including any (or all) of Optium-27, Optium-35, Optium-82, Optium-688 Optium-747, and Optium-97l .
• the composition can include one or more bacteriophages tropic for Kluyvera sp., preferably Kluyvera ascorbata, including any (or all) of Optium-67, Optium-l2l, Optium-502, and Optium-53 l . Additionally, or alternatively, the composition can include one or more bacteriophages tropic for Citrobacter sp., preferably Citrobacter freundii, including any (or all) of Optium-24, Optium-l 17, Optium-2l9, Optium-225 Optium-574, and Optium-7l6.
• the composition can include one or more bacteriophages tropic for Yokenella sp., preferably Yokenella regensburgei, including any (or all) of Optium-46, Optium-l l6, Optium-304, Optium-356, Optium-8l2, and Optium-835.
• the composition includes only one bacteriophage or a plurality of bacteriophages selected from Optium-24, Optium-27, Optium-35, Optium-46, Optium-67, Optium-82, Optium-l l6, Optium-l l7, Optium-l2l, Optium-2l9, Optium-225, Optium-304, Optium-356, Optium-502, Optium-53 l, Optium-574, Optium-688, Optium-7l6, Optium-747, Optium-8l2, Optium-835, and Optium-97l .
• the number and type of phage included within the composition may vary according to the application and obesogenic, inflammatory, and/or other bacteria to be targeted.
• the composition includes at least two, preferably 3-5 bacteriophages having a tropism for the same target bacteria. Having more than one bacteriophage specific for a given bacterium can beneficially increase the effectiveness of the composition.
• the tropism for at least some of the bacteriophages disclosed herein is illustrated in Figure 3. In some instances, an illustration of tropism such as that shown in Figure 3 can be helpful in selecting the number and type of phages to include in a composition having a single bacteriophage or cocktail of bacteriophages. For example, a minimum (or smaller number) of phages can be selected based on their overlapping tropism such that at least two, preferably 3-5 phages, are tropic for each of the target bacteria.
• the bacteriophages are additionally selected for those with greater genetic diversity from each other and/or for the mode of attachment or infection. Targeting the same bacterium with a plurality of genetically diverse phages and/or phages having different modes of attachment or infection can beneficially reduce the likelihood of escape mutants within the bacterial populations.
• the bacteriophages included in the composition can be selected de novo in addition to and/or complementary with the foregoing and can be selected for a narrow tropism against any target obesogenic, inflammatory, and/or other bacteria. Such identification and selection can be undertaken, for example, using the disclosed processes for making the disclosed compositions.
• the bacteriophage can be classified or typed as, or 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) SO 1 -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) Tl4ike, more preferably subcluster B, D, or a new or unknown subcluster type; Jello cluster, preferably subcluster A, B, or a new or unknown subcluster type
• first bacteriophage exhibits a narrow tropism for a single host bacterium
• said first bacteriophage can be useful for highly specific targeting and killing of said single bacterium, whether used (e.g., administered) alone or in combination with one or more additional bacteriophage.
• the one or more additional bacteriophage can have (i) the same or (ii) a different narrow tropism (e.g., a specific infectivity for the same, single bacterial host or a different single bacterial host).
• the one or more additional bacteriophage can have tropism broader than the tropism of said first bacteriophage (e.g., a specific infectivity for a range of bacterial hosts, which may or may not include the bacterial host of the first bacteriophage).
• the first bacteriophage exhibits broad tropism for a plurality of host bacterial hosts
• said first bacteriophage can be useful for broad targeting and killing of said plurality host bacterial hosts, whether used (e.g., administered) alone or in combination with one or more additional bacteriophage.
• the one or more additional bacteriophage can have (i) the same or (ii) a different broad tropism (e.g., a specific infectivity for the same plurality of bacterial hosts or other than the plurality of bacterial hosts).
• the one or more additional bacteriophage can have narrow tropism for a single or narrower range of bacterial hosts.
• each of the bacteriophage listed in Figure 3 can be useful (e.g., as a component of a composition for administration to a mammalian, preferably human, subject) alone or in combination with any one or more of the other bacteriophage listed in Figure 3, for example.
• a targeted therapeutic can be developed, prescribed and/or administered to treat a specific combination of (potentially harmful or detrimental) bacteria in the gut microbiome of the subject.
• a composition that comprises a standard or universal cocktail (or mixture) of phage, which, collectively, target and kill any of a variety of (potentially harmful or detrimental) bacteria that may be present in the gut microbiome of the subject can be developed, prescribed and/or administered.
• the tropism for each bacteriophage listed in Table 3 was determined using a lytic activity assay, as described, for example, in Example 4 below, where the respective bacteriophage was combined with a culture of the specific target microbe. Aliquots of the culture over time were plated to determine the (degree of) presence or absence of the bacterium over time. A reduced bacterial titer corresponded with the bacteriophage being tropic for and killing the bacterium.
