Methods and devices for manipulation of the intestinal microbiota

Abstract

The present disclosure generally provides systems and methods for manipulating bacterial growth, abundance, and diversity in biological samples. More specifically, the present invention relates to systems and methods for manipulating gut microbiota.

Description

Technical Field

[0001] The present disclosure relates generally to the field of systems and methods for manipulating bacterial growth and abundance in biological samples. More specifically, the present invention relates to systems and methods for manipulating gut microbiota. Background Art

[0002] In recent years, a lot of efforts have been invested in understanding the connection between intestinal microbiota and health. It is clear that changing the composition of resident microorganisms in the intestine may have a profound impact on certain health conditions. Based on the scientific evidence and continuous research accumulated so far, it seems that the ability to selectively manage the composition of microorganisms in the GI tract may have an impact on various diseases and is helpful in treatment. In addition, it has been proven that adding specific bacteria as food supplements is insufficient. In order to have an impact, the entire microbial population (which is called the intestinal microbiota) including a wide variety of species inside the GI tract should be considered. Therefore, the ability to control the composition of the intestinal microbiota by enriching a specific microbial population has a strong therapeutic potential. In fact, maintaining the biodiversity in the GI tract has been shown to be beneficial for various food allergies, inflammatory diseases of the GI tract, diabetes and obesity.

[0003] Given the diversity of the gut microbiota and the complexity of the human GI tract as a therapeutic target, there is an unmet need for effective, accessible, easy-to-use therapeutic tools for manipulating the human gut microbiota. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 : illustrates a heat map presenting the profile of gut microbes in fecal samples;

[0005] Figure 2 : illustrates the principal coordinate analysis (PCoA) presenting the microbiota composition;

[0006] Figure 3 : illustrates the rarefaction plot of alpha diversity (community richness) by treatment using Faith phylogenetic diversity estimation;

[0007] Figure 4 : illustrates the rarefaction plot of alpha diversity (community richness) by treatment using observed OTUs (operational taxonomic units);

[0008] Figure 5 : box plots illustrating the normalized relative abundance levels of differentially abundant species comparing treatment samples to controls; and

[0009] Figure 6 : Box plots illustrating the normalized relative abundance levels of differentially abundant bacteria at the family level comparing treatment samples to controls. Summary of the invention

[0010] Therefore, the main object of the present invention is to provide efficient systems and methods for manipulating the human gut microbiota inside and outside the gastrointestinal (GI) tract, and the use of such systems as potential therapeutic tools. The present invention provides methods for manipulating the growth of multiple bacterial species of the gut microbiota, the methods comprising subjecting the multiple bacterial species to at least one regimen, wherein the regimen comprises a set of predetermined conditions, and wherein the set of conditions comprises at least one illumination event at a wavelength in the range of 400 nm to 900 nm.

[0011] The present invention further provides a method for obtaining a sample having a desired profile of bacterial species of the intestinal microbiota, the method comprising:

[0012] a) providing a starting sample of bacterial species of the intestinal microbiota;

[0013] b) determining the profile of said bacterial species in said starting sample of the intestinal microbiota;

[0014] c) selecting at least one protocol designed to manipulate the growth of a bacterial species, wherein the protocol comprises a set of predetermined conditions, and wherein the set of predetermined conditions comprises at least one illumination event at a wavelength in the range of 400 nm to 900 nm;

[0015] d) subjecting said starting sample to the at least one protocol; and optionally,

[0016] e) determining the profile of said bacterial species in said sample.

[0017] The present invention further provides a system for in situ manipulation of human intestinal microbiota, the system comprising:

[0018] a) a sample of human microbiota having a desired spectrum of bacterial species;

[0019] b) a swallowable vehicle configured to move through the GI tract of a human subject, wherein the vehicle comprises at least one light emitting unit and at least one reservoir configured to carry and release a sample of the human microbiota;

[0020] c) at least one protocol for manipulating the gut microbiota, wherein the protocol comprises a set of predetermined conditions, and wherein the set of conditions comprises at least one illumination event at a wavelength in the range of 400 nm to 900 nm;

[0021] d) a software operated controller configured to control the vehicle; and,

[0022] e) Software configured to upload said at least one set of predetermined conditions to said software operated controller.

[0023] The present invention still further provides a method for treating a disorder in a human subject by manipulating the intestinal microbiota, the method comprising:

[0024] a) providing a system for in situ manipulation of a human gut microbiota, wherein the system comprises a swallowable luminescent vehicle configured to move through the GI tract of a human subject, software, a software-operated controller, and a plurality of protocols for manipulating the gut microbiota, wherein each protocol comprises a set of predetermined conditions, and wherein the set of conditions comprises at least one illumination event at a wavelength in the range of 400 nm to 900 nm;

[0025] b) selecting at least one of said plurality of regimens for manipulating the intestinal microbiota that is suitable for treating said disorder;

[0026] c) uploading the at least one protocol to the software operated controller of the vehicle;

[0027] d) swallowing the vehicle; and

[0028] e) delivering said at least one regimen for manipulating the intestinal microbiota at a desired location in the GI tract of said subject.

[0029] The present invention further provides a method of fecal transplantation in a subject, the method comprising:

[0030] a) preparing a stool sample suitable for transplantation into the GI tract of said subject

[0031] b) providing a swallowable vehicle configured to move through the GI tract of a human subject, wherein the vehicle comprises at least one light emitting unit, a software operated controller, and at least one reservoir configured to carry and release the stool sample;

[0032] c) loading the reservoir with a stool sample;

[0033] d) programming the software-operated controller to deliver the stool sample to a desired location in the GI tract;

[0034] e) swallowing the vehicle; and,

[0035] f) releasing said stool sample at said desired location of the GI tract.

