Microbial composition, method for preparing same and use thereof

Abstract

The present invention relates, in some embodiments, to a method for producing a composition comprising a co-culture comprising a plurality of bacteria, having a high similarity% to a sample of origin, and comprising a bacterial load.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 292,536, filed December 22, 2021, entitled "SCREENING SYSTEMS AND ASSAYS USING BACTERIA" and Israeli Patent Application No. 286748, filed September 27, 2021, entitled "MICROBIAL COMPOSITIONS AND METHODS OF PREPARING SAME", the contents of which are incorporated herein by reference in their entirety. Technical field

[0003] The present invention pertains to the field of microbiology and, in particular, to composite microbial compositions and methods for their preparation and use. Background art

[0004] Increasing evidence supports the use of probiotics to promote human health, for example, being beneficial to the immune system, inhibiting infections, etc.

[0005] The human body contains bacterial cells, creating a network of bacteria - human cell interactions that greatly influence our health status and even our behavior patterns. For example, the gastrointestinal tract harbors a rich and diverse microbial community. It is a complex system that provides an environment or niche for communities of many different microorganisms, including a wide variety of bacteria. Microbial flora also inhabit other body regions such as the skin, nails, eyes, oral cavity, upper respiratory tract, and urogenital tract.

[0006] A healthy microbiota includes bacterial colonization through a balanced community that provides the host with a variety of benefits, including resistance to broad - spectrum pathogens, production and absorption of essential nutrients, and appropriately controlled systemic immunity. In the context of "dysbiosis" or disrupted symbiotic relationships, microbiota functions may be lost or disrupted, leading to increased susceptibility to pathogens, altered metabolic profiles, or induction of pro - inflammatory signals that can result in local or systemic inflammation or autoimmunity. Thus, the microbiota, including the gut microbiota, is considered a key factor in the pathogenesis of many diseases and disorders, including but not limited to various intestinal pathogenic infections.

[0007] Current practices for treating intestinal dysbiosis include fecal transplantation from healthy donors. Generally, fecal transplantation, also known as fecal microbiota transplantation (FMT), is a method for reconstructing and / or modulating the gastrointestinal microbiota of a recipient, with the aim of preventing, treating, and / or improving a disease or condition. Studies have shown that FMT can restore health-related bacteria in the lower intestine, thereby treating, for example, Clostridoides difficile (C. diff) infection and preventing its recurrence (recurrent C. diff; rCDI). C. diff is one of the causes of healthcare-associated diarrhea due to severe disruption of the microbiome, for example exacerbated by the use of antibiotics. According to a systematic review [Gupta K, Tappiti M, Nazir AM, et al. (May 05, 2022) Fecal Microbiota Transplant in Recurrent Clostridium Difficile Infections: A Systematic Review. Cureus 14(5): e24754. doi: 10.7759 / cureus.24754], FMT has demonstrated a cure rate of over 90% among C. diff patients who have undergone several FMT treatments. Although FMT is rapidly becoming an accepted treatment for many other diseases associated with disruption of the intestinal microbiome [Baktash A, Terveer EM, Zwittink RD, Hornung BVH, Corver J, Kuijper EJ and Smits WK (2018) Mechanistic Insights in the Success of Fecal Microbiota Transplants for the Treatment of Clostridium difficile Infections. Front. Microbiol. 9: 1242. doi: 10.3389 / fmicb.2018.01242], there are concerns about its safety, low reproducibility, and scalability of preparation.

[0008] There is still a great need for methods for preparing bacterial compositions that have increased similarity to origin samples (e.g., fecal samples) that can be used to treat dysbiosis.

[0009] Advantageously, such methods can reduce the need for redundant collection of the origin sample, reduce safety risks associated with the use of biological samples, and preserve the original microbiota profile while achieving preparation scalability and reproducibility.

[0010] Such compositions can in particular also be used as systems that mimic the in vitro microbial environmental niche of a subject (e.g., the gastrointestinal microbiota of a subject), and are thus capable of providing insights into the modulation of bacterial populations in view of exposure to at least one compound. Summary of the Invention

[0011] In some aspects of the invention, there is provided a method for producing a composition, the composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles, the co-culture comprising: i) at least 30% similarity to an origin sample, and ii) at least 1·E 4 / 1gr of bacterial load of the particles.

[0012] In some aspects of the invention, there is provided a composition produced by a method according to the invention.

[0013] In some aspects of the invention, there is provided an in vitro method for evaluating: the effect of at least one compound on a plurality of bacteria; the effect of a plurality of bacteria on at least one compound; or both.

[0014] In some embodiments, the method comprises the steps of: providing a microorganism comprising a plurality of bacteria derived from an origin sample; contacting the plurality of bacteria with the particles and causing at least a portion of the plurality of bacteria to attach to the particles; and culturing at least a portion of the plurality of bacteria attached to the particles in a growth medium for a period of less than 14 days, wherein the culturing comprises culturing under anaerobic conditions, thereby producing the composition, the composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles, the co-culture comprising: i) at least 30% similarity to an origin sample; and ii) at least 1·E 4 / 1gr of bacterial load of the particles.

[0015] In some embodiments, the method comprises the following steps: providing a plurality of bacteria derived from an origin sample; contacting the plurality of bacteria with particles and causing at least a portion of the plurality of bacteria to adhere to the particles; and culturing the plurality of bacteria that are at least partially adhered to the particles in a growth medium for a period of less than 14 days, wherein the culturing comprises culturing under anaerobic conditions, thereby producing the composition, the composition comprising a co-culture, the co-culture comprising a plurality of bacteria grown in the presence of the particles, the co-culture comprising: i) at least 30% similarity to the origin sample; and ii) at least 1·E 4 / 1gr bacterial load of the particles.

[0016] In some embodiments, the plurality of bacteria are characterized by having different growth, culturing, and / or proliferation conditions selected from the following: metabolic requirements, nutritional requirements, pH, temperature, aerobic, obligate anaerobic, facultative anaerobic, microaerophilic, attachment form, planktonic, growth medium, flow, shaking, stirring, agitation, static, moist, low humidity, and any combination thereof.

[0017] In some embodiments, the culturing is carried out until the similarity of the co-culture to the origin sample reaches greater than or equal to 30%, and the bacterial load is at least 1·E 4 / 1gr of the particles.

[0018] In some embodiments, the culturing period ranges from 6 hours to 14 days.

[0019] In some embodiments, when using any one of the following: next-generation sequencing (NGS) technology, whole-genome sequencing (WGS), or both, and determining the similarity by a measure that takes into account the genetic relatedness of the bacteria, the co-culture comprises at least 30% similarity to the origin sample.

[0020] In some embodiments, the similarity comprises at least 50% weighted Unifrac similarity. In some embodiments, the co-culture comprises at least 50% weighted Unifrac similarity.

[0021] In some embodiments, the co-culture comprises at least 70% similarity to the origin sample, and at least 1·E 8 / 1gr bacterial load of the particles.

[0022] In some embodiments, the similarity comprises at least 70% weighted Unifrac similarity, and the co-culture comprises at least 1·E 8 / 1gr bacterial load of the particles.

[0023] In some embodiments, the similarity includes any of the following: the presence of bacterial taxonomic categories, genetic relatedness, the observed phylogenetic distance between bacteria, bacterial diversity, the abundance of bacteria, relative abundance, or any combination thereof.

[0024] In some embodiments, the similarity between the co-culture and the origin sample includes the similarity between bacterial populations.

[0025] In some embodiments, the origin sample further includes additional microorganisms, and the additional microorganisms include any one of archaea, viruses, fungi, or any combination thereof.

[0026] In some embodiments, the additional microorganisms are contacted with the particles.

[0027] In some embodiments, the additional microorganisms are at least partially attached to the particles.

[0028] In some embodiments, the co-culture further includes the additional microorganisms.

[0029] In some embodiments, the similarity between the co-culture and the origin sample further includes the similarity of at least one of archaea, viruses, fungal populations, or any combination thereof.

[0030] In some embodiments, the plurality of bacteria provided belong to at least 5 bacterial species and / or at least 2 bacterial genera.

[0031] In some embodiments, the composition includes bacteria in a planktonic form and bacteria that are at least partially attached to the particles.

[0032] In some embodiments, the growth medium includes at least two carbon sources.

[0033] In some embodiments, the growth medium includes a carbon source that is from at least two chemical groups selected from monosaccharides, disaccharides, polysaccharides, and any combination thereof.

[0034] In some embodiments, the growth medium includes at least one monosaccharide, at least one disaccharide, and at least one polysaccharide.

[0035] In some embodiments, the contacting step, the culturing step, or both are performed in a single container.

[0036] In some embodiments, the method further comprises separating the bacteria not attached to the particles from the bacteria attached to the particles at at least one time point selected from before, during, after the culturing step, and any combination thereof, thereby producing: (i) a composition comprising bacteria in a planktonic form; and / or (ii) a composition comprising bacteria attached to the particles.

[0037] In some embodiments, the method further comprises mixing composition (i) and composition (ii) in any desired ratio.

[0038] In some embodiments, a plurality of bacteria are provided in a container, and the method further comprises the steps of: adding at least one compound to the container; and determining any of the following characteristics: (i) bacterial diversity; (ii) relative bacterial abundance; (iii) bacterial load; (iv) any other effect of the at least one compound on the plurality of bacteria; (v) any change in the at least one compound; and (vi) any combination of (i) to (v); thereby evaluating: the effect of at least one compound on a plurality of bacteria; the effect of a plurality of bacteria on at least one compound; or both. In some embodiments, any change in the at least one compound includes chemical modification, structural modification, or a combination thereof.

[0039] In some embodiments, the origin sample is selected from: derived from at least one origin; derived from at least one subject; is a microbiome sample, a skin sample, an oral sample, a fecal sample, a vaginal sample; comprises gut microbiota; and any combination thereof.

[0040] In some embodiments, the compound changes: (i) a similarity level of at least 30% at the end of the culturing period, (ii) the bacterial load of 1·E 4 / 1gr particles, or both (i) and (ii).

[0041] In some embodiments, the change includes increasing or decreasing at least one of: the similarity level, the bacterial load, or both.

[0042] In some embodiments, the method is carried out simultaneously in a plurality of individual containers.

[0043] In some embodiments, the bacterial diversity; the relative bacterial abundance; the bacterial load; and / or any other effect of the at least one compound on the plurality of bacteria are compared with the corresponding characteristics in the plurality of bacteria of the origin sample, and a change in the characteristics indicates that the at least one compound has an effect on the plurality of bacteria.

[0044] In some embodiments, the method further includes culturing a control plurality of bacteria not exposed to the compound in a separate container, and wherein the bacterial diversity; the relative bacterial abundance; the bacterial load; and / or other effects of the at least one compound on the plurality of bacteria are compared to the corresponding characteristics in the control plurality of bacteria, and wherein any change in the characteristics indicates an effect of the compound on the plurality of bacteria.

[0045] In some embodiments, the addition and / or determination is performed using bacteria attached to the particles, bacteria not attached to the particles, or both.

[0046] In some embodiments, the composition is a pharmaceutical composition for modulating the microflora of a subject in need thereof.

[0047] In some embodiments, the composition is for an in vitro method to evaluate: the effect of at least one compound on a plurality of bacteria; the effect of a plurality of bacteria on at least one compound; or both.

[0048] In some embodiments, the composition further includes an acceptable carrier or excipient.

[0049] In some embodiments, the method further includes the step of subjecting the bacteria to at least one compound; thereby generating a desired microbial profile, wherein the desired microbial profile includes any one of the following: a predetermined bacterial alpha-diversity, a predetermined beta-diversity, a predetermined relative bacterial abundance; a predetermined bacterial load; or any combination thereof. In some embodiments, subjecting the bacteria to at least one compound changes - for example, decreases or increases - the similarity level of at least 30% at the end of the culture period.

[0050] In some embodiments, the co-culture and / or composition is enriched with bacteria from different sources.

[0051] In some embodiments, the method further includes the step of harvesting the cultured plurality of bacteria. In some embodiments, the harvested cultured plurality of bacteria includes at least 1·E 6 / 1gr, such as the bacterial load per 1gr of particles. In some embodiments, at least 1·E is obtained at the end of the culture period 6 / 1 gr, such as the bacterial load per 1 gr of particles. In some embodiments, the method further comprises the step of harvesting the co-cultured plurality of bacteria and optionally culturing and / or maintaining in the culture system - any additional microorganisms (referred to herein as "biological biomass" or "biological cells") that are, for example, attached and / or non-attached to the particles. In some embodiments, prior to harvesting, the biological biomass is separated from the growth medium or the cell culture fluid - for example, by centrifugation, filtration, and / or by allowing the biological biomass to precipitate within the culture vessel.

[0052] On the other hand, there is provided an in vitro method for evaluating: the effect of at least one compound on a plurality of bacteria; the effect of a plurality of bacteria on at least one compound; or both, the method comprising the steps of: providing a sample comprising a plurality of bacteria, the sample being contained within a container; contacting the plurality of bacteria with particles and causing at least a portion of the plurality of bacteria to attach to the particles; culturing the plurality of bacteria that are at least partially attached to the particles in a growth medium for a period of less than 14 days, wherein the culturing comprises culturing under anaerobic conditions to obtain a co-culture comprising the plurality of bacteria; adding at least one compound to the container; and determining any one of the following: (i) bacterial diversity; (ii) bacterial relative abundance; (iii) bacterial load; (iv) any other effect of the at least one compound on the plurality of bacteria; (v) any change in the structure of the at least one compound; and (vi) any combination of (i) to (v); thereby evaluating: the effect of at least one compound on a plurality of bacteria; the effect of a plurality of bacteria on at least one compound; or both.