• the lytic activity assay is one method for characterizing the lytic activity and/or tropism for identified phages, but other methods and assays exist and are known in the art that can be used in addition to or alternatively from the plaque-based assay.
• a plaque assay or spot assay could be used.
• the bacteriophages incorporated into compositions of the present disclosure preferably do not have genes encoding bacterial toxins that may negatively affect a human, such as, for example, cholerae toxin, botulinum toxin, and diphtheria toxin. Additionally, the bacteriophage is preferably devoid of any gene encoding a bacterial virulence factor or phage integrase genes. This can beneficially prevent the bacteriophage from delivering a virulence factor to its host bacterium, which could then cause a non-pathogen or opportunistic pathogen to become virulent.
• the bacteriophages of various embodiments may include mutant or recombinant strains of an isolated bacteriophage capable of infecting and lysing the obesogenic, inflammatory, and/or other bacterium.
• the mutant of various embodiments can be prepared, for example, by exposing an isolated bacteriophage to a mutagenic agent such as a chemical mutagen or electromagnetic radiation.
• Recombinant bacteriophage strains of various embodiments can be prepared by inserting a polynucleotide into the genome of the isolated bacteriophage strain, where the polynucleotide encodes for an antibacterial protein or therapeutic protein. Examples of antibacterial and therapeutic proteins are disclosed in PCT Application Publication No. WO 2018/174810 and WO 2018/030323, the disclosures of which are incorporated in its entirety by reference herein.
• compositions of the present disclosure include bacteriophages that, when administered to a mammal, preferably a human, decrease the number or concentration of obesogenic, inflammatory, and/or other bacteria within the intestinal microbiome of the mammal.
• the compositions disclosed herein can be included within a kit that additionally includes a probiotic and/or prebiotic. Alone, a probiotic may not be as effective or have as much of a beneficial effect when the bacteria within the probiotic have to compete with native bacteria in the gut. However, when coupled with and co-administered with the compositions disclosed herein, the disclosed probiotics can beneficially improve and/or stabilize the gut microbiome and provide a healthier and often more diverse microbiome.
• the probiotic microbes can then colonize the newly clearly niches throughout the gut and thereby provide a twofold benefit: preventing the recolonization of obesogenic, inflammatory, and/or other bacteria by occupying the newly cleared niches and providing the host with beneficial nutrients and/or metabolites.
• the probiotic includes a plurality of bacteria selected from Bifidobacterium sp. and Lactobacillus sp.
• a probiotic of the present disclosure can include any (or all) of Lactobacillus acidophilus , Lactobacillus brevis, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus lactis , Bifidobacterium bifidum, and/or Bifidobacterium lactis.
• the priobiotic can include any (or all) of Lactobacillus rhamnosus , Lactobacillus reuteri, Bacteroides fragilis, Bifidobacterium breve, Bifidobacterium longum, and/or Bifidobacterium pseudocatenulatum .
• the priobiotic can additionally include one of, a plurality of, or all of: Bifidobacterium infantis, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactococcus cremoris, and/or Enterococcus faecium.
• the priobiotic can include any (or all) of Roseburia hominis , Akkermansia muciniphila , or Faecalibacterium prausnitzii.
• the probiotic can include one or more bacterium of the Lachnospiraceae family.
• each of the disclosed compositions includes a pharmaceutically- acceptable carrier in addition to one or more bacteriophages having a narrow tropism for one or more obesogenic, inflammatory, and/or other bacterium.
• pharmaceutically acceptable means a biologically compatible formulation, gaseous, liquid or solid, or mixture thereof, that is suitable for one or more routes of administration, in vivo delivery, or contact.
• a formulation is compatible in that it does not destroy activity of an active ingredient therein (e.g. , the bacteriophage or bacteriophage cocktail) or induce adverse side effects that outweigh any prophylactic or therapeutic effect or benefit.
• compositions may contain one or more (pharmaceutically-acceptable) carriers or excipients.
• Pharmaceutically acceptable carriers and excipients are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there exists a wide variety of suitable formulations of pharmaceutical compositions (see, e.g. , Remington's Pharmaceutical Sciences, incorporated herein by reference).
• Suitable excipients may be or include carrier molecules and can 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.
• carriers include silicon dioxide (silica, silica gel), carbohydrates or carbohydrate polymers (polysaccharides), cyclodextrins, starches, degraded starches (starch hydrolysates), chemically or physically modified starches, modified celluloses, gum arabic, ghatti gum, tragacanth, karaya, carrageenan, guar gum, locust bean gum, alginates, pectin, inulin or xanthan gum, or hydrolysates of maltodextrins.
• the bacteriophage can be dispersed throughout the carrier.
• the pharmaceutically acceptable excipient or carrier can be suitable for oral consumption.