[0036] Additional features and advantages of the present invention will become apparent from the following drawings and description. DETAILED DESCRIPTION

[0037] Before explaining at least one embodiment of the present invention in detail, it should be understood that the present invention is not limited in its application to the construction and arrangement details of the components described below, in the examples, or in the drawings. The present invention is applicable to other embodiments or to be practiced or implemented in various ways. In addition, it should be understood that the words and terms used herein are for descriptive purposes and should not be considered as limiting.

[0038] The present invention provides a method for manipulating the growth of multiple bacterial species of intestinal microbiota, the method comprising subjecting the multiple bacterial species to at least one regimen, wherein the regimen comprises a set of predetermined conditions, and wherein the conditions of the set comprise at least one illumination event at a wavelength in the range of 400nm to 900nm. As used herein, the term "multiple / species" refers to a large number of different types of microorganisms associated with the intestinal tract without limitation. The term "multiple / species" can be understood in the context of the present invention as the "diversity" of bacterial species in the intestinal tract. As used herein, the term "intestinal microbiota" means to be understood as the total microorganisms resident in the GI tract or intestinal tract and / or associated with the GI tract or intestinal tract in a healthy and / or diseased state without limitation. According to some embodiments, manipulating the growth of the multiple bacterial species of the intestinal microbiota comprises obtaining a sample of the intestinal microbiota from a subject, and subjecting the sample to the at least one regimen, wherein the at least one regimen comprises at least one illumination event at a wavelength in the range of 400nm to 900nm. According to some embodiments, the manipulation of the growth of the plurality of bacterial species of the intestinal microbiota is performed in situ and comprises subjecting at least a portion of the GI tract of the subject to the at least one regimen, the at least one regimen comprising at least one illumination event at a wavelength in the range of 400nm to 900nm. The phrase "at least a portion of the GI tract" is meant to be understood without limitation as a region or portion (small portion or large portion) of the gastrointestinal tract of the subject, including but not limited to the stomach, small intestine or large intestine. For the avoidance of any doubt, more than one portion of the GI tract may be subjected to the method of the present invention. In one embodiment, the manipulation of the growth of the plurality of bacterial species of the intestinal microbiota is performed in situ and comprises subjecting more than one portion of the GI tract of the subject to the at least one regimen, the at least one regimen comprising at least one illumination event at a wavelength in the range of 400nm to 900nm. In another embodiment, a single portion of the GI tract is subjected to one or more regimens. In one embodiment, a single portion of the GI tract is repeatedly subjected to a single regimen. In yet another embodiment, a single portion of the GI tract is subjected to multiple regimens. According to some embodiments, one and / or more portions of the GI tract of a subject may be subjected to one and / or more regimens in any order or any combination suitable for achieving the desired effect. As used herein, the term "manipulation" refers, without limitation, to affecting the composition of a bacterial species population. In one embodiment, the term "manipulation" refers to inhibiting the growth of at least one of a plurality of bacterial species. In another embodiment, the term "manipulation" refers to eliminating at least one bacterial species in a sample. In yet another embodiment, the term "manipulation" refers to enhancing the growth of at least one bacterial species in a sample. As used herein, the term "regimen" refers, without limitation, to a set of predetermined conditions whose combination is designed to result in a desired outcome.In one embodiment, the regimen includes at least one of the following: at least one parameter selected from the group consisting of: illumination dose, wavelength, duration of each illumination, number of illumination events, frequency of illumination events, exposure to oxygen, humidity, exposure to sound waves, pH, exposure to radio frequency, temperature, presence of drugs, presence of bacterial species, presence of biologically active substances, or a combination thereof. As used herein, the term "biologically active substances" refers to signal molecules, biomolecules, genetic and translationally modified nuclear substances, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), organic molecules, inorganic molecules, amino acids, vitamins, polyphenols, steroids, lipophilic poorly soluble drugs, vascular regulators, peptides, neurotransmitters and their analogs, nucleosides, proteins, including but not limited to growth factors, hormones, aptamers, antibodies, cytokines, enzymes and heat shock proteins, cannabinoids, cannabinoid acids such as tetrahydrocannabinolic acid (THCa), cannabidiol acid (CBDa), cannabinolic acid (CBNa), cannabichromene acid (CBCa), tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD) and cannabichromene (CBC) and endocannabinoids (endocannobinoid) and their analogs, or any other molecules that can play a biological function. As used herein, the term "drug" refers to a substance with a therapeutic effect. As used herein, the term "therapeutic effect" refers, without limitation, to a response following any type of treatment, the results of which are judged to be useful or beneficial. This is true whether the results are expected, unexpected, or even undesirable consequences. In one embodiment, the therapeutic effect is selected from the group consisting of: anti-inflammatory effects, anti-fibrotic effects, anti-tumor effects, and neuroprotective effects. Non-limiting drugs for preventing and treating inflammation, fibrosis, and hyperglycolysis such as diabetic nephropathy include ubiquitous phenolic compounds of natural origin, such as ferulic acid, natural phenols, such as resveratrol, rutin, quercetin, phenolic acids, vitamins and allicin, cannabinoids (selected from the group consisting of tetrahydrocannabinolic acid (THCa), cannabigerolic acid (CBDa), cannabinolic acid (CBNa), cannabichromenic acid (CBCa), tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD) and cannabichromene (CBC) and / or their derivatives); and synthetic analogs, dexmedetomidine, (α2-AR) agonists, metabolic protectors, including but not limited to vildagliptin, pristimerin, metformin, pyridoxamine, vasomodulators, epinephrine, rutin, isoxsuprine.A non-limiting list of drugs that exert anti-cancer effects includes, but are not limited to, chemotherapeutic agents, including, but not limited to, cisplatin, carboplatin, chlorambucil, melphalan, nedaplatin, oxaliplatin, triplatin tetranitrate, satraplatin, imatinib, nilotinib, dasatinib, and radicicol; immunomodulators, anti-angiogenic agents, mitogens, nucleoside analogs , DNA intercalating agents, anti-aging agents, dipeptides, epigenetic factors and regulators, metformin, rapamycin, valproic acid or salts, wortmannin, polyamine spermidine, HDAC inhibitor sodium butyrate, butyric acid, sirtuin activator, resveratrol, coenzyme CoQ1, small dicarboxylic acids, aspirin, salicylic acid, benzoic acid, carnitine analogs, human growth hormone, topoisomerase analogs, antibodies, cytokines, folic acid antimetabolites, anti-glycolytic agents (anti-glycolisis agent), inhibitor oligonucleotides of human carcinogenic or proto-oncogenic transcription factors, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, mitotic inhibitors, nucleoside analogs, DNA intercalating agents, topoisomerase analogs, antibodies, cytokines or folic acid anti-metabolites, hexokinase inhibitors, lactate dehydrogenase inhibitors, phosphofructokinase 2 or phosphofructokinase-2-kinase / fructose-2,6-bisphospholipase inhibitors, pyruvate kinase M2 inhibitors, transketolase inhibitors, pyruvate dehydrogenase inhibitors, pyruvate dehydrogenase kinase inhibitors, glucose-6-phosphate dehydrogenase inhibitors, GLUT inhibitors, proton transport inhibitors, monocarboxylate transporter inhibitors, hypoxia-inducible factor 1α inhibitors, AMP-activated protein kinase inhibitors, glutamine inhibitors, asparagine inhibitors, arginine inhibitors, fatty acid synthase inhibitors, ATP-citrate lyase inhibitors, dimethyl malate and malic enzyme 2 inhibitors or any combination thereof. As used herein, the term "sample of intestinal microbiota" refers to a natural sample derived from the GI tract of a human subject without limitation. The samples of the present invention can be collected directly from the GI tract of the subject or from body waste such as feces excreted from the body. In one embodiment, the protocol includes at least one event of illuminating the sample at a wavelength within the range of 400nm to 900nm. In one embodiment, the protocol includes at least one event of illuminating the sample at a wavelength within the range of 420nm to 880nm. In one embodiment, the wavelength is 420-460nm, 640-680nm and 830-870nm.According to some embodiments, the wavelength is 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm, 590nm, 600nm, 610nm, 620nm, 630nm, 640nm, In one embodiment, the scheme includes at least two illumination events. In another embodiment, the scheme includes a series of illumination events. In another embodiment, similar wavelengths are used in each illumination event. In another embodiment, at least two illumination events are carried out at different wavelengths. For clarity, the scheme of the present invention can include a required number of illumination events, which are at different or identical wavelengths and continue for a required period of time.