[0053] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. In addition, these materials, methods, and examples are illustrative only and are not intended to be limiting necessarily.

[0054] Other embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1Including non-metric multidimensional scaling (NMDS), β-diversity (weighted Unifrac) plots that show the microbial diversity of the particle-attached bacterial fraction (marked as octagons), the non-attached bacterial fraction (marked as squares), and the planktonic culture samples (marked as triangles), all obtained simultaneously from the same origin sample (fecal sample; marked as plus). Sampling of the particle-attached bacterial fraction, non-attached fraction, and planktonic culture was performed several times. Each sampling is represented by a point; thus, each system shows several points.

[0056] Figure 2 Including vertical bar graphs that show the weighted Unifrac similarity and bacterial counts (qPCR measurement) of the composition at different time points (T1, T2, and T3) relative to a reference sample during cultivation in a medium containing a single carbon source (glucose) compared to cultivation in a multi-carbon source medium (glucose; maltose; trehalose; starch). The origin fecal sample was considered 100% (not shown on the graph).

[0057] Figure 3 Including vertical bar graphs that show the effect of supplementing trace elements to the glucose medium or multi-carbon medium on the similarity of the composition relative to the reference sample. The trace elements used were: a manganese source; a copper source; and an iron source. Sampling and evaluation of weighted Unifrac similarity and bacterial counts (based on qPCR measurement) were performed at three time points (T1, T2, and T3) during cultivation.

[0058] Figure 4 Including graphs that show the similarity level of the composition relative to the origin reference fecal sample ("origin"). The results are presented as principal coordinates analysis (PCoA; metric multidimensional scaling based on PCA, MDS) plots. Samples containing bacteria were cultivated under aerobic and anaerobic conditions in different media (e.g., brain heart infusion medium (BHI); or pharmaceutical-grade medium for human intestinal bacteria growth). There is an inner square around the anaerobic condition (the anaerobic condition is marked as AN).

[0059] Figure 5Including a vertical bar graph that shows the similarity levels and bacterial counts (presented as a line graph; secondary Y axis; measured based on quantitative PCR (qPCR)) of bacterial fecal samples cultured using different source culture medium substrates under anaerobic conditions. Cultured on a small scale (PG2) or medium-scale production (fermenter / bioreactor PG2) in a culture medium including: glucose; yeast extract; peptone extract; sodium chloride; disodium hydrogen phosphate; or cultured in BHI medium on a small scale. Different time points were detected, and the reference fecal sample was labeled "origin".

[0060] Figure 6 Including a vertical bar graph that shows the similarity and bacterial count levels of co-cultures produced using a multi-carbon source culture medium including: glucose; maltose; trehalose; starch; yeast extract; peptone extract; sodium chloride; disodium hydrogen phosphate; manganese source; copper source; and iron source according to an embodiment of the present invention. The results shown are the combined data of 3 processes using 3 origin samples from the same donor collected on different dates. These samples were independently cultured under medium-scale conditions (6L).

[0061] Figures 7 - 9 Including non-metric multi-dimensional scaling (NMDS) of Bray-Curtis similarity measures for fungal, viral, and bacterial communities present in a composition (labeled as squares) and reference stool samples (labeled as diamonds) produced according to an embodiment of the present invention at the species level. Community analysis was also performed on fecal samples from healthy volunteers (labeled as circles).

[0062] Figure 10 Including a scatter plot that indicates the relative proportion / relative abundance of metabolic pathways tested in a composition produced according to an embodiment of the present invention. The X axis represents the origin sample; while the Y axis represents the produced composition. Each point represents a metabolic super pathway, and its position is on one side of the gray dashed line y = x line, meaning that the pathway is enriched in one of the two comparison samples.

[0063] Figures 11A - 11BIncluding the growth preferences of two bacterial families for the specific phase of the composition (attached to particles or unattached). Each graph represents a different test bacterium. Each point on these graphs represents the difference (delta) between the relative abundance of the test bacterium in the attached phase and its phase abundance in the unattached phase in a sample obtained from the culture vessel at a specific time point. The skewing of these values towards a specific side of the graph indicates the preference of the bacterium for a certain phase. A - shows a preference of the bacterium for the attached part; while B - shows a preference of the bacterium for the unattached part.

[0064] Figures 12A - 12B Including graphs, each graph representing a different β - diversity: A - genus - level diversity recovery; and B - species - level diversity recovery. Each graph consists of three plots, which show the comparison between two specific phases. The upper first and second plots compare the combined phase with the unattached phase and the attached phase respectively. The lower third plot compares the unattached phase with the attached phase. Each point on the graph represents the difference between subsets of two selected test phases (attached, unattached, combined) in a sample obtained from the culture vessel at a specific time point. When a specific phase is compared with another phase, a higher metric value is observed through the skewing of the distribution from the zero value towards a specific side of the graph, and each side represents a different phase. The Wilcoxon Non - Parametric Sign Rank test is used to determine the statistically significant differences between each pair of test phases and is marked with an asterisk (*). The asterisk is shown on the side of significant enrichment.

[0065] Figure 13 Including a heatmap showing the relative abundance of Akkermansia. The data is depicted in colors, and the darker the color, the higher the relative abundance of the bacteria in the test sample or composition (starting from white - indicating the absence of bacteria; and up to 1: black, indicating the maximum relative abundance). The X - axis - represents the specific sampling time points of bacterial fermentation; while the Y - axis - represents the origin samples used for culturing. Single origins Ori1, Ori2, and Ori3. Pooled origins: Ori1 + Ori2 + Ori3; or Ori2 + Ori3.

[0066] Figure 14 Including a flowchart showing, by way of non - limiting example, the steps of the method disclosed herein in some of its embodiments.

[0067] Figure 15 Including a flowchart showing, by way of non - limiting example, the steps of the method disclosed herein in some of its embodiments.

[0068] Figure 16A flowchart including steps of the method disclosed herein in some of its embodiments shown by way of non - limiting examples.

[0069] Figure 17 Including non - metric multi - dimensional scaling (NMDS), β - diversity (Bray - Curtis) plots showing microbial diversity among four systems generated from four different fecal samples (from four different donors; labeled as donor 1 - donor 4); donor 1 provided samples at three different independent time points (labeled as experiment 1 - experiment 3); namely, several systems were generated simultaneously: four systems from four different donors; and another two systems were generated from experiment 1 and experiment 2 of donor 1 (a total of six in vitro culture systems). The particle - attached bacterial phase was sampled several times. Each sampling is shown by a point in the figure; thus, each system / donor has several points shown.

[0070] Figure 18 Including a box bar graph showing the average effect of compound_A on the bacterial counts (based on qPCR measurements) of four different systems generated from four fecal samples compared to systems based on untreated control microbiomes. Measurements were taken at three different time points (TP1 - TP3) during the culture. Note that the bacterial load results for all systems (four treated systems and four untreated systems - control systems) are shown on the same graph. The X - axis measures the number of genomic copies per 1 μl and relates to the bacterial count measurements by qPCR. This figure provides a non - limiting example of a method for evaluating the effect of a compound on multiple bacteria.

[0071] Figure 19 A - Figure 19 D includes a box bar graph showing the effect of compound_A on the α - diversity of four different systems generated from four different donors compared to systems based on untreated control microbiomes. Measurements were taken at three time points during the culture cycle of the particle - attached bacterial phase. The α - diversity of the pre - culture fecal samples is shown in each figure ("origin"). This figure provides a non - limiting example of a method for evaluating the effect of a compound on multiple bacteria.

[0072] Figure 20 A - Figure 20D includes a non-metric multidimensional scaling (NMDS) plot that shows the effect of Compound_A on the β-diversity (Bray-Curtis) of the microbial community compared to the control. Measurements were taken at three time points during the culture cycle of the particle-attached bacterial phase. Each plot shows the diversity of different systems that were generated from different donors at three consecutive time points during the culture cycle (all time points are shown on the same plot). This plot provides a non-limiting example of a method for evaluating the effect of a compound on multiple bacteria.

[0073] Figure 21 Includes a heatmap showing different bacterial genera affected by the exposure of multiple bacteria cultured in an in vitro system to Compound_A. Each experiment was conducted simultaneously in four treatment systems (Samples 1 - 4). The darker the color, the greater the change. Patterned colors indicate that the change due to exposure is a decrease, while non-patterned colors indicate that the change due to exposure is an increase. Analysis was performed compared to the untreated control bacterial population. The intensity of the color indicates the fold change level (see the indicator level in the right rectangle); the average color indicator (Av.) is also shown. A cross (×) indicates that the genus is not significant in the sample.

[0074] Figure 22 Includes a plot showing the effect of Compound_A and its derivative (Compound_A’) on the relative abundance of the bacterial population compared to the untreated bacterial population of the control system. The relative abundance is shown at the genus taxon level; and all systems were generated from the same fecal sample. Sampling was done at five time points. This plot provides a non-limiting example of a method for evaluating the effect of a compound on multiple bacteria.

[0075] Figure 23 Includes a plot showing the relative abundance of nine selected genera in the particle-attached bacterial fraction and the unattached bacterial fraction after exposure to Compound_D. The upper plot shows the treated fraction and the lower plot shows the relative abundance of the untreated system.

[0076] Figure 24 Includes a plot showing the relative abundance of four selected bacteria (labeled: "Bacteria 1 - Bacteria 4") in the generated control screening system and after the addition of Compound_B. The relative abundance of each bacteria in the whole bacterial population was measured in both the particle-attached bacteria ("attached") phase and the planktonic ("plankton") phase. Measurements were taken at one time point (T1) in the control system and two time points (labeled: T1 and T2) in the treated system. Detailed Description

[0077] Preparation and Use Methods

[0078] According to some embodiments, provided is a method for producing a composition, the composition comprising a co-culture, the co-culture comprising a plurality of bacteria grown in the presence of particles.

[0079] According to some embodiments, provided is a method for producing a composition, the composition comprising a co-culture, the co-culture comprising a plurality of bacteria and particles.

[0080] According to some embodiments, provided is a method for obtaining a composition characterized by or having an increased production of intestinal bacteria.

[0081] According to some embodiments, provided is an in vitro system / model (e.g., an in vitro gastrointestinal system) for evaluating the effect of at least one compound on the bacterial population and / or microbiome population of a subject; and / or the effect of the bacterial population and / or microbiome population of a subject on at least one compound.

[0082] According to some embodiments, provided is an in vitro system / model for optionally studying and / or modulating the physiology, metabolic activity, prebiotic properties, probiotic properties, or pathogenic properties of the flora of a subject after addition of at least one compound.

[0083] According to some embodiments, provided is an in vitro method for evaluating the mutual influence of at least one compound with a plurality of bacteria (e.g., the bacterial population of a subject, and / or the microbiome population).

[0084] According to some embodiments, provided is a method and system for culturing a bacterial population (e.g., a plurality of intestinal bacteria).

[0085] According to some embodiments, provided is a composition obtained by the methods and / or systems disclosed herein.

[0086] The methods, compositions, and in vitro systems / models according to the present invention have several advantages in some embodiments, as shown and detailed below. In some embodiments, the method according to the present invention includes co-culturing different bacteria in a single container (e.g., under the same culture conditions). In some embodiments, the method according to the present invention is capable of co-culturing different bacteria using a culture medium comprising pharmaceutical-grade ingredients (e.g., components suitable for human consumption). In some embodiments, the method according to the present invention is capable of producing a composite microbial composition and / or is capable of producing a composition comprising a co-culture, the co-culture comprising a plurality of bacteria grown in the presence of particles, comprising at least 30% similarity to the origin sample, and having at least 1·E 4 / 1gr particles (e.g., 1·E 6The bacterial load of (e.g., 1 gr particles). In some embodiments, where the origin sample is a fecal sample, a controlled industrial method for developing a composition that will obtain a preserved (e.g., of a healthy donor) gut microbiome profile can advantageously address the scalability, reproducibility, and safety issues in the preparation of fecal microbiota transplantation (FMT). In some embodiments, the in vitro culture system according to the present invention - which includes a culturing step with a growth or culture medium in the presence of particles as detailed herein - substantially maintains a high similarity (e.g., bacterial diversity and / or bacterial relative abundance) with the biological sample used to generate the system, and thus can be used as an in vitro model of the bacterial population and / or microbiome population of the mimic object. In some embodiments, the system according to the present invention mimics the microbiota of the object (e.g., the microbiota of the gastrointestinal tract), and thus is used to determine bacterial population regulation or changes - once exposed to, supplemented with, or in contact with at least one compound / element. In some embodiments, the methods and systems according to the present invention can be used as high-throughput screening assays, and simultaneously compare the effects of different treatments / compounds (e.g., different bacteria, different drug derivatives, different prebiotics, etc.) on the bacterial population and / or microbiome population of the object. In some embodiments, the methods and systems according to the present invention are capable of identifying regulated bacteria (at any taxonomic level) that can serve as therapeutic targets for enhancing the efficacy of drugs (e.g., through drug-probiotic or drug-prebiotic combination therapies aimed at restoring / preserving the original or healthier microbiome profile). In some embodiments, the methods and systems according to the present invention are capable of evaluating the effect of at least one compound on the microbiome of the object (e.g., any change in the chemical structure of the administered compound). In some embodiments, the methods and systems according to the present invention can be used as part of personalized medicine (e.g., by using the microbiota or microbiome of the object as the origin sample to generate the system), where the effect of a compound of interest (e.g., a drug) on a co-culture of multiple bacteria including the microbiota of the mimic object is examined. In some embodiments, using the system according to the method of the present invention provides reproducible results, and thus the effect of a compound on multiple bacteria that normally colonize the environmental niche and / or multiple bacteria of the microbiome of the mimic object under relevant environmental conditions can be accurately determined, regardless of the origin of the biological sample (e.g., the object) used to establish the model. In some embodiments and without wishing to be bound by any theory, for example, compared to a system that only includes planktonic bacteria (cultured in the presence of particles and / or in the absence of particles) or adherent forms of bacteria, the system according to the present invention - which includes both planktonic and adherent forms of bacteria - better represents the natural behavior of the bacterial population in the object. In some embodiments, the system is an independent system and thus is not affected by the complexity of other physiological and / or metabolic processes present in the human body.