• compositions described herein may be formulated in any form suitable for the intended method of administration.
• tablets, troches, lozenges, aqueous or oil suspensions, non-aqueous solutions, dispersible powders or granules (including micronized particles or nanoparticles), emulsions, hard or soft capsules, syrups, or elixirs may be prepared.
• Orally consumable products can also include a semi-solid food, solid food, a semi-solid or solid spoonable food, confectionary, drink, or dairy product.
• the dairy product of various embodiments is ice cream, milk, milk powder, yogurt, kefir, or quark.
• Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents, and preserving agents to provide a palatable preparation.
• compositions particularly suitable for use in conjunction with tablets include, for example, inert diluents, such as celluloses, calcium or sodium carbonate, lactose, calcium or sodium phosphate; disintegrating agents, such as cross-linked povidone, maize starch, or alginic acid; binding agents, such as povidone, starch, gelatin, or acacia; and lubricating agents, such as magnesium stearate, stearic acid, or talc.
• inert diluents such as celluloses, calcium or sodium carbonate, lactose, calcium or sodium phosphate
• disintegrating agents such as cross-linked povidone, maize starch, or alginic acid
• binding agents such as povidone, starch, gelatin, or acacia
• lubricating agents such as magnesium stearate, stearic acid, or talc.
• Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
• a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
• Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example celluloses, lactose, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with non-aqueous or oil medium, such as glycerin, propylene glycol, polyethylene glycol, peanut oil, liquid paraffin or olive oil.
• an inert solid diluent for example celluloses, lactose, calcium phosphate or kaolin
• non-aqueous or oil medium such as glycerin, propylene glycol, polyethylene glycol, peanut oil, liquid paraffin or olive oil.
• compositions may be formulated as suspensions comprising a compound of the embodiments in admixture with at least one pharmaceutically acceptable excipient suitable for the manufacture of a suspension.
• compositions may be formulated as dispersible powders and granules suitable for preparation of a suspension by the addition of suitable excipients.
• Excipients suitable for use in connection with suspensions include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, dispersing, or wetting agents such as a naturally occurring phosphatide (e.g ., lecithin), a condensation product of an alkyl ene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g, heptadecaethyleneoxycethanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g, polyoxyethylene sorbitan monooleate); polysaccharides and polysaccharide-like compounds (e.g, dextran sulfate); glycoaminoglycans and glycos and glyco
• the suspensions may also contain one or more preservatives such as acetic acid, methyl and/or n-propyl p-hydroxy- benzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin.
• preservatives such as acetic acid, methyl and/or n-propyl p-hydroxy- benzoate
• coloring agents such as acetic acid, methyl and/or n-propyl p-hydroxy- benzoate
• flavoring agents such as sucrose or saccharin.
• sweetening agents such as sucrose or saccharin.
• the pharmaceutical compositions may also be in the form of oil-in water emulsions.
• the oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these.
• Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth; naturally occurring phosphatides, such as soybean 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 and flavoring agents.
• Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol, or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring, or a coloring agent.
• sweetening agents such as glycerol, sorbitol, or sucrose.
• Such formulations may also contain a demulcent, a preservative, a flavoring, or a coloring agent.
• Cosolvents and adjuvants may be added to the formulation.
• cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
• Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.
• compositions and/or formulations disclosed herein contain a total amount of one or more bacteriophages (collectively or individually) sufficient to achieve the intended effect (e.g, reduce the number or concentration of obesogenic, inflammatory, and/or other bacteria in the intestinal microbiome of the subject).
• compositions may, for convenience, be prepared or provided as a unit dosage form and can be packaged in unit dosage forms for ease of administration and uniformity of dosage.
• a “unit dosage form” as used herein refers to a physically discrete unit suited as unitary dosages for the subject to be treated.
• Each unit containing a predetermined quantity of the one or more bacteriophage optionally in association with a pharmaceutically-acceptable carrier (e.g, excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce the desired effect (e.g ., reduce the number or concentration of obesogenic, inflammatory, and/or other bacteria in the intestinal microbiome of the subject).
• a pharmaceutically-acceptable carrier e.g, excipient, diluent, vehicle or filling agent
• Unit dosage forms can contain a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of an administered compound.
• Unit dosage forms also include, for example, capsules, troches, cachets, lozenges, tablets, ampules and vials, which may include a composition in a freeze-dried or lyophilized state.
• a sterile liquid carrier for example, can be added prior to administration or delivery in vivo.
• Unit dosage forms additionally include, for example, ampules and vials with liquid compositions disposed therein.
• the individual unit dosage forms can be included in multi-dose kits or containers.
• a dosage (or administration) of the composition can one or more doses.
• a dose of the composition can include, for example, greater than or equal to lxlO 4 PFU/mL or PFU/mg of composition of one or more bacteriophage, in accordance with one or more aspects or embodiments of the present disclosure.