[0039] Any combination of illumination at the indicated wavelengths and other parameters forms an integral part of the present invention. In one embodiment, the protocol is designed to produce specific spectra of multiple bacterial species in a sample. Figure 1, which illustrates an exemplary embodiment of a heat map of the intestinal microbial profile in a stool sample at the genus level after an illumination event relative to wavelength. The horizontal lines represent the relative abundance of the main genera that exhibit at least 50% change relative to the control. The bacterial abundance of each genus is expressed in standard deviation units of the internal standard based on the row and is expressed by color base. Hierarchical clustering is based on the Euclidean distance measure and the Word's agglomeration method. The side bars show the taxonomic classification of different genera at the phylum, class, order, and family levels. A non-limiting list of intestinal microorganisms in the sample includes the following bacterial species: Roseburia; Sutterella; Prevotella; Haemophilus; Anaerotruncus; Geobacter; Neisseria; Pseudomonas; Paraprevotella; Anaerostipes; Dehalobacterium; Coprococcus eutactus; Ruminococcus; Bifidobacterium longum; Dorea formicigenerans; Bacteroides caccae; and Parabacteroides gordonii. For the sake of clarity, and to avoid any doubt, any intestinal microorganism is an integral part of the present invention. According to some embodiments, the genus Roseburia (which consists of obligate Gram-positive anaerobic bacterial species and includes five species: intestinal Roseburia (R.intestinalis), human Roseburia (R.hominis), glucose-eating Roseburia (R.inulinivorans), fecal Roseburia (R.faecis) and cecal Roseburia (R.cecicola)) is increased in the 440nm treatment. According to some embodiments, the genus Sutterella (which represents Gram-negative anaerobic bacteria and includes three species: parvirubra Sutterella, stercoricanis Sutterella and Walder Sutterella (S.wadsworthensis)) is increased in the 660nm and 880nm treatments. According to some embodiments, the genus Prevotella (which includes almost 40 different species with large genomic diversity) is increased in the 660nm and 880nm treatments. According to some embodiments, Geobacter, Neisseria, and Pseudomonas were reduced under all lighting treatments.

[0040] In one embodiment, the scheme is designed to enrich at least one microbial genus in vitro and / or in vivo and / or in situ. As used herein, the term "to enrich" refers to the increase in the ratio of a specific bacterial genus and / or species in a sample without limitation. In one embodiment, the scheme is designed to enrich at least one bacterial species. In one embodiment, the scheme is designed to enrich at least one bacterial genus. In one embodiment, the scheme is designed to increase the growth of at least one bacterial species and / or genus and inhibit the growth of at least one bacterial species and / or genus at the same time. In another embodiment, the scheme is designed to increase the growth of more than one bacterial species and / or genus and inhibit the growth of more than one bacterial species and / or genus at the same time. In one embodiment, the scheme is designed to eliminate at least one bacterial species and / or genus. In one embodiment, the scheme is designed to reduce at least one bacterial species and / or genus. In one embodiment, the scheme is designed to obtain a human intestinal microbial population sample with a desired spectrum of bacterial species and / or genus. In one embodiment, the scheme. In one embodiment of the present invention, the protocol is designed to selectively enhance, reduce or eliminate microbial species in a sample to obtain a human intestinal microbiota sample having a desired profile. As used herein, the term "profile" refers to information about quantitative sample characteristics based on the bacterial species populations that constitute the intestinal microbiota sample without limitation.