[0087] In some embodiments, the phrase "bacteria grown in the presence of particles" refers to bacteria cultured in vitro in the presence of particles. In some embodiments, "a composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles" includes: a composition comprising particle-attached bacteria, e.g., substantially free of planktonic bacteria cultured in the presence of particles; a composition comprising planktonic bacteria cultured in the presence of particles, e.g., substantially free of bacteria attached to particles; and a composition comprising particle-attached bacteria and planktonic bacteria cultured in the presence of particles. In some embodiments, the method may include the step of separating particle-attached bacteria from the particles. In some embodiments, the composition may be supplemented with bacteria cultured in the absence of particles.

[0088] In some embodiments, culturing includes contacting the bacteria with the particles. In some embodiments, the bacteria contacting the particles are at least partially attached to the particles. In some embodiments, the bacteria contacting the particles are completely attached to the particles. In some embodiments, the bacteria contacting the particles are not attached to the particles. In some embodiments, the bacteria contacting the particles and not attached to the particles include planktonic bacteria.

[0089] In some embodiments, the term "population(s)" (e.g., bacterial population or bacterial community) with respect to microorganisms may be interchangeable with the term "community" and refers to two or more groups (e.g., two or more bacterial groups or microbial groups) that coexist symbiotically.

[0090] In some embodiments, the term "composition" includes the terms "in vitro system(s) or model(s)", "in vitro culture system(s) / model(s)", "in vitro biological system model", etc. In some embodiments, at least one compound may be added at any step of producing the composition.

[0091] In some embodiments, the phrase "better represents the natural behavior of the bacterial population in the subject" refers to a system of a bacterial population having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% and any range therebetween of similarity to the bacterial population in the subject.

[0092] In some embodiments, the phrase "better represents the natural behavior of bacterial populations in an object" refers to a system of bacterial populations that includes at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or even 100% and any range therebetween of similarity to the bacterial growth forms (e.g., planktonic state and / or adherent state) in the object. In some embodiments, the term "planktonic" includes bacteria in a non-adherent / non-attached state.

[0093] In some embodiments, a system or co-culture comprising the plurality of bacteria mimics the microbiota of an object. In some embodiments, the phrase "mimics the microbiota / microflora of an object" refers to a community of commensal bacteria and / or commensal microorganisms that replicates the relevant environmental conditions of the object's microbiome, e.g., exhibits similar in vivo functions, physiology, metabolic activity, and / or probiotic properties of the object's microflora. In some embodiments, the system mimics different regions of the gastrointestinal tract, e.g., with respect to environmental conditions such as pH levels, physical and / or chemical conditions, enzymes and their concentrations, etc.

[0094] As used herein, the terms "microbiome", "microflora" and "microbiota" are interchangeable and refer to a collection of microorganisms (including but not limited to bacteria, viruses, fungi (such as yeast)) found to reside in, constitute, or known to reside in an environmental niche. In some embodiments, the term "microbiome" also includes the structural components and / or metabolites / signal molecules of the microbial community; and / or the surrounding environmental conditions in the environmental niche. In some embodiments, the plurality of bacteria are co-cultured with a collection of microorganisms found or known to reside in the environmental niche of the object. Non-limiting examples of environmental niches include but are not limited to: intestine; skin; eye; bronchus; mouth (e.g., saliva); upper respiratory tract; urogenital tract; and vaginal tissue, to name a few. In some embodiments, the term "microbiota" refers to a bacterial population, e.g., excluding non-bacterial populations.

[0095] In some embodiments, the plurality of bacteria used in accordance with the present invention are derived from an origin sample. As used herein, the term "derived" may be interchangeable with the terms "originated" and "obtained", and refers to the source from which the plurality of bacteria for culturing / fermentation are obtained. In some embodiments, the origin sample includes a collection of microorganisms (e.g., bacteria, viruses, fungi (such as yeast)) found to reside in, constitute, or known to reside in an environmental niche. In some embodiments, the origin sample includes bacteria and further includes additional microorganisms, e.g., at least one taxonomic group of any one of archaea, viruses, fungi, or any combination thereof. In some embodiments, prior to culturing, the plurality of bacteria are isolated from the source / origin, e.g., separated from other microorganisms or microbes present in the sample. In some embodiments, the other microorganisms or microbes are co-cultured and / or maintained with the plurality of bacteria in a culture system. In some embodiments, the term "virus" is or includes a bacteriophage.

[0096] In some embodiments, the term "culturing" includes the term "fermentation" and refers to the in vitro maintenance, proliferation, and / or growth of microorganisms (e.g., bacteria) in various buffers and / or media under laboratory or industrial conditions. Suitable media can be selected by those skilled in the art, and examples of such media include, but are not limited to, YCFA (Yeast Casitone Fatty Acid), BHI (Brain Heart Infusion), GAM (Gifu Anaerobic Medium), TSB (Tryptic Soy Broth), TYG (Tryptic Yeast Extract Glucose), FAB (Fastidious Anaerobic Broth), etc. In some embodiments, the medium includes pharmaceutical-grade ingredients. In some embodiments, the medium includes food-grade ingredients. In some embodiments, the medium includes components suitable for veterinary use. The term "fermentation" has its ordinary meaning in the art. In some embodiments, the term "fermentation" is used herein to refer to a microbial metabolic process, including the conversion of sugar(s) into acid and / or gas using, for example, bacteria.

[0097] In some embodiments, the collection of additional microorganisms or microbes present in the origin sample is co-cultured with the plurality of bacteria. In some embodiments, the other microorganisms or microbes are added or mixed into the culture system separately. In some embodiments, any one of archaea, viruses, and / or fungi is maintained in the co-culture, e.g., preserved and / or present, without any change in its quantity and / or the relative abundance of its taxonomic groups relative to the origin sample. In some embodiments, "preserved and / or present" refers to a population similar to the origin sample as defined herein. In some embodiments, the term "virus" includes a bacteriophage.

[0098] In some embodiments, as used herein, the term "co-culture" or "co-cultured" refers to the maintenance, proliferation, and / or growth of the plurality of bacteria and optionally one or more of the additional microorganisms or microbes in the culture system described herein.

[0099] In some embodiments, the origin sample comprising the plurality of bacteria is similar to the microbiota of the subject (such a sample is referred to herein as a "similar sample"), e.g., a healthy subject or a subject suffering from a disease, disorder, or condition such as dysbiosis. In some embodiments, a "similar sample" is a sample that includes at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or even 100% identity or similarity to the microbiota of the subject and / or to the plurality of bacteria derived from / originating from the subject (e.g., as measured by alpha- or beta-bacterial diversity; bacterial relative abundance; and / or bacterial load). In some embodiments, a "similar sample" is generated by mixing, e.g., bacteria (e.g., single strains and / or multiple bacteria and / or co-cultured bacteria) in the environmental niches of the subject (e.g., in the intestine, eye, mouth, skin, bronchi, vagina, upper respiratory tract, urogenital tract, etc.) based on a desired microbiome profile. In some embodiments, a "similar sample" further includes mixing other microbes, e.g., archaea, viruses, fungi, or any combination thereof.

[0100] In some embodiments, the origin sample includes a predetermined target or desired microbial population, e.g., a bacterial population. In some embodiments, the origin sample is used "as is" or as a starting material for the methods described herein after being processed. In some embodiments, the processing of the sample includes any one of the following: dilution, e.g., dilution with a buffer, a medium, or a combination thereof; homogenization; partial or complete removal of non-flora substances, coarse particulate matter, fibers, or any combination thereof; and / or any other method known in the field of sample processing (e.g., fecal sample processing). In some embodiments, the composition is characterized by having at least 30% similarity to the predetermined target population.

[0101] In some embodiments, the origin sample is a synthetic sample. In some embodiments, the origin sample is a biological sample. In some embodiments, the plurality of bacteria are derived from at least one origin. In some embodiments, the plurality of bacteria are derived from a combination of at least 2, at least 3, at least 4, at least 5 or more origins / sources. In some embodiments, the plurality of bacteria are sampled from or derived from a donor (such as a human subject) having a desired microbial population in the sampling environment. In some embodiments, the sample is derived from a healthy subject, a non-healthy subject, and / or a subject suffering from dysbiosis (e.g., in a certain environmental niche). In some embodiments, the sample is derived from more than one bacterial source. In some embodiments, the plurality of bacteria are derived from at least one environment. In some embodiments, the plurality of bacteria are derived from a sample. In some embodiments, the plurality of bacteria are at least partially derived from a sample. In some embodiments, the sample, composition, and / or co-culture is enriched with bacteria and optionally other microorganisms from different sources or origins.

[0102] In some embodiments, the term "synthetic sample" refers to a sample comprising a bacterial community that is artificially created by combining / mixing selected (two or more) bacterial species - e.g., which may typically colonize a predetermined environmental niche. In some embodiments, the synthetic sample is or comprises a selected bacterial group at any taxonomic level based on the relative abundance of the bacterial group in the desired microbiota of different subjects and / or different environmental niches.

[0103] In some embodiments, the sample used in the method or system according to the invention is or comprises any one of the following: stored microbiota, stored microbiome sample, a plurality of bacterial populations, bacterial colonies, particle-attached bacteria, and / or planktonic bacteria, or any combination thereof. In some embodiments, the sample is frozen or lyophilized before being used in the method or system according to the invention. In some embodiments, the sample is stored at a temperature below 8°C (e.g., in the temperature range of 2 - 8°C).

[0104] In some embodiments, the method according to the invention comprises the step of providing a composition comprising a co-culture, the co-culture comprising a plurality of bacteria produced according to the invention (e.g., in frozen or lyophilized form), which is used, for example, in an evaluation method and / or as a starting material for producing a composition according to the invention.

[0105] In some embodiments, the plurality of bacteria is or comprises at least one bacterial population selected from the group consisting of: fecal bacterial population, intestinal bacterial population, ocular bacterial population, oral bacterial population (e.g., salivary bacterial population), skin bacterial population, bronchial bacterial population, vaginal bacterial population, upper respiratory bacterial population, urogenital bacterial population, or any combination thereof. In some embodiments, the plurality of bacteria is or comprises at least one bacterial population selected from the group consisting of: soil bacterial population, groundwater bacterial population, open water bacterial population, or any combination thereof. In some embodiments, the plurality of bacteria is or comprises an intestinal microbiota.

[0106] In some embodiments, the origin sample comprising the plurality of bacteria is derived from a subject, e.g., from a fecal sample, oral sample (e.g., saliva sample), skin sample, ocular sample, bronchial sample, vaginal sample, or any combination thereof. In some embodiments, the sample comprising the plurality of bacteria is derived from at least 1, at least 2, at least 3, at least 4, at least 5, or more origins, e.g., the sample is a pool derived from several subjects.

[0107] In some embodiments, a microbiota (e.g., a bacterial population) is isolated from non-microbiota material. In some embodiments, where the source is a fecal sample, the fecal microbiota is at least partially separated from the non-microbiota material, coarse particulate material, and / or fibers of the fecal material. The separation can be carried out, for example, by homogenization, centrifugation, filtration, and / or by any other method known to those skilled in the art. In some embodiments, the fecal sample used in the method according to the invention comprises fibers originating from feces. In some embodiments, the bacterial population is at least partially separated from undigested particles or material.

[0108] In some embodiments, the sample comprises a soil sample.

[0109] In some embodiments, the sample is derived from a plant (s).

[0110] As used herein, the term "plurality" means any integer equal to or greater than 2. In some embodiments, the plurality of bacteria comprises at least two different bacteria. In another embodiment, the plurality of bacteria comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 or more different bacteria. In some embodiments, the plurality of bacteria is from one or more taxonomic classifications.

[0111] In some embodiments, the term "multiple bacteria" refers to a population of bacteria that includes at least two different strains or bacterial species. In some embodiments, the bacterial population includes from two different types of bacteria up to one thousand different types of bacteria, or more.