• Alternative doses can include greater than or equal to lxlO 5 , lxlO 6 , lxlO 7 , lxlO 8 , lxlO 9 , or lxlO 10 PFU/mL or PFU/mg.
• Suitable dosage size or amounts can range from 1 pL to 500 mL or more (e.g., of fluid or semi-solid composition), 1 pg to 5000 mg or more (e.g., of solid or semi-solid composition), or other amount as known in the art.
• compositions can be administered in accordance with the methods at any frequency as a single bolus or multiple dose e.g., one, two, three, four, five, or more times hourly, daily, or weekly or for as long as appropriate.
• Exemplary frequencies are typically from 1-3 times, 2 -times or once, daily; for example, once per day for 30 days or indefinitely.
• Timing of administration can be dictated by the desired physiological characteristic to be affected, such as decreasing or inducing weight loss, decreasing inflammation, etc. The skilled artisan will appreciate the factors that may influence the dosage, frequency, and timing required to provide an amount sufficient or effective for providing the desired effect or benefit. Dosage and administration can be adjusted to provide sufficient levels of the one or more bacteriophage (collectively or individually) or to maintain the desired effect.
• the composition can be administered or received as part of a treatment protocol.
• the treatment protocol can include a first treatment period (or phase).
• the first treatment phase can include a first dosage of the composition, administered or received in accordance with 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 daily, every other day, once weekly, twice weekly, etc.).
• the first dosage can include any suitable dose (amount) disclosed herein.
• the first dose (amount) of the first dosage in the first treatment phase can be or include a (relatively high) initial treatment dose (e.g., greater than or equal to lxlO 6 , lxlO 7 , lxlO 8 , lxlO 9 , or lxl O 10 PFU/mL or PFU/mg.
• the first dosage phase (or schedule thereof) 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.).
• the treatment protocol can include a second treatment period (or phase).
• the second treatment phase can include a second dosage of the composition, administered or received in accordance with a second dosage schedule.
• the second dosage schedule can be, for example, a weekly dosage schedule or any other suitable dosage schedule (e.g., daily, every other day, twice weekly, etc.).
• the second dosage can include any suitable dose (amount) disclosed herein.
• the second treatment phase can include a (lower) maintenance treatment dose (e.g., greater than or equal to lxlO 4 , lxlO 5 , lxlO 6 , lxlO 7 , or lxlO 8 PFU/mL or PFU/mg.
• the second dosage can include a lower concentration of phage that the first dosage.
• the second treatment phase can include a less frequent dosage schedule than the first treatment phase.
• the second dosage phase (or schedule thereof) 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).
• composition containing one or more bacteriophages is co- administered with a probiotic and/or prebiotic.
• co-administration means concurrently or administering one substance followed by beginning the administration of a 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, a range bounded by any two of the aforementioned numbers, and/or about any of the aforementioned numbers. In some embodiments, co-administration is concurrent administration.
• Embodiments of the present disclosure additionally include processes for making the compositions disclosed herein and methods for administering said compositions to decrease the number or concentration of obesogenic, inflammatory, and/or other bacteria in the intestinal microbiome of a mammal, preferably a human.
• An exemplary process for preparing the bacteriophage component of compositions disclosed herein includes isolating the bacteriophage from an environmental source and characterizing the bacteriophage.
• An exemplary, detailed process is provided in Example 3 below.
• “characterizing” the bacteriophage consists of measuring its lytic activity to ensure that the bacteriophage is a lytic phage and not a lysogenic phage, measuring the host range to ensure the phage has a narrow tropism that includes a desired bacterial target, passing it through mice to ensure that the bacteriophage survives conditions of the mammalian gastrointestinal tract, and sequencing the bacteriophage genome to ensure that the phage is not harboring any bacterial toxin or virulence factor or integrase gene, which may indicate that the phage can be lysogenic.
• any bacteriophage that fails any one criterion is excluded from further consideration.
• genomic sequencing of the bacteriophage is performed to determine how closely related each of the various phages are, particularly when those phages are tropic for the same bacterium. This additional, sometimes optional step, can beneficially allow for the selection and combination of phages that are as unrelated to each other as possible and to decrease the likelihood that the targeted bacteria can develop resistance against the selected phages.
• additional phages specific for each additional putative obesogenic, inflammatory, and/or other bacterium can be included that are each validated as lytic, having a narrow and specific tropism, able to survive conditions in the mammalian GI tract, and unlikely to promote bacterial escape mutants resistant to the cocktail of phages.
• the selected bacteriophage is prepared in any manner to be incorporated as a component of a food stuff, feed additive, or liquid additive and can be in various forms such as, for example, a liquid state or a dried state including as a powder.
• the bacteriophage is dried by air drying method, natural drying method, a spray drying method, a freeze-drying method, or the like.