[0041] The present invention further provides a method for obtaining a sample having a desired profile of bacterial species of the intestinal microbiota, the method comprising:

[0042] a) providing a starting sample of bacterial species of the intestinal microbiota;

[0043] b) determining the profile of said bacterial species in said starting sample of the intestinal microbiota;

[0044] c) selecting at least one protocol designed to manipulate the growth of a bacterial species, wherein the protocol comprises a set of predetermined conditions, and wherein the set of predetermined conditions comprises at least one illumination event at a wavelength in the range of 400 nm to 900 nm;

[0045] d) subjecting said starting sample to the at least one protocol; and optionally,

[0046] e) determining the profile of said bacterial species in said sample.

[0047] In one embodiment, the at least one scheme includes at least two illumination events. In another embodiment, the at least one scheme includes a series of multiple illumination events. In one embodiment, the wavelength of illumination is similar in each event. In one embodiment, the wavelength of illumination is similar in each event. In one embodiment, the wavelength of illumination is similar in each event. In another embodiment, the illumination event is carried out at at least two different wavelengths. In one embodiment, the at least one scheme is designed to enhance the growth of at least one bacterial species colony in the starting sample. In another embodiment, the at least one scheme is designed to inhibit the growth of at least one bacterial species colony in the starting sample. In another embodiment, the at least one scheme is designed to eliminate the growth of at least one bacterial species colony in the starting sample. In one embodiment, the starting sample is subjected to at least two schemes. In another embodiment, the at least two schemes are designed to manipulate the growth of different bacterial species colonies in the starting sample. In one embodiment, the wavelength is 420-460nm, 640-680nm and 830-870nm. According to some embodiments, the wavelength is 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm, 590nm, 600nm, 610nm, 620nm, 630nm, 640nm, 0nm, 650nm, 660nm, 670nm, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm, 770n m, 780nm, 790nm, 800nm, 810nm, 820nm, 830nm, 840nm, 850nm, 860nm, 870nm, 880nm, 890nm and 900nm.

[0048] In one embodiment, the at least one regimen further comprises at least one parameter selected from the group consisting of: illumination dose, wavelength, duration of each illumination, number of illumination events, frequency of illumination events, exposure to oxygen, humidity, exposure to sound waves, pH, exposure to radio frequency, temperature, presence of drugs, presence of bacterial species, and presence of biologically active substances.

[0049] According to some embodiments, the present invention provides a system for in situ manipulation of human intestinal microbiota, the system comprising:

[0050] a) a sample of human microbiota having a desired spectrum of bacterial species;

[0051] b) a swallowable vehicle configured to move through the GI tract of a human subject, wherein the vehicle comprises at least one light emitting unit and at least one reservoir configured to carry and release a sample of the human microbiota;

[0052] c) at least one protocol for manipulating the gut microbiota, wherein the protocol comprises a set of predetermined conditions, and wherein the set of conditions comprises at least one illumination event at a wavelength in the range of 400 nm to 900 nm;

[0053] d) a software operated controller configured to control the vehicle; and,

[0054] e) Software configured to upload said at least one set of predetermined conditions to said software operated controller.

[0055] In one embodiment, the software-operated controller is configured to perform at least one of the following functions: regulating the function of the light-emitting unit, regulating the movement of the vehicle, identifying the position of the vehicle, instructing the vehicle to reach a desired position of the GI tract, delivering the at least one regimen for manipulating the intestinal microbiota at the desired position, and actuating the release of the cargo from the at least one reservoir at the desired position. A plurality of light-emitting elements are included, and the plurality of light-emitting elements are configured to emit light at a wavelength in the range of 400nm to 900nm. In one embodiment, the light-emitting element is a light-emitting diode (LED) or a laser beam. In one embodiment, the light-emitting element is a light-emitting diode (LED). According to some embodiments, the system further includes at least one thermoelectric element in communication with the controller, wherein the thermoelectric element is configured to control a local temperature. In one embodiment, the at least one thermoelectric element is configured to increase or decrease a local temperature. In another embodiment, the swallowable vehicle includes at least two thermoelectric elements. In one embodiment, the swallowable vehicle of the present invention further comprises a second reservoir, which is in communication with the controller and is loaded with a liquid medium and is designed to release the liquid medium in a desired location of the GI tract. In one embodiment, the release of the liquid medium from the second reservoir at the desired location results in a change in humidity. In one embodiment, the liquid medium is water or saline. In one embodiment, the reservoir comprises a dispenser.

[0056] According to some embodiments, the controller is configured to deliver two or more regimens for manipulating the intestinal microbiota at the desired location. In one embodiment, the two or more regimens are delivered to the same location of the GI tract of the human subject. In yet another embodiment, the two or more regimens are delivered to different locations of the GI tract. In one embodiment, the controller is configured to deliver multiple regimens. In one embodiment, the multiple regimens are delivered to different locations of the GI tract of the subject.

[0057] The present invention further provides a method for treating a disorder in a human subject by manipulating the intestinal microbiota, the method comprising:

[0058] a) providing a system for in situ manipulation of a human gut microbiota, wherein the system comprises a swallowable luminescent vehicle configured to move through the GI tract of a human subject, software, a software-operated controller, and a plurality of protocols for manipulating the gut microbiota, wherein each protocol comprises a set of predetermined conditions, and wherein the set of conditions comprises at least one illumination event at a wavelength in the range of 400 nm to 900 nm;

[0059] b) selecting at least one of said plurality of regimens for manipulating the intestinal microbiota that is suitable for treating said disorder;

[0060] c) uploading the at least one protocol to the software operated controller of the vehicle;

[0061] d) swallowing the vehicle; and

[0062] e) delivering said at least one regimen for manipulating the intestinal microbiota at a desired location in the GI tract of said subject.