[0112] In some embodiments, the term "different" refers to bacteria characterized by having different growth, culture, and / or proliferation conditions. In one embodiment, the phrase "bacteria characterized by having different growth, culture, and / or proliferation conditions" refers to bacteria that require different optimal growth, culture, and / or proliferation conditions. In some embodiments, the different growth, culture, and / or proliferation conditions are at least two optimal conditions, where the first condition allows for substantially optimal growth, culture, and / or proliferation of a first bacterium, and the second condition allows for substantially optimal growth, culture, and / or proliferation of a second bacterium.

[0113] In some embodiments, the different growth, culture, and / or proliferation conditions include any of the following: metabolic requirements, nutritional requirements, pH, temperature, aerobic, obligate anaerobic, facultative anaerobic, microaerophilic, attached form, planktonic, growth medium, flow, shaking, stirring, agitation, static, moisture, low humidity, and any combination thereof. In some embodiments, the growth, culture, and / or proliferation conditions include at least one obligate anaerobic bacterium and at least one facultative anaerobic bacterium. In some embodiments, the different growth, culture, and / or proliferation conditions include at least two bacterial populations selected from the following: obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles.

[0114] In some embodiments, the different growth, culture, and / or proliferation conditions are biological parameters selected from the following: metabolic requirements, nutritional requirements, growth medium, and any combination thereof.

[0115] In some embodiments, the different growth, culture, and / or proliferation conditions are metabolic requirements selected from the following: aerobic, obligate anaerobic, facultative anaerobic, microaerophilic, and any combination thereof.

[0116] In some embodiments, the different growth, culture, and / or proliferation conditions are physical parameters selected from the following: temperature, moisture, low humidity, flow, shaking, stirring, agitation, static, and any combination thereof.

[0117] In some embodiments, the different growth, culture, and / or proliferation conditions are chemical parameters selected from the following: pH, redox potential, gas composition, dissolved gases, and any combination thereof.

[0118] In some embodiments, the different growth, culture, and / or proliferation conditions are fermentation techniques selected from the following: fed-batch, semi-batch, batch, continuous, and any combination thereof.

[0119] In some embodiments, the different growth, cultivation, and / or proliferation conditions are bacterial growth forms selected from the following: planktonic, adherent forms, and any combination thereof.

[0120] In some embodiments, the term "metabolic requirements" includes any of the following: a source of carbon and energy, a source of essential elements (e.g., H, O, N), macronutrients, micronutrients, vitamins, hormones, growth factors, CO2 levels, oxygen levels, light, metabolic precursors or substrates, or any combination thereof.

[0121] In some embodiments, the composition is produced by a method comprising: providing a plurality of bacteria derived from an origin sample; contacting the plurality of bacteria with particles and causing at least a portion of the plurality of bacteria to adhere to the particles; and culturing the plurality of bacteria at least partially adhered to the particles in a growth medium for a period of less than 14 days. In some embodiments, the culturing comprises culturing under anaerobic conditions. In some embodiments, the culturing comprises culturing under aerobic conditions.

[0122] In some embodiments, the composition is produced by a method comprising: providing a plurality of bacteria derived from an origin sample; culturing the plurality of bacteria in a growth medium for a period of less than 14 days, the culturing comprising culturing under anaerobic conditions.

[0123] In some embodiments, the composition is produced by a method comprising: providing a plurality of bacteria derived from an origin sample; culturing the plurality of bacteria in a growth medium for a period of less than 14 days, the culturing comprising culturing under aerobic conditions.

[0124] In some embodiments, the growth medium comprises a carbon source and a nitrogen source in a molar / molar ratio / atom / atom ratio ranging from 50:1 to 1:50, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the carbon-nitrogen ratio or the ratio between the carbon types represented (w / w or m / m) is based on the dry form.

[0125] In some embodiments, the phrases "providing a plurality of bacteria derived from an origin sample" and "providing a sample comprising a plurality of bacteria" are interchangeable.

[0126] In some embodiments, the plurality of bacteria or a sample comprising them is provided within a container, and the method further comprises the steps of: adding at least one compound to the container; and determining any one of the following: (i) bacterial diversity; (ii) bacterial relative abundance; (iii) bacterial load; (iv) any other effect of the at least one compound on the plurality of bacteria; (v) any change (e.g., structural) of the at least one compound; and (vi) any combination of (i) to (v); thereby evaluating: the effect of at least one compound on the plurality of bacteria; the effect of the plurality of bacteria on at least one compound; or both.

[0127] In some embodiments, the in vitro evaluation methods and other uses according to the invention as defined herein can be implemented by using any one of the systems / compositions described herein (e.g., in the presence of particles, in the absence of particles, under culture conditions including anaerobic conditions, under culture conditions including aerobic conditions, or any combination thereof). In some embodiments, the evaluation can be carried out in a system comprising particles, in a system without particles, and / or in both systems, for example, as detailed above and below. In some embodiments, the compound(s) can be added before and / or after adding the particles to the culture system, for example, as detailed above and below. The evaluation and / or determination steps can be carried out in any bacterial part / phase produced according to the invention.

[0128] The phrase "contacting the plurality of bacteria with the particles" is used herein in its broadest sense and refers to any type of combined action - for example, bringing the plurality of bacteria into proximity with the particles such that the bacteria can attach to / adhere to the particles. In some embodiments, the contact is carried out in solution. In some embodiments, the contact comprises combining the bacteria, the particles, and the solution in any order, any combination, and / or sub-combination, including premixing any two of these elements before adding the third element. For example, the bacteria are inoculated into a solution and then cultured in a solution comprising particles. In some embodiments, a particle blend is prepared in solution as in the previous step and then combined with the plurality of bacteria.

[0129] In some embodiments, the contact is carried out in a solution selected from saline, phosphate buffered saline, growth medium, or any combination thereof. In some embodiments, the contact comprises inoculating a liquid (e.g., buffer, medium, or a combination thereof) containing particles with the plurality of bacteria; and incubating the particles and the plurality of bacteria together for a time sufficient for the plurality of bacteria to at least partially attach to the particles. In some embodiments, the contact is or comprises culturing the bacteria in the presence of the particles. In some embodiments, the contact and the culturing are carried out simultaneously or as a subsequent step.

[0130] In some embodiments, the time sufficient for at least a portion of the plurality of bacteria to attach or adhere to the particles is from 2 hours to 48 hours. In some embodiments, the time range sufficient for at least a portion of the plurality of bacteria to attach / adhere to the particles is: 2 hours to 12 hours, 2 hours to 24 hours, 6 hours to 24 hours, 10 hours to 24 hours, 15 hours to 20 hours, 20 hours to 40 hours, 6 hours to 40 hours, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0131] In some embodiments, the cultivation includes fed-batch cultivation or semi-fed-batch cultivation. For example, some or all nutrients are provided to the culture during the cultivation process. In some embodiments, the fed-batch culture includes fixed-volume fed-batch, variable-volume fed-batch, or any combination thereof.

[0132] As used herein, the term "fed-batch cultivation" refers to a cultivation method in which components, such as nutrients (e.g., carbon sources and / or nitrogen sources), are provided to the culture at at least one time point after the start of the cultivation / cultivation process. In some embodiments, the cultivation is batch cultivation, e.g., nutrients are provided at the start of the cultivation process. In some embodiments, the cultivation is a combination of fed-batch cultivation and batch cultivation. For example, one nutrient is provided at the start of the cultivation, and another nutrient is provided during the cultivation process.

[0133] In some embodiments, the growth medium includes one or more nitrogen sources. In some embodiments, the nitrogen source is or includes at least one of the following: yeast extract, peptone, yeast peptone, enriched yeast peptone, casein, guar gum peptone, synthetic amino acid medium, wheat peptone, potato peptone, ammonium salts (multiple salts, such as ammonium carbonate, ammonium chloride, and ammonium nitrate), or any combination thereof.

[0134] In some embodiments, the cultivation is or includes cultivation under anaerobic conditions.

[0135] In some embodiments, the term "anaerobic conditions" refers to conditions in which the free oxygen is less than 500 ppm, 450 ppm, 400 ppm, 350 ppm, 300 ppm, 250 ppm, 200 ppm, 150 ppm, 100 ppm, 50 ppm, or 10 ppm, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, anaerobic conditions include conditions where no free oxygen is provided. In some embodiments, anaerobic conditions include conditions lacking free oxygen.

[0136] In some embodiments, the term "aerobic conditions" refers to conditions that include the presence of molecular oxygen. In some embodiments, the oxygen concentration is above 20% (v / v of the total gas present during the culture).

[0137] In some embodiments, the method for producing a composition according to the present invention comprises the step of in vitro culturing a complex bacterial population characterized by having different growth, culturing, and / or proliferation conditions, thereby producing a composition comprising a co-culture containing the complex bacterial population.

[0138] In some embodiments, the produced composition or co-culture includes at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% similarity to the origin sample, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0139] In some embodiments, the bacterial load of the produced composition or co-culture is at least 1·E 4 to at least 1·E 9 range. In some embodiments, the produced composition or co-culture includes or is characterized by having the following bacterial loads: at least 1·E 4 / 1 gr of particles, at least 1·E 5 / 1 gr of particles, at least 1·E 6 / 1 gr of particles, at least 1·E 7 / 1 gr of particles, at least 1·E 8 / 1 gr of particles, at least 1·E 9 / 1 gr of particles, at least 1·E 10 / 1 gr of particles, at least 1·E 11 / 1 gr of particles, at least 1·E 12 / 1 gr of particles, at least 1·E 14 / 1 gr of particles, at least 1·E 16 / 1 gr of particles or at least 1·E 20 / 1 gr of particles, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0140] In some embodiments, the culturing is carried out until the co-culture has a similarity of greater than or equal to 30% to the origin sample, and the bacterial load is in the range of 1·E 4 to 1·E 9 per 1 gr of particles.

[0141] In some embodiments, a culture period of less than 14 days includes: up to 13 days, up to 12 days, up to 11 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 1 day, up to 5 hours, up to 3 hours, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0142] In some embodiments, a culture period of fewer than 14 days includes: 1 to 13 days, 2 to 13 days, 5 to 13 days, 2 to 10 days, 6 to 12 days, 4 to 11 days, 8 to 13 days, 2 to 9 days, 2 to 5 days, 10 hours to 4 days, 12 hours to 48 hours, 5 hours to 6 days, 6 hours to 5 days, 6 hours to 14 days, 3 hours to 6 days, 3 hours to 5 days, 12 hours to 24 hours, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0143] In some embodiments, the method includes a culture period ranging from 6 hours to 6 days, thereby producing a composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles, the co-culture comprising: i) at least 50% similarity to the origin sample, and ii) at least 1·E 6 / 1gr bacterial load of the particles.

[0144] In some embodiments, the method includes culturing for a period ranging from 12 hours to 5 days in a growth medium comprising at least two types of carbon sources selected from monosaccharides, disaccharides, and polysaccharides, thereby producing a composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles, the co-culture comprising: i) at least 70% weighted Unifrac similarity to the origin sample, and ii) at least 1·E 8 / 1gr bacterial load of the particles.

[0145] In some embodiments, culturing includes culturing under any of the following conditions: static, flow, stirring, shaking, agitating, or any combination thereof. In some embodiments, culturing includes stirring the plurality of bacteria, for example, at 50 to 750 revolutions per minute (RPM), 50 to 650 RPM, 100 to 750 RPM, 100 to 700 RPM, 150 to 700 RPM, 200 to 750 RPM, 130 to 690 RPM, 90 to 720 RPM, 70 to 550 RPM, 110 to 710 RPM, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0146] In some embodiments, the terms "flowing", "agitating", "shaking", and "stirring" refer to conditions that cause movement or motion of the liquid phase within the culture vessel. In some embodiments, the movement is axial, radial, mixed, distributive, or any combination thereof. The movement or motion can be effected by using mechanical means (e.g., impellers, moving platforms, oscillators, shakers); manually; automatically, or any combination thereof. In some embodiments, the term "static conditions" refers to conditions in which no agitation or any other movement action (manually, automatically, and / or mechanically) is performed on the liquid phase.

[0147] In some embodiments, culturing comprises subjecting the plurality of bacteria to a temperature within the range of 32 - 39 °C, 32 - 38 °C, 33 - 38 °C, 34 - 38 °C, 35 - 38 °C, 36 - 38 °C, or 37 - 38 °C, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0148] In some embodiments, culturing comprises subjecting the plurality of bacteria to a pH within the range of 3.0 - 9.0 or within the range of 4.0 - 8.0, e.g., 3.0, 4.0, 5.0, 6.0, 6.2, 6.3, 6.4, 6.5, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.0, 7.1, 7.2, 8.0, 9.0, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0149] In some embodiments, culturing comprises subjecting the plurality of bacteria to a pH of 6.2 to 7.2, 6.2 to 7.1, 6.2 to 7.0, 6.3 to 7.2, 6.4 to 7.2, 6.5 to 7.1, 6.6 to 7.2, or 6.8 to 7.2, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0150] In some embodiments, prior to culturing the plurality of bacteria, the sample is diluted at a weight / volume (w / v) ratio within the range of 1:1 to 1:300 (such as but not limited to 1:5 to 1:60, 1:5 to 1:50, 1:10 to 1:25, 1:7 to 1:28, 1:9 to 1:30, 1:15 to 1:20, 1:6 to 1:24, 1:12 to 1:26, or 1:20 to 1:30, or any value and range therebetween) (e.g., with a buffer, a culture medium, or a combination thereof). Each possibility represents a separate embodiment of the invention.