• the preparation of the bacteriophage strain can also serve to enhance the properties of the composition including stability.
• the term "foodstuff is understood to be any substance or product which in the processed, partially processed, or unprocessed state are intended to be, or reasonably expected to be, ingested by humans.
• Foodstuff can also include drinks, chewing gum, and any substance—including water— intentionally added to the foodstuff during its manufacture, preparation or treatment.
• feed is understood to cover all forms of animal food. Foodstuffs can also be used as feeds.
• the bacteriophage is prepared in any manner to be incorporated as a component of a nutritional supplement to promote or support weight management and control, support metabolic health, and/or support a healthy balance in the gut microbiota, and can be in various forms such as, for example, a liquid state or a dried state including a powder.
• the term “nutritional supplement” is understood to cover substances or substance compositions which are intended as agents having properties to support or promote health, but not to treat, diagnose, prevent, or cure any disease.
• the compositions disclosed herein can be administered alone or co- administered with a probiotic. The administration dosage and schedule can be determined based on the desired effect and as known in the art.
• A“naturally-occurring” bacteriophage is a phage isolated from a natural or human-made environment that has not been modified by genetic engineering.
• A“recombinant bacteriophage” is a phage that comprises a genome that has been genetically modified by insertion of a heterologous nucleic acid sequence into the genome or by removal of a nucleic acid sequence from the genome.
• the genome of a naturally-occurring phage may be modified by recombinant DNA technology to introduce a heterologous nucleic acid sequence into the genome at a defined site. Additionally, or alternatively, the genome of a naturally-occurring phage may be modified by recombinant DNA technology to remove nucleic acid sequences that, for example, encode bacterial virulence factors (e.g ., toxins).
• bacterial virulence factors e.g ., toxins
• co-administration refers to concurrent, sequential, and/or combined administration of two or more components.
• two components can be co- administered by administering each component in a separate dosage concurrently, simultaneously, or sequentially (e.g., distinct administrations separated by a period of time).
• the period of time can be very short (e.g, substantially immediately following a first administration) or it may be longer (e.g, 10-60 seconds later, 1-60 minutes later, 1-24 hours later, 1-7 days later, or any value or range of values therebetween).
• Concurrent or simultaneous administration can include overlapping administration timeframes for the two or more components or administration of a combination product comprising a mixture of the two or more components.
• the term“cocktail,”“bacteriophage cocktail,“phage cocktail,” or similar is intended to be understood as a composition that includes two or more bacteriophages.
• the composition may have a proportional or disproportional number or concentration of phages, and the phages comprising the cocktail may have overlapping or non-overlapping tropisms.
• the cocktail can be in a dry form or suspended in a pharmaceutically-acceptable carrier.
• the term“gut,” as used herein, is synonymous with“digestive tract,”“gastrointestinal tract,” or similar and is intended to include the system of modified epithelial cells, mucus, and associated environment and secreted factors spanning between the mammalian mouth and anus, including the intervening organs ( e.g ., stomach, small intestine, and large intestine).
• microbiome may refer generally to the collective genomes of the microbiota or to the microorganisms themselves and may be used synonymously with the term microbiota.
• microbiota generally refers to the population, collection, and/or totality of microbes in a defined environment, habitat, or ecological community, and typically includes a plurality of genera, species, or strains of commensal, symbiotic, beneficial, and/or opportunistic pathogenic microorganisms (e.g., bacteria, archaea, fungae, protists, and/or viruses), and typically including their genetic elements (genomes).
• the term“microbiota” or“microbiome” is generally made with reference to the population of microbes inhabiting the intestinal tract of a mammal (i.e. the intestinal microbiome).
• the term“obesogenic bacterium,”“obesogenic bacteria,” or similar term is intended to encompass those bacteria whose presence, concentration, or imbalance within the intestinal microbiome of a mammalian subject, preferably humans, correlate with, promote, or induce weight gain in the mammalian subject and/or whose removal or reduction within the intestinal microbiome of a mammalian subject, preferably humans, correlate with, promote, or induce weight loss in the mammalian subject.
• a bacterium within the intestinal microbiome of a mammalian subject may be understood to be an obesogenic bacterium if when targeted by a specific bacteriophage (e.g, via administration of a tropic phage or phage cocktail), a concomitant reduction in the concentration of the bacterium correlates with, promotes, or induces weight loss in the mammalian subject (e.g, over time).
• the term“inflammatory bacterium,”“inflammatory bacteria,” or similar term is intended to encompass those bacteria whose presence, concentration, or imbalance within the intestinal microbiome of a mammalian subject, preferably humans, correlates with, promotes, or induces inflammation, preferably low-level, systemic inflammation in the mammalian subject and/or whose removal or reduction within the intestinal microbiome of a mammalian subject, preferably humans, correlates with, promotes, or induces a decrease in such inflammation and/or inflammatory markers in the mammalian subject.