[0063] In one embodiment, the swallowable carrier comprises at least one reservoir configured to carry and release cargo. In another embodiment, further comprising the step of loading a sample derived from a human intestinal microbiota having a desired spectrum of bacterial species into the at least one reservoir. In another embodiment, the step of loading the sample derived from the intestinal microbiota into the reservoir is before step d. In yet another embodiment, the method further comprises the step of releasing the sample derived from the human intestinal microbiota or a portion thereof at the desired location of the GI tract of the subject. In one embodiment, the step of releasing the sample is before step e. In another embodiment, the step of releasing the sample is after step e. In one embodiment, the scheme includes multiple lighting events. In one embodiment, the software-operated controller is configured to perform at least one of the following functions: regulating the function of the light-emitting unit, regulating the movement of the carrier, identifying the position of the carrier, indicating that the carrier has reached the desired location of the GI tract, delivering the at least one scheme for manipulating the intestinal microbiota at the desired location, and actuating the release of the cargo from the at least one reservoir at the desired location. In yet another embodiment, the regimen further comprises at least one parameter selected from the group consisting of: illumination dose, wavelength, duration of each illumination, number of illumination events, frequency of illumination events, exposure to oxygen, humidity, exposure to sound waves, pH, exposure to radiofrequency, temperature, presence of drugs, presence of bacterial species, and presence of bioactive substances. According to some embodiments, a non-limiting list of disorders of the present invention includes: inflammatory bowel disease, Crohn's disease, type II diabetes, HIV, GI tract malignancies, ulcerative colitis, proctitis, overweight, irritable bowel syndrome, Alzheimer's, depression, celiac disease, specific food allergies, Clostridium Difficile infection, diarrhea, chronic constipation, and dysbacteriosis.

[0064] The present invention further provides a method of performing a fecal transplant in a subject, the method comprising:

[0065] a) preparing a stool sample suitable for transplantation into the GI tract of said subject;

[0066] b) providing a swallowable vehicle configured to move through the GI tract of a human subject, wherein the vehicle comprises at least one light emitting unit, a software operated controller, and at least one reservoir configured to carry and release the stool sample;

[0067] c) loading the reservoir with a stool sample;

[0068] d) programming the software-operated controller to deliver the stool sample to a desired location in the GI tract;

[0069] e) swallowing the vehicle; and,

[0070] f) releasing said stool sample at said desired location of the GI tract.

[0071] As used herein, the term "preparing a stool sample" refers, without limitation, to subjecting a stool sample to at least one protocol of the present invention to obtain a sample having a desired spectrum of bacterial species. In one embodiment, the fecal transplant method further comprises the step of delivering at least one protocol for manipulating the intestinal microbiota. In one embodiment, the protocol includes at least one event of illuminating the GI tract of the subject at a wavelength in the range of 400nm to 900nm. In one embodiment, the protocol includes multiple illumination events. According to one embodiment, the protocol includes at least one parameter selected from the group consisting of: illumination dose, wavelength, duration of each illumination, number of illumination events, frequency of illumination events, exposure to oxygen, humidity, exposure to sound waves, pH, exposure to radio frequency, temperature, presence of drugs, presence of bacterial species, presence of biologically active substances, or a combination thereof.

[0072] According to some embodiments, the subject suffers from a disorder characterized by an impaired intestinal microbiota. A non-limiting list of disorders characterized by an impaired intestinal microbiota includes: inflammatory bowel disease, Crohn's disease, type II diabetes, HIV, GI tract malignancies, ulcerative colitis, proctitis, overweight, irritable bowel syndrome, Alzheimer's, depression, celiac disease, specific food allergies, Clostridium difficile infection, diarrhea, chronic constipation, and dysbacteriosis.

[0073] Example 1: Effects of tested low-level light therapy (LLLT) on fecal microbiome composition using several lighting regimens.

[0074] The aim of this study was to test the effects of low-level light therapy at three different wavelengths on the composition of the fecal microbiota in vitro.

[0075] Three experimental frameworks were used to conduct this study:

[0076] 1. Long-term lighting exposure with different test regimens to evaluate changes in microbial composition after treatment.

[0077] 2. Combined short-term experiments evaluating bacterial composition and inflammation-related functions.

[0078] 3. Experiments with toxin-producing strains of Clostridium difficile to evaluate the specific effects of LLLT on bacterial growth.

[0079] Frame 1: Long-term experiment with longitudinal lighting exposure

[0080] The test protocol with respect to illumination wavelength and exposure time is detailed in Table 2. Illumination was performed with 5 exposures, of which 2 exposures were performed each day (same time each day with at least 8 hours interval between exposures, i.e. 8:00am and 16:00pm), for 2.5 days.

[0081] plan wavelength Exposure time (seconds) 1 440nm 50 2 660nm 60 3 850mm 110 4 Comparison

[0082] Table 2: Different test lighting scenarios*

[0083] Sample size: 10 stool samples tested for each protocol. Total 40 tested samples + original pre-plating uncultured samples.

[0084] Experimental Procedure:

[0085] 1. Preparation of Fecal Samples

[0086] a. Collect stool samples (preferentially from individuals who have not been treated with antibiotics in the past year) in sterile specimens and immediately freeze at -20°C. When transporting to the laboratory, keep stool samples in anaerobic jars with gas bags in containers with ice or dry ice.

[0087] b. In the laboratory, the samples were stored at -80°C.

[0088] 2. Culture of Fecal Samples

[0089] a. For incubation, place samples in an anaerobic chamber and adjust to room temperature.

[0090] b. Inside the anaerobic chamber, 0.2 g of fecal sample was placed in 1800 μl of pre-reduced liquid medium (BHI with supplements).