[0151] In some embodiments, the growth medium comprises one type of carbon source. In some embodiments, the medium comprises at least two types of carbon sources. In some embodiments, the medium comprises carbon sources selected from at least two groups of monosaccharides, disaccharides, and polysaccharides. In some embodiments, the growth medium further comprises at least one monosaccharide, at least one disaccharide, and at least one polysaccharide.

[0152] In some embodiments, the at least one monosaccharide, the at least one disaccharide, and the at least one polysaccharide are present in the growth medium in the following weight / weight / weight (w / w / w) ratios: 1:0:0, 1:1:0, 0:1:1, 0:1:0, 0:0:1, 1:0:1, 1:1:1, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0153] In some embodiments, the at least one monosaccharide, the at least one disaccharide, and the at least one polysaccharide are present in the growth medium in a weight / weight / weight (w / w / w) ratio of X:Y:Z, where the range of any one of the following is within the range of 0 to 10 (or any value and range therebetween): X and Y; X and Z; and / or Y and Z. Each possibility represents a separate embodiment of the present invention.

[0154] In some embodiments, the monosaccharide is selected from: glucose (dextrose), fructose (levulose), galactose, or any combination thereof.

[0155] In some embodiments, the disaccharide is selected from: sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, isomaltose, nigrosin, maltulose, mannotriose, xylobiose, or any combination thereof.

[0156] In some embodiments, the term "polysaccharide" encompasses any polymer of carbohydrates composed of monosaccharide units linked to each other by glycosidic bonds. In some embodiments, the polysaccharide is selected from: alginates, starch, cellulose, pectin, arabinoxylans, glycogen, galactan, inulin, or any combination thereof. In some embodiments, the polysaccharide comprises synthetic polysaccharides. In some embodiments, synthetic polysaccharides encompass any non-naturally occurring polysaccharides.

[0157] In some embodiments, the growth medium further comprises trace elements selected from: iron source, zinc source, copper source, manganese source, selenium, iodine, fluorine source, molybdenum source, cobalt source, chromium source, nickel source, their soluble salts, or any combination thereof.

[0158] In some embodiments, the method further comprises the step of substantially separating bacteria not attached to the particles from bacteria attached to the particles (e.g., by filtration, washing, and / or vortexing), thereby producing: (i) a composition comprising bacteria in a planktonic form; and / or (ii) a composition comprising bacteria attached to particles. In some embodiments, the method further comprises the step of substantially removing bacteria not attached / non-adherent to the particle(s) from the culture system and / or from the growth medium (e.g., by filtration, washing, and / or vortexing). In some embodiments, the non-attached / non-adherent bacteria are or comprise planktonic bacteria. In some embodiments, the non-attached bacteria are devoid of sessile bacteria. In some embodiments, the separation or removal is performed at least once at a time point selected from: before and / or during the culturing step. In some embodiments, the separation or removal is performed at the end of the culturing step.

[0159] In some embodiments, the term "substantially separated" as used herein means that the non-attached bacteria are less than 50% (e.g., less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less, or any value and range therebetween) in a composition comprising particle-attached bacteria and / or the particle-attached bacteria are less than 50% (e.g., less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less, or any value and range therebetween) in a composition comprising non-attached bacteria.

[0160] In some embodiments, the phrase "substantially removing bacteria not attached / non-adherent to the particle(s)" means removing at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the non-attached / non-adherent bacteria present in the co-culture, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0161] In some embodiments, the evaluation method is implemented in a system using bacteria in a planktonic form according to the present invention. In some embodiments, the evaluation method is initially implemented in a system comprising bacteria in a planktonic form - e.g., before the contacting step and / or before the plurality of bacteria attach to the particles. In some embodiments, after at least partial attachment (e.g., during the culturing step), the method is implemented in a system comprising any one of: particle-attached bacteria; bacteria in a planktonic form; or both.

[0162] In some embodiments, the method further comprises the steps of: separating the plurality of bacteria cultured in the presence of the particles into different culture systems to produce: i) a culture system comprising planktonic bacteria initially cultured in the presence of the particles; and ii) a culture system comprising bacteria attached to the particles. In some such embodiments, the compound can be added to any of the following: the culture system prior to the separation step; system (i); system (ii); or any combination thereof. Evaluation can be carried out separately in each system to which the compound has been added.

[0163] In some embodiments, after at least partial attachment, the culture comprising the plurality of bacteria can be divided into several containers, e.g., for evaluating different compounds / conditions. In some embodiments, the plurality of bacteria are cultured under the same culture conditions.

[0164] In some embodiments, the contacting step, the culturing step, or both are carried out within a container. In some embodiments, the contacting step, the culturing step, or both are carried out in a single / one container, interconnected containers, or communicating containers.

[0165] In some embodiments, the container comprises any compartment that is compatible with or configured to culture and / or maintain the growth, proliferation, activity, and / or viability of bacteria.

[0166] In some embodiments, the term "container" refers to any receptacle in which bacteria can be cultured using conventional fermentation techniques, e.g., bioreactor(s), flask(s), test tube(s), microtiter dish(es), well plate(s), multi-well plate assembly(ies), petri dish(es), etc.

[0167] In some embodiments, the contacting step, the culturing step, and / or the adding step are carried out in a sequential manner in a single / one container.

[0168] In some embodiments, the container comprises interconnected containers or communicating containers. In some embodiments, the terms "interconnected containers" and "communicating containers" refer to a plurality of compartments or containers that comprise a homogeneous liquid that is sufficiently connected below the upper surface of the liquid.

[0169] In some embodiments, the in vitro model simulates different regions in the gastrointestinal (GI) tract. In some such embodiments, several single containers - each including an independent system - can be positioned in a sequential manner and interconnected, for example, by tubes or conduits that are alternately positioned above the upper surface of a liquid and below the lower surface of the liquid. Each container can represent the environmental conditions of the gastrointestinal tract. The GI motility and / or functionality can be simulated, for example, by using mechanical means. In some embodiments, liquids and / or gases are transferred through the interconnected tubes or conduits.

[0170] In some embodiments, during culturing, the plurality of bacteria are not separated into different containers. In some embodiments, the method is performed in a plurality of containers. In some embodiments, the plurality of bacteria are separated into different containers before and / or during culturing, for example, for simultaneously evaluating the effect of a compound on the plurality of bacteria. In some embodiments, the plurality of bacteria are cultured in different containers under the same culturing conditions. In some embodiments, the culturing conditions are selected from: biological parameters (e.g., metabolic requirements, nutritional requirements, growth medium); metabolic requirements (e.g., aerobic, obligate anaerobic, facultative anaerobic, microaerophilic); physical parameters (e.g., temperature, moisture, low humidity, flow, shaking, stirring, agitation, static); chemical parameters (e.g., pH, redox potential, gas composition, dissolved gases); fermentation techniques (e.g., fed-batch, semi-batch, batch, continuous); bacterial growth forms (e.g., planktonic, attached form), and any combination thereof.

[0171] In some embodiments, the evaluation method comprises the following steps: (a) providing a fecal sample of a subject; optionally, diluting the fecal sample (e.g., at a ratio ranging from 1:5 (g / ml) to 1:200 (g / ml)), homogenizing, and filtering the diluted sample; (b) inoculating particles with the fecal sample of step (a) at a ratio, for example, ranging from 1:2 (g / ml) to 1:10 (g / ml), and incubating or culturing the particles inoculated with the fecal sample for a period of less than 14 days, thereby producing a co-culture comprising a plurality of bacteria; (c) contacting the bacterial population with at least one compound of interest; and (d) determining any one of the following: (i) bacterial diversity; (ii) bacterial relative abundance; (iii) bacterial load; (iv) the effect of the at least one compound on the plurality of bacteria; (v) any change (e.g., chemical and / or structural change) of the at least one compound, and (vi) any combination of (i) to (v). In some embodiments, the dilution is performed in a buffer, a medium, or a combination thereof.

[0172] In some embodiments, a pooled sample derived from or obtained from more than one origin is used as part of an assay method or system to determine whether a compound of interest is suitable for treatment in a subject in need thereof. As a non-limiting example, at least two bacterial populations representing different sources (e.g., gut and skin) can be used to test the effect of a compound of interest and to determine whether the compound of interest is suitable for treating a disease or disorder associated with the first microbial population - provided that it does not alter the second microbial population. The following is an exemplary assay, according to which a compound that is identified as altering not only a healthy gut microbiota or correcting an altered gut microbiota, but also a healthy skin microbiota, is determined to be unsuitable for treating a gut-related disease or disorder. Optionally, a compound that is identified as correcting an altered gut microbiota without altering a healthy skin microbiota is determined to be suitable for treating a gut-related disease or disorder.

[0173] In some embodiments, as part of an assay method or culture system, samples of different origins (e.g., from different donors, from the same donor but from different niches, aggregates of origin samples mixed in different proportions, or any combination thereof) are cultured in different containers under the various conditions of the present application, e.g., to compare the effects of a compound on samples of different origins.

[0174] Reference Figure 14, which describes non-limiting examples of the settings of the method of the present invention. First, a sample comprising a plurality of bacteria is provided (e.g., a microbial population comprising a sample originating from at least one subject or originating population) (step 200); subsequently, the sample can be diluted (step 220). The sample can be diluted in any solution (such as PBS) and / or any suitable culture medium that still maintains the natural environment of the bacteria and is suitable for growth. The dilution can be carried out within any suitable range for the preservation and / or growth of bacteria, such as 1 gr sample: 1 mL solution - 1 gr sample - 10 L solution; 1 gr sample: 10 mL solution - 1 gr sample - 5 L solution; or 1 gr sample: 10 mL solution - 1 gr sample - 1 L solution. Then, the sample or the diluted sample is cultured with the particle or particles under conditions that cause at least a portion of the bacteria to attach to the particle or particles (step 240). Subsequently, the bacterial population that is at least partially attached to the particle or particles is contacted with the compound of interest for a period of time and under conditions sufficient for the compound of interest to exert its activity on the bacteria (step 260). Subsequently, the effect of the compound of interest on the bacteria is determined (step 280). As determined in step 280, the output of the effect of the compound of interest on the bacteria can be presented as bacterial load, bacterial diversity, bacterial relative abundance, bacterial abundance, and / or others (e.g., bacterial viability, ratio of particle-attached or adhered bacteria to planktonic bacteria, gene expression profile, toxin production, etc.). In addition, or alternatively, the effect of the bacterial population on the compound can be investigated by determining the change in the concentration of the compound of interest (e.g., due to consumption and / or degradation of the compound) and / or any change in its structure - for example, by metabolomics, analytical chemistry, biochemistry, genomics, or by any other suitable method known in the art.

[0175] Reference Figure 15, which describes a non - limiting example of the setup of the method of the present invention. First, an origin sample comprising a plurality of bacteria (e.g., a microbial population comprising a sample originating from at least one subject or origin population) is provided (step 200); subsequently, the sample can be diluted and contacted with a molecule of interest (steps 220 + 260). As disclosed herein, the sample can be diluted in any solution suitable for bacterial preservation and / or growth and subjected to a time and conditions sufficient for the compound of interest to exert its activity on the bacterial population. Then, the sample or the diluted sample is cultured with the particle or the plurality of particles under conditions that allow the bacteria to at least partially attach to the particle or particles (step 240). Subsequently, the effect of the compound of interest on the bacteria is determined (step 280). The output of the effect of the compound of interest on the bacteria, as determined in step 280, can be presented as bacterial load, bacterial diversity, bacterial relative abundance, bacterial abundance, and / or others (e.g., bacterial viability, ratio of particle - attached or adherent bacteria to planktonic bacteria, gene expression profile, toxin production, etc.). Additionally, or alternatively, the effect of the bacterial population on the compound can be examined by determining any change in the concentration and / or structure of the compound of interest - for example, by metabolomics, analytical chemistry, biochemistry, genomics, or by any other suitable method known in the art.

[0176] Reference Figure 16 , which describes a non - limiting example of the setup of the method of the present invention. First, a sample comprising a plurality of bacteria (e.g., a microbial population comprising a sample originating from at least one subject or origin population) is provided (step 200); subsequently, the sample can be diluted (step 220). As described herein, the sample can be diluted in any solution suitable for preservation and / or growth. Then the sample or the diluted sample is cultured with a particle or a plurality of particles under conditions that allow the bacteria to at least partially attach to the particle(s), and is contacted with the compound of interest substantially simultaneously. The bacteria and the compound can be contacted for a period of time under conditions sufficient for the compound of interest to exert its activity on the bacterial population (steps 240 + 260). Subsequently, the effect of the compound of interest on the bacteria is determined (step 280). The output of the effect of the compound of interest on the bacteria, as determined in step 280, can be presented as bacterial load, bacterial diversity, bacterial relative abundance, bacterial abundance, and / or others (e.g., bacterial viability, ratio of particle - attached or adherent bacteria to planktonic bacteria, gene expression profile, toxin production, etc.). Additionally, or alternatively, the effect of the bacterial population on the compound can be examined by determining any change in the concentration and / or structure of the compound of interest - for example, by metabolomics, analytical chemistry, biochemistry, genomics, or by any other suitable method known in the art.