• a bacterium within the intestinal microbiome of a mammalian subject may be understood to be an inflammatory bacterium if, when targeted by a specific bacteriophage (e.g, via administration of a narrowly tropic phage or phage cocktail), a concomitant reduction in the concentration of the bacterium correlates with, promotes, or induces a decrease in (low-level systemic) inflammation and/or inflammatory markers in the mammalian subject (e.g ., over time).
• prebiotic generally refers to a component (e.g., an energy source or food, food ingredient, dietary supplement, etc.) that when consumed by a mammalian subject stimulates the growth, diversity, or activity of at least a subset of microbes within the subject’s microbiome.
• the prebiotic can be selected to include a preferred food source for one or more bacteria within the subject’s intestinal microbiome and/or within a co-administered probiotic to encourage the growth or activity of that population of microbes.
• probiotic generally refers to one or more live microbes associated with neutral or beneficial effects in the mammalian gastrointestinal tract.
• An exemplary probiotic can include one or more of Lactobacillus sp., Bifidobacterium sp., Saccharomyces sp., and/or a component (e.g., a food, food ingredient, dietary supplement, etc.) that includes the same.
• a probiotic can assist in maintaining or restoring beneficial levels, diversity, or activity, of the microbiome.
• sequence identity refers to a specified percentage of residues in two nucleic acid or amino acid sequences that are identical when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection.
• sequences differ in conservative substitutions the percent sequence identity may 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 this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
• the term“subject,” as used herein, is synonymous with the terms“patient,”“individual,” and similar terms, and generally refers to individual or organism of any mammalian species, (1) preferably including humans, whether or not under the care of a healthcare provider (e.g, a physician, nurse, or medical assistant or volunteer), and (ii) non-human mammals, whether or not under the care of a veterinarian or other veterinary professional, assistant, or volunteer, such as, but not limited to, dogs, cats, horses, cattle, rodents, or other domesticated or wild mammal.
• Table 2 (below) includes a list of each sequenced bacteriophage with its corresponding SEQ ID NO. Table 2.
• Figure 1 is a graphical representation showing improved oral glucose tolerance in mice with intestinal microbiotas colonized with Enterobacter cloacae strain B29 treated with bacteriophages as compared to control mice with intestinal microbiotas colonized with Enterobacter cloacae strain B29 but not treated with bacteriophages.
• the phage cocktail inhibits B29-induced glucose intolerance.
• mice Prior to colonization with the human pathogen E. cloacae strain B29, mice are treated with oral antibiotics to deplete their native gut microbiota.
• mice receive daily doses of B29 by oral gavage and are placed on a high fat diet (60% of total energy from fat, 20% from carbohydrates, and 20% from protein) to induce weigh gain.
• the treatment group receives 7 daily doses of phage cocktail by oral gavage.
• the treatment group black line receives bacteriophages in drinking water.
• the bacteriophage cocktail included greater than or equal to lxlO 9 PFU of Optium-l25, Optium-4l7, Optium-l8, Optium-l l8, Optium-86, Optium-7l2, Optium-l l3, Optium-34, and Optium-38.
• the 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 the indicated time points over the next 2 hours. Blood sugar levels are significantly improved in the phage-treated group (p ⁇ 0.05).
• a four-week test/trial was conducted using 17 individuals (10 male and 7 female) between the ages of 25 and 59 years old taking a single daily dose of a bacteriophage cocktail.
• the cocktail included greater than or equal to lxlO 8 PFU of Optium-l2, Optium-l8, Optium-38, Optium-79, Optium-l25, Optium-l26, Optium-2l2, Optium-7l2, Optium-7l9, Optium-7l2 and Optium-8l9, having a combined tropism for Enter obacter cloacae , Escherichia coli, and Klebsiella pneumoniae.
• each bacteriophage was isolated at a titer greater than or equal to l .OxlO 9 phages.
• An exemplary method for identifying and characterizing bacteriophages for inclusion within a composition for reducing the number or concentration of a bacterium associated with the intestinal microbiome of a mammal includes at least the following method acts.
• the method can include: (1) collecting environment sample(s); (2) setting up enrichment culture; (3) isolating phage from the enrichment culture; (4) purifying the phage; (5) phage titer test to high concentration; (6) characterizing the phage ( e.g ., using electron microscope and genomic DNA isolation and sequencing); (7) performing a restriction enzyme assay; (8) performing a lytic activity assay; and (9) optionally preparing a phage frozen stock.
• the method can include collect an environment sample from the soil, sewage water, or other environmental source and setting up an enrichment culture.
• the enrichment culture can include, for example, 25 mL LB broth, 1 mL target bacterium, and 1 mL of the environment sample.