[0091] c. The fecal material was suspended by vortexing for 5 min.

[0092] d. The fecal suspension was allowed to stand at room temperature for 5 min to allow the insoluble components to settle.

[0093] e. 50 μl of the suspension were then plated on agar plates (size 35 mm) followed by incubation at 37° C. under anaerobic conditions for 3 days, where they were exposed to lighting treatments as detailed above, twice a day.

[0094] f. Incubate samples in the dark between illumination exposures.

[0095] g. 500 μl of the starting stool suspension was stored as a frozen stock at -80°C and prepared by adding glycerol to a final concentration of 25% glycerol (equal volumes of bacterial suspension in growth medium, and 1:1 pre-reduced glycerol with PBS) for optional bacterial composition assessment.

[0096] 3. Lighting treatment

[0097] The lighting was performed in 5 exposures in the anaerobic chamber, where in each day, 2 exposures were performed (at the same time of each day, with at least 8 hours interval between exposures, i.e. 8:00am and 16:00pm), for 2.5 days. After the 5th exposure, a 6-hour duration was required between lighting and bacterial harvesting. The lighting device was kept at a specific predetermined distance from the plate (using a special holder) so that all plate surfaces were equally exposed.

[0098] 4. Bacterial Harvest at the End Point

[0099] a. 0.5 ml of liquid medium (BHI) was added to each plate and bacterial colonies were scraped off and suspended in the liquid medium.

[0100] b. 250 μl of harvested bacteria were added to an equal volume of 1:1 glycerol with PBS (25% glycerol final concentration) and frozen at -80°C for RNA extraction.

[0101] c. Collect and store the remainder of the fecal suspension in the same manner as detailed in stage 4.b for the next experiment of co-cultivation.

[0102] Framework 2: Short-term experiments with post-treatment evaluation of bacteria-host function

[0103] To isolate the direct effect of the lighting-treated fecal microbiota, the effects of untreated and treated fecal microbiota (obtained in Experiment 1) on epithelial cells were tested using co-cultures.

[0104] 1. Pre-growth of HT-29 cells:

[0105] a. Suspend 1 ml of thawed HT-29 stock solution (concentration 3-4*106 cells / ml) in 9 ml pre-warmed McCoy medium (supplemented with 10% (v / v) inactivated FCS (fetal calf serum) and 2 mM glutamine) to wash out DMSO.

[0106] b. The cells were then centrifuged at 125 g for 10 min at room temperature.

[0107] c. Aspirate the medium and resuspend in 10 ml McCoy medium (supplemented with 10% (v / v) FCS and 2 mM glutamine).

[0108] d. Plate the cells in a T-25cm 2 The cells were placed in flasks at 5% CO 2 Incubate at 37°C in an air atmosphere for 2-4 days.

[0109] e. Harvest cells when reaching 80%-90% confluent growth by aspirating the media and adding 1 ml of trypsin (0.25% w / v trypsin-0.53 mM EDTA) followed by incubation at 37° C. for 5 min to detach the cells from the flask. Visualize cell detachment under a microscope.

[0110] f. To stop trypsin activity, add 1 ml of McCoy medium (supplemented with 10% (v / v) FCS and 2 mM glutamine).

[0111] g. Then add medium accordingly to obtain 5x10 6 The final concentration was cells / ml.

[0112] 2. Co-culture

[0113] a. HT29 colon epithelial cells were cultured at 5x10 4 cells / cm 2 The cells were seeded at a density of 1.5 μg / ml in 24-well plates in 1 ml McCoy medium (supplemented with 10% inactivated FCS and 2 mM glutamine) and incubated at 37°C in 5% CO 2 and 95% air for 24 h.

[0114] b. For co-cultivation, the medium was changed to reducing medium and 100 μl of the fecal bacterial suspension from Experiment 1 was added to each well of a 24-well plate.

[0115] c. The co-culture was incubated under anaerobic conditions at 37°C for 6 hours.

[0116] d. At the end of the experiment:

[0117] a) Collect bacterial cell suspension from each well (HT29 will adhere to the bottom).Add 500 μl of bacterial suspension to an equal volume of 1:1 glycerol with PBS (25% glycerol final concentration) and freeze at -80°C for RNA extraction.

[0118] b) Then, 350 μl of cell lysis reagent (RLT buffer + beta mercaptoethanol) used in the first step of the RNA extraction procedure (QIAGEN) was added to each well. The cells were scraped from the wells for transfer to the lysis buffer.

[0119] c) Immediately proceed to the next extraction step according to the manufacturer's instructions. The extracted RNA was stored at -80°C for evaluation of inflammatory cytokine expression using real-time PCR.

[0120] Frame 3: Effects of lighting treatments on the growth of toxigenic Clostridium difficile.

[0121] C: Clostridium difficile strain selection

[0122] Strains:

[0123] Toxigenic strains of C. difficile expressing both the tcdA and tcdB genes required for the production of toxin A and toxin B:

[0124] Clostridium difficile (Prevot) Lawson et al. ( 43255 TM ) belongs to toxin type 0.

[0125] Comparison of C. difficile inhibition to reference beneficial species such as Bifidobacterium longum, which has been reported to reduce mucosal inflammatory markers in IBD (can be grown anaerobically on MRS at 37°C) 3

[0126] 1. Recover Clostridium difficile stock solution according to ATCC instructions.

[0127] 2. C. difficile was plated in brain heart infusion broth (BHIS-Brain Heart Infusion Supplement) supplemented with yeast extract (0.5%, wt / vol) and l-cysteine ​​(10%, wt / vol) and incubated at 37°C for 48 h under anaerobic conditions as previously reported. 4 ,5.

[0128] 3. Lighting treatment:

[0129] a. C. difficile culture was adjusted to an optical density at 600 nm (OD600) of 0.5 (mid-exponential phase).