[0177] In some embodiments, in an early step of the method, for example, adding a compound before the bacteria are at least partially attached to the particles advantageously enables determination of the effect of the compound on the original bacterial profile (e.g., the bacterial profile of the original subject). In some embodiments, the early stage includes adding the compound before introducing the particles into the culture vessel (e.g., contacting the compound with the origin sample), adding the compound during inoculation, or any combination thereof. In some embodiments, the bacteria are initially attached to the particles before adding the compound, advantageously enabling the bacteria to recover and form a more resilient / less sensitive bacterial population before being exposed to the compound.

[0178] In some embodiments, the method according to the invention comprises contacting the plurality of bacteria with at least one compound of interest. In some embodiments, the method according to the invention comprises contacting the plurality of bacteria with at least 2, at least 3, at least 4, at least 5 or more compounds of interest simultaneously or sequentially.

[0179] In some embodiments, the term "at least one compound" includes any element, condition and / or treatment, or a plurality or list of elements, conditions and / or treatments. In some embodiments, the at least one compound is or comprises: small molecules, drugs, chemicals, peptides, polypeptides, proteins, carbohydrates, prebiotics, bacteriophages, bacteria, fungi, physical parameters, or any combination thereof. In some embodiments, the conditions include any culture and / or growth conditions, such as different growth media, different carbon sources, different nitrogen sources, etc.

[0180] In some embodiments, the term "physical parameter" refers to any measurable physical property, or a plurality or list of measurable physical properties, including but not limited to: temperature; pH; stress factors; flow; shaking; stirring; humidity; gases, e.g., O2, CO2, N2; and so on.

[0181] In some embodiments, the at least one compound is or comprises a microorganism not present in the origin sample. In some embodiments, the at least one compound comprises a drug. In some embodiments, the drug is a drug approved for use or consumption by a subject.

[0182] In some embodiments, the method comprises contacting the plurality of bacteria with a drug. In some embodiments, the plurality of bacteria are contacted with a dose of the drug approved for consumption by a subject. In some embodiments, the plurality of bacteria are contacted with an effective amount of the drug approved for consumption by a subject (such as but not limited to eliciting a therapeutic effect of the drug). In some embodiments, the dose used is lower or higher than the approved dose. In some embodiments, any derivative or salt of the approved drug is used. In some embodiments, the approved drug comprises any of its derivatives, analogs or salts, or any combination thereof.

[0183] In some embodiments, an "approved dose" is a clinically approved dose. In some embodiments, clinical approval is clinical approval for consumption or use by a human subject.

[0184] The types of clinically approved drugs and their administration are common and will be apparent to those of ordinary skill in the art.

[0185] In some embodiments, the subject is a eukaryote. In some embodiments, the subject is a plant. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0186] The phrases "add at least one compound to the container" or "expose the bacteria to at least one compound" are used herein in their broadest sense and refer to any type of combined action that causes the plurality of bacteria to be exposed to the compound, for example. The plurality of bacteria can be exposed to, subjected to, or contacted with the compound at any step of the method.

[0187] In some embodiments, the at least one compound is added to the container before, substantially together with, or shortly after adding the sample or diluted sample to the container. Particles can be added before, substantially together with, or after adding the compound. In some embodiments, the at least one compound is added to the container after adding the sample and substantially together with or shortly after adding the particles. In some embodiments, the bacterial population is exposed to the at least one compound during a culturing step. In some embodiments, the bacterial population is exposed to the at least one compound during the process of the plurality of bacteria contacting the particle(s), as a previous step, during a culturing step, or any combination thereof. In some embodiments, the plurality of bacteria are exposed to the at least one compound only before the plurality of bacteria attach to the particle; the exposure continues throughout the culturing period or both. In some embodiments, the compound can contact a sample comprising the plurality of bacteria, a diluted sample, a co-culture comprising the plurality of bacteria, particle-attached bacteria, bacteria in a planktonic form (e.g., bacteria growing in the presence of particles), or any combination thereof.

[0188] In some embodiments, the evaluation method includes the steps of inoculating particles with a sample (or diluted sample) and contacting the particles inoculated with the sample with the at least one compound of interest.

[0189] In some embodiments, the step of determining or evaluating at least one of the following is performed at any point in time throughout the steps of the method (e.g., during cultivation): (i) bacterial diversity; (ii) bacterial relative abundance; (iii) bacterial load; (iv) any other effect of the at least one compound on the plurality of bacteria; or (v) any change in the at least one compound (e.g., chemical and / or structural change). In some embodiments, the determination is made before and / or after the plurality of bacteria come into contact with the particles. In some embodiments, the step of determining at least one of the following is performed at any point in time during cultivation (e.g., 1, 5, 10, 15, 20 hours of cultivation; on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, and / or 14th day of cultivation): (i) bacterial diversity; (ii) bacterial relative abundance; (iii) bacterial load; (iv) any other effect of the at least one compound on the plurality of bacteria; or (v) any change in the at least one compound.

[0190] In some embodiments, the method further comprises transferring the plurality of bacteria to a different container at least once at a time point selected from: (i) during the contacting step; (ii) during the course of the cultivation cycle; (iii) before the step of adding the at least one compound, (iv) after the step of adding the at least one compound, or (v) any combination of (i) to (iv).

[0191] In some embodiments, the determination step is performed using a co - culture comprising the plurality of bacteria, the plurality of bacteria attached to particles, bacteria in a planktonic form, or any combination thereof. In some embodiments, the effects of the compound are compared between any of the following: a co - culture comprising the plurality of bacteria, the plurality of bacteria attached to particles, bacteria in a planktonic form.

[0192] In some embodiments, the bacterial diversity of the treated bacteria; the relative abundance of the bacteria; the bacterial load; and / or any other effect of the at least one compound on the plurality of bacteria (of the treated bacteria) is compared with the corresponding characteristics in an origin sample or any cultured sample, and a change / alteration in any characteristic indicates that the at least one compound has an effect on the plurality of bacteria.

[0193] In some embodiments, any other effect of the at least one compound on the plurality of bacteria includes: the ratio of particle - attached bacteria to planktonic bacteria, the abundance of specific bacteria / plurality of bacteria, gene expression profiles, protein and / or metabolite production, toxin production, the suitability of the compound for generating a desired / predetermined microbial profile, the suitability of the compound for enhancing the attachment of specific bacteria to particles and / or enhancing the planktonic growth form of specific bacteria in a plurality of bacterial populations; or a combination thereof.

[0194] In some embodiments, the method according to the invention is carried out simultaneously in several containers. In some embodiments, several containers can be used simultaneously in a high-throughput assay. In some embodiments, the method further comprises culturing a "control plurality of bacteria" not exposed to the compound in a separate container. In some embodiments, such a high-throughput assay is carried out in a system generated from the same origin sample, for example, in order to determine the optimal treatment or efficacy for a microbial population. In some embodiments, the bacterial diversity; the relative abundance of the bacteria; the bacterial load; any other effects of the at least one compound and / or any changes in the at least one compound at different treatments are compared with each other and / or with the corresponding characteristics in the control plurality of bacteria. In some embodiments, any change or alteration indicates an effect of the at least one compound on the plurality of bacteria. In some embodiments, the effect is determined at several time points during and / or after the exposure of the plurality of bacteria to the compound. In some embodiments, for determining the effect of the compound on the plurality of bacteria, the comparison between different characteristics is carried out at the same time point.

[0195] In some embodiments, the term "any change or alteration" includes: an increase, decrease, transformation, regulation, change and / or deviation of the microbial profile and / or bacterial load compared to a reference sample (e.g., the origin sample, the untreated control plurality of bacteria or any combination thereof).

[0196] In some embodiments, at the end of the culture period, the cultured control system includes at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% similarity, or any value and range therebetween, with the origin sample used to generate the system. Each possibility represents a separate embodiment of the invention. In some embodiments, the produced control co-culture comprising the plurality of bacteria includes at least 30%, at least 40%, at least 50%, 50 - 85% similarity, 60 - 90% similarity, 70 - 85% similarity, 75 - 99% similarity, 80 - 94% similarity, 85 - 100% similarity, 90 - 97% similarity or 95 - 100% similarity, or any value and range therebetween, with the provided origin sample. Each possibility represents a separate embodiment of the invention.

[0197] In some embodiments, the effect of a compound on the plurality of bacteria is examined by comparing the level of similarity, bacterial load, and / or any other effect between the plurality of treated bacteria and any of the following: (i) a control / cultured (e.g., untreated) plurality of bacteria; (ii) a sample provided by origin, (iii) a different plurality of treated bacteria, (iv) a co-culture comprising a plurality of bacteria, or any combination thereof. The effect can be examined by comparing different bacterial forms or fractions / phases (e.g., particle-attached bacteria, unattached bacteria, or a combination thereof).

[0198] In some embodiments, at the end of the culture period, the bacterial load of an untreated control system (i.e., a system that is generated and not exposed to the compound(s) or element(s)) ranges from at least 1·E per 1 gr of particles 4 to at least 1·E 9 , or any value and range therebetween. In some embodiments, at the end of the culture period, the untreated control system comprises or is characterized by having the following bacterial load: at least 1·E 4 / 1 gr of particles, at least 1·E 5 / 1 gr of particles, at least 1·E 6 / 1 gr of particles, at least 1·E 7 / 1 gr of particles, at least 1·E 8 / 1 gr of particles, at least 1·E 9 / 1 gr of particles, at least 1·E 10 / 1 gr of particles, at least 1·E 11 / 1 gr of particles, at least 1·E 12 / 1 gr of particles, at least 1·E 14 / 1 gr of particles, at least 1·E 16 / 1 gr of particles or at least 1·E 20 / 1 gr of particles, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0199] In some embodiments, adding; contacting or exposing the plurality of bacteria to the at least one compound is carried out under conditions suitable for the at least one compound to exert its effect on the bacterial population.

[0200] In some embodiments, the suitable conditions are selected from: pH level, moisture, low humidity, or any combination thereof. In some embodiments, the suitable conditions include subjecting the bacteria to any one of the following: flow, shaking, stirring, agitation, static conditions, or any combination thereof.

[0201] The compositions, systems, and methods according to the present invention are particularly useful in some embodiments for: analyzing metabolites produced by the microbiota of an object; exploring how or how a specific microbial profile population can be regulated / altered in view of exposure to different compounds / treatments; how the microbial population of an object affects different compounds; identifying compounds that induce microbial profile regulation / alteration (e.g., a desired / target microbial profile); identifying effective combination treatments that can exert synergistic or additive effects, e.g., drug-probiotic, probiotic-prebiotic, drug-prebiotic therapies; using as a platform for producing compositions with different desired microbial profiles - e.g., by adding different compounds to a culture system and / or subjecting the plurality of bacteria to different conditions; for evaluating the suitability of compounds / conditions for generating a desired microbial profile; using as a platform for producing compositions rich in bacteria (e.g., specific bacteria attached to particles) - e.g., by adding compounds to the system / subjecting the plurality of bacteria to different conditions that promote or enhance the growth of bacteria attached to particles and / or the bacterial attachment to particles; for evaluating the suitability of compounds / conditions for enhancing the growth of specific bacteria (e.g., in an attached form) from a plurality of bacterial populations; identifying bacterial strains that are favorable for attachment to particles, strains that are favorable for the planktonic form, and strains that can grow in both forms, e.g., under specific conditions. In some embodiments, the conditions in the system can simulate different regions of the gastrointestinal tract, thereby identifying bacteria that are favorable for specific growth forms in an object.

[0202] In some embodiments, the favorable growth form of a specific bacteria is determined by: separating bacteria that are not attached to the particle(s) from bacteria attached to the particle (e.g., by filtration and / or by allowing the particle to sediment), and determining the abundance of bacteria in each fraction (e.g., based on next-generation sequencing (NGS) technology and / or whole-genome sequencing (WGS)). In some embodiments, a bacteria is defined as being favorable for a specific growth form when its abundance is higher in one of the fractions.

[0203] In some embodiments, the term "microbial profile" includes bacterial diversity (e.g., α- or β-diversity); bacterial relative abundance; and / or bacterial load.

[0204] In some embodiments, the phrase "generating a desired or different microbial profile" refers to forming a plurality of bacteria having a designed / pre-determined bacterial diversity (e.g., α- or β-diversity); bacterial relative abundance; and / or bacterial load (e.g., suitable for treating a disease, disorder, or condition).

[0205] In some embodiments, the terms “modified” / “modify” or “modulate” / “modulating” include increasing or decreasing the level of similarity of the microbiome profile (e.g., compared to the origin or other reference microbiome profile).

[0206] In some embodiments, the term “modifies” includes correcting the bacterial load, alpha-diversity, relative abundance, and / or beta-diversity of a non-healthy microbiota to be similar to that of a healthy subject by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, 100%, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0207] In some embodiments, “modifying” includes diverting the bacterial load, alpha-diversity, relative abundance, and / or beta-diversity of a “non-responding patient” to be similar to that of a “responding patient” by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, 100%, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, a “responding patient” includes a subject who achieves a response, e.g., a subject in remission and / or a subject who no longer has a disease, disorder, or condition after treatment. In some embodiments, a “non-responding patient” includes a subject in whom the disease, disorder, or condition does not show a decrease or improvement after treatment.