• the inoculated enrichment culture is then incubated (e.g., at 35 °C for 2 days with aeration).
• the liquid can be transferred from the flask into a cylindrical or conical tube for centrifugation (e.g, at 8000 RPM for 20 minutes).
• the supernatant can then be filtered (e.g, using a 0.45 pm filter) into another sterile conical tube and optionally refrigerated.
• the phage can subsequently be isolated from the enrichment culture.
• the filtered enrichment culture can be mixed and serially diluted (e.g, by factors of 10) to a final concentration of lxl O 7 PFU/mL.
• An overnight culture of the target bacterium can then be inoculated into each tubes of the dilution series (e.g, using 500 pL of the bacterial culture).
• the inoculated tubes are then incubated at 35°C for 40 minutes.
• each sample is mixed with 5mL of melted top agar and plated (e.g, onto petri dishes). The plated samples are incubated for 24 h or until plaque formation is visible.
• the phages are purified by picking a single plaque with a sterile needle and transferring to sterile media (e.g, LB broth).
• sterile media e.g, LB broth
• the inoculated media is mixed, incubated, and phages picked from a resulting plaque are filtered as before prior to serial dilution.
• An overnight culture of the target bacterium can then be inoculated into each tubes of the dilution series (e.g, using 500 pL of the bacterial culture).
• the inoculated tubes are then incubated at 35 °C for 40 minutes.
• each sample is mixed with 5mL of melted top agar and plated (e.g, onto petri dishes).
• the plated samples are incubated for 24 h or until plaque formation is visible. This phage purification process may be repeated two or more times.
• the phage titer is determined. This can allow isolation of phage to an adequate high concentration in preparation for further analysis.
• the phage titer assay can be conducted, for example, by mixing 20 mL LB broth, 0.5 mL of an overnight culture of the target bacterium, and plaque picking from the last phage purification round that had been suspended in 100 uL LB broth. The flask is then incubated at 35 °C for 2 days with medium shaking. Following this incubation period, the sample is centrifuged (e.g ., 8000 rpm for 10-15 minutes) and supernatant filtered (e.g., through a 0.45 pm filter). The filtered supernatant can then be used, as above, to generate plaques for determining the concentration of phage within the filtered lysate.
• the final concentration of the phage lysate is greater than or equal to lxlO 8 PFU/mL.
• the phage can then be characterized. This includes isolating the phage DNA using methods and phage DNA isolation kits, as known in the art.
• the isolated phage DNA can be sequenced using any method or suitable technology known in the art. If characterized using electron microscopy, 10 pL of phage lysate is combined with 10 pL of tungsten heavy metal solution and placed on an electron microscopy grid and viewed via electron microscopy, as known in the art.
• the electron microscopy data can be used to determine phage morphology and structural classification.
• the sequenced phage DNA can be used to confirm the lack of any bacterial toxin and/or virulence factor and/or integrase gene, and to compare with other phage DNA having a similar tropism for a relative genetic similarity.
• An exemplary method for determining lytic activity and tropism for identified bacteriophages includes at least the following method acts.
• a target bacterium such as Enter obacter cloacae strain B29
• CFU/mL colony-forming units per milliliter
• phage titered a concentration of lxlO 8 PFU/mL
• concentration of lxlO 8 PFU/mL giving a multiplicity of infection of 100.
• One milliliter of sample was removed from the flask, serial diluted, spread over LB-agar plates, and incubated overnight. The number of surviving bacteria was determined by colony count the next day.
• An additional 1 mL of each sample was removed from respective cultures every 2 hours during the first 12 hours after inoculation then once more at the 24 th hour after inoculation to detect whether the phage was lytic and whether bacteria develop resistance within the first 24 hours.
• each phage was tested by measuring its ability to lyse a range of bacteria, from closely to distantly related bacterial strains. For example, Table 3 illustrates host range testing of 9 bacteriophages that were initially isolated based on their ability to lyse Enter obacter cloacae strain B29. Each phage was tested against two other E.
• cloacae strains namely ATCC 13047 and ATCC 23855, as well as more distantly related Shigella hoydii (ATCC 9207), Klebsiella pneumoniae (ATCC 10031), Salmonella enterica Serovar Typhimurium LT2, Escherichia coli strain 814, Serratia marcescens , and Pseudomonas aeruginosa.
• a spot test was performed by dropping 100 pL of the respective phage solution containing lxlO 9 PFU/mL onto a lawn of specified-target bacterium and incubating overnight at 37 °C.
• the results are illustrated in Table 3 where a positive indication corresponds to clearing of bacteria in the location where the phages were dropped or“spotted” on the plate.
• the clearance indicates lysis of the target bacterium and therefore tropism for the same.
• the different phages display tropism for different bacterial strains, while maintaining tropism for many (or all) of Enterobacter cloacae strains tested.