[0130] b. Split the conditioned cultures into 4 sample falcons, one falcon for each tested lighting treatment.

[0131] c. Pour the conditioned culture into sterile petri dishes with a high surface area to obtain a low bacterial suspension height (approximately 0.5 cm) under sterile conditions in an anaerobic chamber.

[0132] d. Lighting exposures were performed as detailed in the following table (a single treatment with exposures of varying durations is recommended if possible):

[0133] plan wavelength Exposure time (seconds) 1 440nm 50. 2 660nm 60 3 850nm 110 4 Comparison

[0134] Table 3: Different test lighting treatments in C. difficile experiments

[0135] a. Measure the optical density (at 600 nm) of C. difficile cultures after illumination treatment and controls periodically during anaerobic incubation at 37° C. for 48 hours to assess growth (using a plate reader if available).

[0136] b. At 0, 12, 24, 36, and 48 hours after illumination exposure, 500 μl of C. difficile culture was serially diluted and plated (100 μl) on BHIS agar plates and OD was measured in parallel for evaluation of CFU (colony forming units) ml -1 Incubate the plates anaerobically at 37°C for 48 hours. Determine CFU / ml / OD 600 and extrapolated to other OD measurements to allow for OD-based CFU quantification.

[0137] result:

[0138] Figure 2 It is demonstrated that, compared to the control, treatment (660nm and 850nm) induces significant changes in microbial composition. (A) PCoA (principal coordinate analysis) presenting microbial composition is used to evaluate microbial composition heterogeneity based on Bray-Curtis distance. Each point represents the bacterial composition spectrum of different test treatment schemes: 440nm wavelength (blue); 660nm (orange); 850nm (red) and untreated samples (dark green) as a control. Numbers indicate different feces donors. (B) Box plots comparing PC1 and PC2 coordinates between different treatments. The results show that, compared to the control, treatment (660nm and 850nm) induces significant changes in microbial composition, as indicated by the significant differences in PC2 coordinate values.

[0139] Figure 3 The use of α diversity (community richness) sparse plots by treatment proves that treatment increases the microbial diversity of IBD samples. Microbial α diversity was assessed using sparse plots to compare samples and controls from different treatments using Faith phylogenetic diversity estimates. Error bars correspond to the differences between samples in each treatment group. (A) 440nm treatment induces an increase in microbial diversity in IBD samples. (B) 660nm treatment induces an increase in microbial diversity in IBD samples. (C) 850nm treatment has no significant effect on α diversity. Due to the already high baseline diversity, healthy samples were unaffected. The results show that 440nm and 660nm treatments induce an increase in the microbial diversity of samples from IBD patients to similar levels of samples from healthy donors.

[0140] Figure 4Prove that treatment increases the microbial diversity of IBD samples. Microbial α diversity was assessed using a rarefaction plot to compare samples and controls from different treatments using observed OTU (operational taxonomic units) estimates (which represent the counts of unique OTUs in each sample). Error bars correspond to the differences between samples in each treatment group. (A) 440nm treatment induces an increase in microbial diversity in IBD samples. (B) 660nm treatment has no significant effect on α diversity. (C) 850nm treatment has no significant effect on α diversity.

[0141] Healthy samples were not affected due to already high baseline diversity. The results indicate that 440 nm and 660 nm treatment induced an increase in microbial diversity in samples from IBD patients to similar levels as samples from healthy donors.

[0142] Figure 5 Treatment of differentially abundant species is illustrated. Box plots comparing standardized relative abundance levels of differentially abundant species between treated samples and controls. Standardization is performed by multiplying the first calculation of the relative abundance of each species within each sample by a factor of 1000. Only samples with non-zero counts in at least 30% of the samples in the control untreated group were included. The differential abundance was estimated using a paired Wilcoxon test between treated samples and controls. The results show a significant increase in the abundance of Lachnospiraceae sp. and Ruminococcus lactaris after treatment at 440nm and 660nm. Lachnospiraceae and Ruminococcus lactaris are known to be positively correlated with healthy individuals. Some species of Lachnospiraceae are butyrate producers; butyrate production in the human intestine is highly significant because it promotes Treg cell differentiation, which can ultimately inhibit pro-inflammatory responses.

[0143] Figure 6 The treatment differential abundance-family level is illustrated. Compare the box plots of the standardized relative abundance levels of differential abundance bacteria on the family level between the treated samples and the control. Standardization is performed by multiplying the first calculation of the relative abundance of each species in each sample by a factor of 1000. Only samples with non-zero counts in the control untreated group, at least 30% of the samples were included. The paired Wilcoxon test between the treated samples and the control was used to estimate the differential abundance. The results indicate a significant increase in the abundance of Lachnospiraceae after 440nm and 660nm treatment. Lachnospiraceae is an important producer of short-chain fatty acids.

[0144] The terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the present invention. Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an" and "the" are intended to also include plural forms. It should be further understood that the terms "comprises" or "comprising" indicate the presence of stated features, integers, steps, operations, elements, or parts and / or groups or combinations thereof when used in this specification, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, parts and / or groups or combinations thereof. As used herein, the terms "comprising", "includes", "including", "having" and variations thereof mean "including but not limited to". The term "consisting of" means "including and limited to".

[0145] As used herein, the term "and / or" includes any and all possible combinations or one or more of the associated listed items, as well as no combination when interpreted in the alternative ("or").

[0146] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. It should also be understood that terms (such as those defined in commonly used dictionaries) should be interpreted as having a meaning consistent with their meaning in the context of this specification and claims, and should not be interpreted in an idealized or overly formal sense unless explicitly defined herein. For the sake of brevity and / or clarity, well-known functions or constructions may not be described in detail.