[0208] In some embodiments, “reducing” or “reduce” is reducing by at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, or 100%, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0209] In some embodiments, “reducing” or “reduce” is reducing by 5 - 50%, 25 - 75%, or 10 - 100%, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0210] In some embodiments, "increasing" or "increase" is an increase of at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 250%, at least 500%, at least 750% or at least 1,000%, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, "increasing" or "increase" is an increase of 5 - 50%, 25 - 75%, 10 - 100%, 50 - 350%, 100 - 400%, 150 - 550%, 450 - 785% or 200 - 1,000%, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0211] In some embodiments, the phrase "composition enriched in bacteria (one or more) attached to particles" refers to a composition that includes a greater ratio of particle - attached bacteria to planktonic bacteria compared to a control. In some embodiments, the control includes a composition that is cultured under substantially the same conditions and does not undergo a compound that promotes or enhances the growth of bacteria attached to particles and / or the attachment of bacteria to particles. In some embodiments, the ratio is at least 1.1 - fold, at least 1.5 - fold, at least 2 - fold, at least 2.5 - fold, at least 3 - fold, at least 3.5 - fold, at least 4 - fold, at least 4.5 - fold, at least 5 - fold, at least 5.5 - fold, at least 6 - fold, at least 6.5 - fold, at least 7 - fold, at least 7.5 - fold or at least 8 - fold, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0212] According to some embodiments, there is provided a composition produced by the method according to the present invention. In some embodiments, the composition includes: bacteria attached to particles and / or bacteria in planktonic form, e.g., bacteria cultured / grown in the presence of particles. In some embodiments, the composition includes a co - culture comprising said plurality of bacteria. In some embodiments, the composition includes bacteria attached or adhered to particles. In some embodiments, the composition includes bacteria in planktonic form cultured / grown in the presence of particles. In some embodiments, the composition and / or harvested co - culture produced according to the present invention is enriched or supplemented with uncultured bacteria (e.g., derived from a biological sample or bacterial collection or repository (e.g., ATCC, DSMZ)), and / or bacteria cultured in the absence of particles. In some embodiments, the composition further includes additional microorganisms, e.g., at least one of archaea, viruses, fungi or any combination thereof. In some embodiments, the composition includes at least one bacterial species present in a form partially attached to said particles and at least one bacterial species of planktonic bacteria.

[0213] In some embodiments, the composition is a synthetic composition. In some embodiments, the term "synthetic composition" refers to a composition comprising bacteria grown or cultured in vitro. In some embodiments, the synthetic composition includes artificial compositions. In some embodiments, the synthetic composition is an artificial composition, such as, but not limited to, a composition prepared or produced in a laboratory and / or a preparation site or institution. In some embodiments, the synthetic composition does not include a composition isolated or obtained from nature per se. In some embodiments, the composition is frozen, spray-dried, or freeze-dried. In some embodiments, the composition is in the form of a dry powder. In some embodiments, the composition includes: a cryoprotectant, a lyoprotectant, an antioxidant, or any combination thereof. In some embodiments, the composition includes at least one metabolite produced in vitro by at least one bacterium.

[0214] In some embodiments, the composition includes bacteria in an aggregated form. In some embodiments, the terms "aggregated form of bacteria" and "bacterial clump" are interchangeable and refer to the collection of individual bacterial particles into a single body.

[0215] In some embodiments, the composition includes bacteria in the form of a biofilm. In some embodiments, the biofilm is in the form of a dry biofilm, e.g., a solid form, e.g., a powder form. In some embodiments, the term "biofilm" refers to a bacterial community embedded within a matrix including, e.g., self-produced extracellular polysaccharides that adhere to the surface of particles. In some embodiments, planktonic bacteria are adhered to, trapped, incorporated, or embedded under, on, or within the biofilm.

[0216] In some embodiments, the co-culture comprising the plurality of bacteria includes particle-attached bacteria. In some embodiments, the co-culture includes at least one bacterium in a planktonic form. In some embodiments, the co-culture includes or is a mixture of particle-attached bacteria and bacteria in a planktonic form.

[0217] In some embodiments, the method according to the present invention includes the step of removing bacteria not attached to the particles or separating bacteria not attached to the particles from the bacteria attached to the particles, thereby producing: (i) a composition comprising or enriched in bacteria attached to the particles and substantially free of planktonic bacteria cultured in the presence of the particles; and / or (ii) a composition comprising or enriched in planktonic bacteria cultured in the presence of the particles and substantially free of bacteria attached to the particles. In some embodiments, the method includes the step of mixing compositions (i) and (ii) in any desired ratio, e.g., thereby producing a final composition rich in a specific bacterium that favors a specific growth form (e.g., planktonic or attached form) and thus present in a higher abundance in such separated compositions.

[0218] In some embodiments, a "composition comprising or enriched in bacteria attached to particles and substantially free of planktonic bacteria cultured in the presence of the particles" refers to a composition comprising bacteria in an attached growth form (e.g., greater than 51%, such as at least 55%, at least 60%, at least 70% or 100%, or any value and range therebetween) compared to planktonic bacteria cultured in the presence of the particles.

[0219] In some embodiments, the phrase "composition comprising or enriched in planktonic bacteria cultured in the presence of the particles and substantially free of bacteria attached to the particles" refers to a composition comprising planktonic bacteria cultured in the presence of the particles (e.g., greater than 51%, such as at least 55%, at least 60%, at least 70% or 100%, or any value and range therebetween) compared to bacteria in an attached growth form.

[0220] In some embodiments, the composition may comprise bacteria cultured in other growth forms (e.g., planktonic culture).

[0221] In some embodiments, the composition according to the present invention is administered to a subject immediately after culturing. In some embodiments, the composition is administered after storage (e.g., at room temperature, at a temperature in the range of 2 - 8 °C or at a temperature below -18 °C). In some embodiments, the composition is provided in a solid form (e.g., freeze-dried, spray-dried or frozen). In some embodiments, the solid (e.g., freeze-dried or spray-dried) compositions disclosed herein are stable for at least three months at room temperature (e.g., at a temperature selected from about 20, 21, 22, 23, 24 and 25 °C (or any value and range therebetween)). In this context, the term "solid" refers to the physical state of the substance.

[0222] In some embodiments, the composition is a solid composition.

[0223] In some embodiments, the composition is a spray-dried or freeze-dried composition.

[0224] Generally, the terms "lyophilized" and "freeze-dried" are interchangeable and refer to the process of freezing a solution and then reducing the concentration of water (e.g., by sublimation to a level that does not support biological and / or chemical reactions). The resulting lyophilized composition can be stored long-term while maintaining its stability. In some embodiments, the lyophilized composition can be used as a powder. In some embodiments, the powder or composition can be formulated in a suitable delivery vehicle and can also be reconstituted by adding a semi-liquid or liquid solution. The volume added during the reconstitution process can be similar to, lower than or higher than the initial volume of the solution before the lyophilization process.

[0225] In some embodiments, the composition comprises a carrier or excipient. In some embodiments, the carrier is a veterinary, agricultural, and / or pharmaceutically acceptable carrier. The terms "carrier" and "excipient" are used interchangeably herein.

[0226] In some embodiments, there is provided a pharmaceutical composition comprising the composition disclosed herein and an acceptable carrier or excipient.

[0227] In some embodiments, the term "pharmaceutical composition" includes the terms "dietary composition" and "nutritional composition".

[0228] In some embodiments, the terms "dietary composition", "nutritional composition", and "nutraceutical composition" refer to compositions suitable for consumption as a food supplement, e.g., for supplementing a normal diet, correcting nutritional deficiencies, maintaining an adequate intake of certain nutrients, and / or for supporting specific physiological functions.

[0229] As used herein, the terms "carrier", "excipient", or "adjuvant" refer to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable" carrier, solvent, diluent, excipient, and vehicle generally refers to non-toxic, inert solids, semi-solids, liquid fillers, diluents, encapsulating materials, any type of formulation adjuncts, or simply a sterile aqueous medium such as saline. In some embodiments, the term "pharmaceutically acceptable carrier" refers to any diluent or vehicle suitable for use in humans or other animals. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes and stearin; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol solutions and phosphate buffer solutions, and other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances that can serve as carriers herein include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifying agents (e.g., carbomer, hydroxypropylcellulose, sodium lauryl sulfate), and other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate may also be present, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives. Any non-toxic, inert, and effective carrier can be used to formulate the compositions contemplated herein.In this regard, suitable pharmaceutically acceptable carriers, excipients, and diluents are known to those skilled in the art, such as those described in The Merck Index, 13th Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, 10th Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management (the entire contents of which are hereby incorporated by reference in their entirety). Examples of pharmaceutically acceptable excipients, carriers, and diluents for use in the present compositions include distilled water, normal saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21. stEd., Lippincott Williams&Wilkins, Philadelphia, Pa., (2005) (each of which is incorporated herein by reference in its entirety). The compositions described herein can also be incorporated into artificially created structures such as liposomes, ISCOMS, slow release particles, and other vehicles. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes used with the peptides described herein are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and sterols such as cholesterol. For example, Coligan, J.E. et al, Current Protocols in Protein Science, 1999, John Wiley&Sons, Inc., New York reviews various methods for preparing liposomes, and also see U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0230] The vehicle can altogether account for from about 0.1% to about 99.99999% of the weight of the pharmaceutical compositions presented herein.

[0231] In some embodiments, the composition is for medical use. In some embodiments, the composition is for agricultural use. In some embodiments, the composition is for veterinary use.

[0232] In some embodiments, there is provided a method for preventing or treating a disease, disorder, or condition (e.g., dysbiosis) and / or for modulating the microbiota in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition according to the present invention.

[0233] The terms "therapeutically effective amount" and "effective amount" refer to the amount required to prevent, ameliorate, and / or treat a disease, disorder, or condition. The effective dose can vary depending on the sex, age, and weight of the subject, the disease or condition and its severity, and any other factors recognized by those skilled in the art.

[0234] In some embodiments, the pharmaceutical composition is for treating or preventing dysbiosis in a subject in need thereof. In some embodiments, the compositions disclosed herein are for preparing a medicament for treating or preventing dysbiosis in a subject in need thereof.

[0235] In some embodiments, the pharmaceutical composition is for modulating the microbiota in a subject in need thereof, e.g., for treating or preventing Clostridium difficile infection, ulcerative colitis, or atopic dermatitis in a subject in need thereof.

[0236] In some embodiments, the term "modulating the microbiota" includes reversing established bacteria typically associated with a disease, health condition, or clinical symptom and / or achieving colonization of beneficial bacteria on the surface of any tissue and / or any body part of an organism - including but not limited to any outer or inner surface of the body and the deeper layers of the skin typically associated with a disease, health condition, or clinical symptom.

[0237] As used herein, the term "dysbiosis" is characterized by an alteration, imbalance, impairment, and / or dysfunction of the microbiota and / or microbiome on the surface of any tissue and / or any body part of an organism, including but not limited to any outer or inner surface of the body and the deeper layers of the skin typically associated with a disease, health condition, or clinical symptom. Such an imbalance can exist in any microbial community, including but not limited to gastrointestinal, skin, oral, bronchial, vaginal, or rectal dysbiosis, etc. In some embodiments, the dysbiosis is tumor dysbiosis.

[0238] In some embodiments, the term "surface of any body part of an organism" refers to the outer surface of the body visible to the naked eye, e.g., the skin of the face, throat, scalp, chest, back, ears, neck, hands, elbows, knees, and other skin sites, and also refers to the inner surface of the body, e.g., which is part of the individual's internal anatomy, such as but not limited to the oral cavity, gastrointestinal tract, and lower genital tract (including but not limited to the vagina).

[0239] In some embodiments, dysbiosis includes an imbalance in the microbial flora in a matrix or culture medium. In some embodiments, the matrix or culture medium comprises or is soil.

[0240] In some embodiments, the altered microbiota refers to a microbial population that has deviated from the homeostatic microbiota - such as in a non - healthy subject. In some embodiments, compared to the healthy microbiota, the altered microbiota includes different bacterial diversity, bacterial relative abundance, and / or bacterial load. As used herein, the term "healthy subject" refers to a subject having the natural flora characteristic of a healthy subject. In some embodiments, the altered microbiota includes pathogenic microorganisms. In some embodiments, the altered microbiota is associated with a medical condition and / or is harmful to the health of the subject (e.g., a human subject).

[0241] In some embodiments, the subject is a mammal. In some embodiments, the subject is an animal. In some embodiments, the subject is a human subject. The subject can be male or female.

[0242] The pharmaceutical composition can take any physical form required for proper administration. The composition can be administered in any suitable form, including but not limited to liquid form, gel form, semi-liquid (e.g., liquid (such as viscous liquid) including certain solids) form, semi-solid (solids including certain liquids) form, or solid form. The composition can be provided in, for example, tablet form, vaginal suppository form, cream form, suppository form, capsule form, liquid form, food form, chewable form, non-chewable form, buccal form, sublingual form, sustained release form, non-sustained release form, extended release form, or non-extended release form.