• the control group were given 0.2 mL sterile Luria broth (LB) via oral gavage once.
• Test groups were given lxlO 10 PFU/mL of phage in 0.2 mL of LB broth via oral gavage once.
• Phages were grown in and purified from host bacteria, and titer determined by standard plaque assay before diluting the phage to the stated concentration in LB.
• fecal samples were collected from all mice. Fecal samples were also collected from all mice every 6 hours until the 24th hour after gavage.
• each fecal sample was resuspended in phosphate buffered saline and filtered through a 0.45 pm filter to isolate phage.
• the filtered solution was grown with host bacteria in a standard plaque-forming assay to calculate phage concentration in the fecal sample and thus determine how well the phage had survived the digestive tract.
• Table 5 shows survivability of the 9 bacteriophages disclosed in Tables 3 and 4 above after passage through the mouse gastrointestinal tract. As shown in Table 1, the different phages are capable of surviving within the digestive tract for an extended period of time.
• Figure 5 is a graphical representation showing arrest of weight gain in mice colonized with Enterobacter cloacae strain B29 and subsequently treated with bacteriophages as compared to control mice with intestinal microbiomes colonized with Enterobacter cloacae strain B29 but untreated with bacteriophage. As shown in Figure 5, the administered phage cocktail inhibited E. cloacae strain B29-induced weight gain.
• mice received daily doses of E. cloacae strain B29 by oral gavage and were placed on a high fat diet to induce weight gain.
• the treatment group (solid line with triangles) received daily doses of the phage cocktail by oral gavage for 7 days.
• the phage treatment was repeated at week 5.
• Body weight was monitored weekly throughout the study and was shown to be significantly different in the phage-treated group as compared to the control group at weeks 5 and 6 (p ⁇ 0.05).
• the study included 6 groups of 8 mice each.
• the study groups included:
• Group 1 Obese control: mice with normal gut microbiota; developed obesity.
• Group 2 Lean control: mice with antibiotic-depleted gut microbiota; did not develop obesity.
• Group 3 Obese control #2: mice with antibiotic-depleted gut microbiota inoculated with E. cloacae strain B29 to reconstitute gut microbiota; developed obesity.
• Group 4 Experimental Group #1 : mice administered phage cocktail at beginning of week 3 during antibiotic-depletion, one week prior to first inoculation with E. cloacae strain B29; administration of phage cocktail correlates with prevention of E. cloacae strain B29 engraftment and prevented development of obesity.
• Group 5 Experimental Group #2: mice administered phage cocktail at the beginning of week 7 after E. cloacae strain B29 had engrafted but before obesity developed; phage treatment correlated with prevented development of obesity, reduced E. cloacae strain B29 levels, and reduced inflammation.
• mice administer phage cocktail at the beginning of week 17 after obesity had developed; phage administration correlated with decreased low-level systemic inflammation, weight loss, and reduction of E. cloacae strain B29 levels in the mouse gut microbiome.
• the study was designed to measure the efficacy of phage treatment when administered before exposure to E. cloacae strain B29, after exposure to E. cloacae strain B29 but before obesity develops, and after obesity develops due to colonization of E. cloacae strain B29 within the mouse gut microbiota.
• Broad-spectrum antibiotics were administered to Groups 2-6 (see group descriptions above) to deplete their native gut microbiota during weeks 1-3, then their microbiotas were reconstituted (except group 2) by twice-daily oral gavage with lxlO 10 CFU/mL of E. cloacae strain B29 during week 4. All mice were fed a high fat diet at the start of week 5 and remained on that diet for the duration of the study.
• TNF-a tumor necrosis factor a
• IL- 1b interleukin 1b
• IL-6 interleukin 6
• TLR-4 toll like receptor 4
• IKKe I-kappa-B kinase epsilon
• ELISA assays were used to measure serum levels of proteins that influence blood sugar and appetite (e.g ., insuin, leptin, and adiponectin), and inflammation (e.g., serum amyloid A and LPS-binding protein).
• systems, devices, products, kits, methods, and/or processes, according to certain embodiments of the present disclosure may include, incorporate, or otherwise comprise properties, features (e.g., components, members, elements, parts, and/or portions) described in other embodiments disclosed and/or described herein. Accordingly, the various features of certain embodiments can be compatible with, combined with, included in, and/or incorporated into other embodiments of the present disclosure. Thus, disclosure of certain features relative to a specific embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment. Rather, it will be appreciated that other embodiments can also include said features, members, elements, parts, and/or portions without necessarily departing from the scope of the present disclosure.
• any steps recited in any method or process described herein and/or recited in the claims can be executed in any suitable order and are not necessarily limited to the order described and/or recited, unless otherwise stated (explicitly or implicitly). Such steps can, however, also be performed in a specific order or any suitable order in certain embodiments of the present disclosure.

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