[0147] It should be understood that when an element is referred to as being "on another element," "attached" to another element, "operably coupled" to another element, "operably engaged" with another element, "connected" to another element, "coupled to" another element, "contacting" another element, etc., it can be directly on, attached to, connected to, operably coupled to, operably engaged with, coupled to, and / or contacting the other element, or there may be intervening elements. In contrast, when an element is referred to as being "directly in contact with" another element, there are no intervening elements.

[0148] It should be understood that terms such as, for example, "process," "compute," "determine," "establish," "analyze," "examine," and the like may refer to operations and / or processes of a computer, computing platform, computing system, or other electronic computing device that manipulate and / or convert physical (e.g., electronic) quantities represented as registers and / or memories of a computer into other data similarly represented as physical quantities within registers and / or memories of a computer or other information non-transitory storage media that may store instructions for performing the operations and / or processes.

[0149] It should be understood that although the terms first, second, etc. may be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. Instead, these terms are only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section.

[0150] Certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination, or as appropriate, in any other described embodiment of the invention.

[0151] Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0152] Throughout this application, various embodiments of the present invention can be presented in range format.It should be understood that the description of range format is only for convenience and simplicity, and should not be construed as an inflexible limitation to the scope of the invention.Therefore, the description of the scope should be considered as having all possible sub-ranges with definite disclosure and the individual numerical values ​​within the scope.For example, the description of the scope such as from 1 to 6 should be considered as having the sub-range with definite disclosure, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., and the individual numerals within the scope, such as 1, 2, 3, 4, 5 and 6.No matter how the breadth of the scope, this is applicable.

[0153] Whenever a numerical range is indicated herein, it is intended to include any cited numerical value (fractional or integer) within the indicated range. The phrases "range between a first indicated number and a second indicated number" and "range from a first indicated number to a second indicated number" are used interchangeably herein and are meant to include the first indicated number and the second indicated number and all fractional and integer values ​​therebetween.

[0154] Whenever the terms "plurality" and "a plurality" are used, they are meant to include, for example, "multiple" or "two or more". The terms "plurality" and "a plurality" may be used throughout this specification to describe two or more components, devices, elements, units, parameters, etc. A term group may include one or more items when used herein. Unless explicitly stated, the method embodiments described herein are not limited to a particular order or sequence. In addition, some of the method embodiments or elements thereof may be performed simultaneously, at the same point in time, or in parallel.

[0155] As used herein, the term "method" refers to ways, means, techniques and procedures for accomplishing a given task, including but not limited to those ways, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine, or those readily developable from known ways, means, techniques and procedures by practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine.

[0156] Reference to a "patient" or "subject" is intended to include any mammal. As used herein, "mammal" refers to any animal classified as a mammal, including but not limited to humans, laboratory animals (including monkeys, rats, mice, and guinea pigs), livestock and farm animals, and zoo, sports or pet animals, such as dogs, horses, cats, cows, etc.

[0157] As used herein, "treating" or "treatment" of a disease includes: preventing the disease, i.e., resulting in the absence of clinical symptoms of the disease in a mammal that is exposed to or susceptible to the disease but does not yet experience or display symptoms of the disease; inhibiting the disease, i.e., halting or reducing the development of the disease or its clinical symptoms; or alleviating the disease, i.e., resulting in regression of the disease or its clinical symptoms.

[0158] In case of conflict, the present patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Throughout this application, various publications, published patent applications, and published patents are cited.

[0159] It should be understood by those skilled in the art that the present invention is not limited to what has been particularly shown and described above. Rather, the scope of the present invention is defined by the appended claims and includes both the combination and sub-combination of the various features described above, as well as changes and modifications thereof that will occur to those skilled in the art after reading the foregoing description.

Claims

1. A system for in situ manipulation of human intestinal microbiota, the system comprising: a) a sample of human microbiota having a desired spectrum of bacterial species; b) a swallowable vehicle configured to move through the GI tract of a human subject, wherein the vehicle comprises at least one light emitting unit and at least one reservoir configured to carry and release a sample of the human microbiota; c) at least one protocol for manipulating the gut microbiota, wherein the protocol comprises at least one set of predetermined conditions, and wherein the set of conditions comprises at least one illumination event at a wavelength in the range of 420-460 nm, 640-680 nm, or 830-870 nm; d) a software operated controller configured to control the vehicle; and, e) Software configured to upload said at least one set of predetermined conditions to said software operated controller.

2. The system of claim 1, wherein the software-operated controller is configured to perform at least one of the following functions: regulating the function of the light emitting unit, regulating the movement of the vehicle, identifying the position of the vehicle, instructing the vehicle to reach a desired location of the GI tract, delivering the at least one regimen for manipulating the intestinal microbiota at the desired location, and actuating the release of the sample of the human microbiota from the at least one reservoir at the desired location.

3. The system according to claim 1 or 2, wherein the light emitting unit comprises a plurality of light emitting elements configured to emit light at a wavelength within the range of 420-460 nm, 640-680 nm or 830-870 nm. The system according to claim 3 , wherein the light emitting element is an LED or a laser beam. 5 . The system of claim 1 , wherein the system further comprises at least one thermoelectric element in communication with the controller, wherein the thermoelectric element is configured to control a local temperature.

6. The system of claim 2, wherein the vehicle further comprises a second reservoir in communication with the controller, the reservoir being loaded with a liquid medium.

7. The system of claim 6, wherein the liquid medium is released from the second reservoir at the desired location.

8. The system of claim 6, wherein the liquid medium is water or saline.

9. The system of claim 2, wherein the controller is configured to deliver two or more regimens for manipulating the intestinal microbiota at the desired location.

10. The system of claim 9, wherein the two or more regimens are delivered at the same location of the GI tract.

11. The system of claim 9, wherein the two or more regimens are delivered at different locations of the GI tract.

12. The system of claim 9, wherein the controller is configured to deliver multiple regimens.

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