[0243] In some embodiments, the composition is formulated for administration by a method selected from: oral, rectal, parenteral, mucosal, vaginal, nasal, local, topical, pulmonary, ocular, oral, buccal administration, or any combination thereof.

[0244] Pharmaceutically acceptable carriers suitable for preparing unit dosage forms of the compositions described herein for oral administration are well known in the art.

[0245] In some embodiments, the similarity includes at least one property selected from: functionality, e.g., the ability of a bacterium to affect the metabolic processes of a subject and / or improve any disease, disorder, or condition of the subject; potential metabolic pathways / paths, e.g., the possibility of producing, synthesizing, consuming, and / or utilizing certain metabolites / organic compounds; relative abundance; alpha-diversity; recovered operational taxonomic units (recovered OTUs) or recovered amplicon sequence variants (ASVs); beta-diversity; and any combination thereof.

[0246] In some embodiments, the term "similarity" includes the similarity between the bacterial community or population present in a co-culture and the bacterial community or population present in the originating sample. In some embodiments, the term "similarity" includes the similarity between other microorganisms present in the composition, including at least one of the following: archaea; viruses, e.g., bacteriophages; fungi; or any combination thereof. Similarity can be determined by any method known to those skilled in the art.

[0247] In some embodiments, similarity is determined by an alpha-diversity metric. In some embodiments, the alpha-diversity metric includes any of the following: observed species, total genera found, Shannon, Chao1, Simpson, or any combination thereof. In some embodiments, alpha-diversity is a measure that considers the diversity or richness of taxa (e.g., bacterial taxa) in a sample. In some embodiments, alpha-diversity is compared between two communities (or samples). In some embodiments, when referring to an alpha-diversity similarity metric, the diversity or richness of taxa in a composition or co-culture is compared to the diversity or richness in the origin sample—e.g., at any taxonomic level. In some embodiments, Shannon, Chao1, and Simpson refer to diversity, as described in Kim BR, Shin J, Guevarra R, Lee JH, Kim DW, Seol KH, Lee JH, Kim HB, Isaacson R. Deciphering Diversity Indices for a Better Understanding of Microbial Communities. J Microbiol Biotechnol. 2017 Dec 28;27(12):2089-2093. doi:10.4014 / jmb.1709.09027. PMID:29032640.

[0248] In some embodiments, similarity is determined by a beta-diversity metric. In some embodiments, the beta-diversity metric includes any of the following: Jaccard, unweighted Unifrac similarity, weighted Unifrac similarity, generalized Unifrac similarity, Bray-Curtis similarity, or any combination thereof. In some embodiments, generalized Unifrac similarity is implemented as elaborated in Jun et al. "Associating microbiome composition with environmental covariates using generalized UniFrac distances". Bioinformatics. 2012 Aug 15;28(16):2106–2113.

[0249] In some embodiments, the similarity is determined by unweighted Unifrac similarity. In some embodiments, the similarity is determined by weighted Unifrac similarity. In some embodiments, "unweighted Unifrac similarity" ("unweighted Unifrac") and "weighted Unifrac similarity" ("weighted Unifrac") refer to a measure of similarity between two communities (or samples) that takes into account both: I. presence / absence data of bacterial populations; and II. phylogenetic relatedness of the bacteria. Weighted Unifrac similarity also takes into account the relative abundances of the bacterial populations.

[0250] In some embodiments, the similarity is determined by Bray-Curtis. In some embodiments, the "Bray-Curtis dissimilarity index" is a measure of non-phylogenetic beta-diversity similarity between two communities (or samples) that takes into account the abundances of bacterial populations in the samples. In other embodiments, compared to other similarity metrics that take into account the phylogenetic relatedness of the bacteria (e.g., weighted Unifrac similarity, unweighted Unifrac similarity), using a non-phylogenetic beta-diversity metric can result in lower similarity values.

[0251] In some embodiments, when using next-generation sequencing (NGS) technology and / or whole-genome sequencing (WGS) to determine similarity by a metric that takes into account the phylogenetic relatedness of the bacteria, the co-culture includes at least 30% similarity to the origin sample. In some embodiments, the metrics that take into account phylogenetic relatedness include any of the following: diversity recovery, weighted Unifrac similarity, unweighted Unifrac similarity, generalized Unifrac similarity, or any combination thereof. This analysis can be performed at any taxonomic level. It should be understood that various similarity metrics can be employed in accordance with the present invention; however, some methods can result in lower similarity values - for example, using a non-phylogenetic beta-diversity metric obtained according to embodiments of the present invention results in lower similarity values (e.g., less than 30%) compared to other tested similarity metrics that take into account the phylogenetic relatedness of the bacteria.

[0252] In some embodiments, the similarity is determined by relative abundance. In some embodiments, the term "relative abundance" is the percentage of a specific taxon (e.g., a bacterial taxon) relative to the total abundance of the taxa, and is used herein to compare the distribution of genera and / or any other bacterial taxonomic level among a community (e.g., a bacterial community) in a sample.

[0253] In some embodiments, α- and / or β-diversity similarities are calculated from the recovered OTUs and / or recovered ASVs. In some embodiments, the terms "recovered OTU", "recovered ASV", or "diversity recovery" refer to the percentage of taxa observed in a test sample relative to the total taxa observed in another sample (e.g., a fecal origin sample).

[0254] In some embodiments, the similarity is compared to a processed origin sample (e.g., homogenized and / or filtered).

[0255] The similarity index can be based on next-generation sequencing (NGS) technologies (e.g., ribosomal 16S RNA or coding DNA) and / or whole-genome sequences (WGS), which will be apparent to those of ordinary skill in the art. The similarity can be determined at any taxonomic level (e.g., phylum, family, genus, species, and / or strain).

[0256] In some embodiments, the origin sample comprises a predefined bacterial population, and the co-culture is characterized by having at least 30% similarity to the predefined bacterial population. In some embodiments, the term "predefined" refers to a customized origin composition that includes a plurality of bacterial populations selected according to specific needs - e.g., suitable for treating a subject suffering from intestinal dysbiosis. In some embodiments, the term "predefined" refers to a bacterial population that typically colonizes an environmental niche. In some embodiments, the origin sample further comprises additional microorganisms (e.g., selected from archaea, viruses, fungi, or any combination thereof), and the co-culture is characterized by having at least 30% similarity to the additional microbiota present in the sample.

[0257] In some embodiments, the plurality of bacteria in the plurality of provided bacterial or origin samples belong to at least 5 to up to 600 bacterial species determined by 16S NGS technology (e.g., at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200 or more bacterial species), and / or belong to at least 2 to up to 250 bacterial genera determined by 16S NGS technology (e.g., at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80 or more bacterial genera), or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0258] In some embodiments, the plurality of bacteria in the plurality of provided bacterial or origin samples belong to at least 35 to up to 80 observed genera and / or belong to at least 120 to up to 300 observed species (determined by 16S NGS technology), or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0259] In some embodiments, the composition or co - culture has at least 30% similarity in terms of the diversity or richness of the plurality of bacteria.

[0260] In some embodiments, the co - culture comprising the plurality of bacteria has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% similarity to the origin sample, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0261] In some embodiments, the bacterial load is determined by any method known to those skilled in the art, such as but not limited to viable bacterial colony - forming units (CFU), quantitative polymerase chain reaction (qPCR), flow cytometry, live - dead staining, propidium monoazide qPCR (PMA - qPCR), microscopy, metabolic assays, spectrophotometry or any combination thereof. The methods for determining the bacterial load (such as the methods disclosed herein) are common and will be obvious to those skilled in the art.

[0262] In some embodiments, the term "bacterial load" is interchangeable with the term "bacterial count". In some embodiments, the co-culture or composition comprises live bacteria, and the bacterial count is measured by CFU.

[0263] In some embodiments, the bacterial load in the composition produced according to the invention is calculated per gr of the introduced medium or particles in the container in dry form. In some embodiments, the bacterial load of the particles for contact with said plurality of bacteria is calculated per gr in dry form. In some embodiments, when measuring the bacterial load of the co-culture in the composition, the weight of the particles added to the culture system is taken into account. In some embodiments, when measuring the bacterial load of the co-culture in the composition, the weight of the particles in the final composition is taken into account.

[0264] In some embodiments, the term "particle" includes a plurality of particles of one type or several types, and refers to a substance / material suitable for, configured for, or adapted for at least one bacterium to adhere / attach and / or grow thereon. In some embodiments, the particle comprises a surface to which bacteria can adhere.

[0265] In some embodiments, in the context of a particle, the term "surface" is interchangeable with the term "surface area" and refers to the outer surface of the particle. In some embodiments, the particle is porous. In such embodiments, the term "surface" includes the outer surface of the porous structure of the particle. In some embodiments, the particle is non-porous. In some embodiments, the particle is a mixture of porous particles and non-porous particles. In some embodiments, the term "porous" refers to void (i.e., "empty") spaces in the material. In some embodiments, the term "porous" includes having a non-uniform surface area.

[0266] As used herein, the terms "particle(s)", "nanoparticle(s)", "microparticle(s)", "nanosphere(s)" and "microsphere(s)" are used interchangeably.

[0267] In some embodiments, the particle does not originate from the originating sample. In some embodiments where the origin is a fecal sample, the particle is not the undigested fiber of the feces. In some embodiments, the originating sample does not contain the particles disclosed herein.

[0268] In some embodiments, the particles used according to the invention are water-insoluble active agents. In some embodiments, the term "water-insoluble active agent" refers to particles having a solubility in water at 25 °C of less than 5 mg / ml, preferably less than 1 mg / ml, and most preferably less than 0.1 mg / ml.

[0269] In some embodiments, the particles used according to the present invention are water-insoluble at physiological pH. In some embodiments, the particles used according to the present invention are water-insoluble at a pH in the range of about 1.0 to 8.0.

[0270] In some embodiments, "particles" include particles having a diameter in the range of 5 microns to 1 cm. In some embodiments, the particles range from 1 micron to 50 mm. In some embodiments, the diameter of the particles is at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60 microns, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1 cm, or any range or value therebetween. Each possibility represents a separate embodiment of the present invention.

[0271] In some embodiments, the average diameter of the particles is in the range of 1 to 1,500 microns. In some embodiments, the average diameter of the particles is in the range of 50 to 1,200 microns, 50 to 1,100 microns, 50 to 1,000 microns, 55 to 1,200 microns, 55 to 1,000 microns, 57 to 1,200 microns or 60 to 1000 microns, including any range therebetween. Each possibility represents a separate embodiment of the present invention.

[0272] In some embodiments, the diameter is the average diameter. In some embodiments, the diameter is the maximum diameter. In some embodiments, the diameter is the minimum diameter.

[0273] In some embodiments, the term "particles" includes any particles of any shape, e.g., within the dimensions embodied. In some embodiments, the particles can be round, amorphous, irregular, spherical, oval, flower-shaped, cubic, spherical, elongated, rod-shaped, having any other shape or any combination thereof.

[0274] In some embodiments, the particles are selected from: microcrystalline cellulose (MCC), dicalcium phosphate (DCP), seeds, polysaccharides or any combination thereof. In some embodiments, the particles include multiple particles of one type. In some embodiments, the particles include multiple particles of several types.

[0275] In some embodiments, the particles include a combination of calcium and cellulose. In some embodiments, the particles include a combination of phosphate and cellulose. In some embodiments, the particles include a combination of calcium, phosphate and cellulose. In some embodiments, the particles include MCC and DCP, or consist of MCC and DCP.

[0276] In some embodiments, the particles are fully immersed in the culture medium. In some embodiments, the particles are at least partially immersed in the culture medium.

[0277] In some embodiments, the particles comprise MCC and DCP, and the weight / weight (w / w) ratio ranges from 5:1 (w / w) to 1:5 (w / w), 4:1 (w / w) to 1:4 (w / w), 3:1 (w / w) to 1:3 (w / w), 2:1 (w / w) to 1:2 (w / w), 1:1 (w / w), 5:1 (w / w) to 1:4 (w / w), 5:1 (w / w) to 1:3 (w / w), 5:1 (w / w) to 1:2 (w / w), 5:1 (w / w) to 1:1 (w / w), 4:1 (w / w) to 1:5 (w / w), 4:1 (w / w) to 1:1 (w / w), 3:1 (w / w) to 1:5 (w / w), 2:1 (w / w) to 1:5 (w / w), or 1:1 (w / w) to 1:5 (w / w). Each possibility represents a separate embodiment of the present invention. In some embodiments, all w / w ratios associated with the particles herein are based on the anhydrous form.

[0278] In some embodiments, the seeds are selected from: cranberries, passion fruits, herbs, oats, or any combination thereof.

[0279] In some embodiments, the particles comprise food-grade particles or consist of food-grade particles. In some embodiments, the food-grade particles comprise polysaccharides, fat crystals, proteins, or any combination thereof, or consist of polysaccharides, fat crystals, proteins, or any combination thereof. In some embodiments, the food-grade particles comprising fat crystals are selected from: glyceryl monooleate, glyceryl stearyl citrate, or a combination thereof. In some embodiments, the food-grade particles comprising polysaccharides are selected from: corn starch, starch nanocrystals, cellulose nanocrystals, microcrystalline cellulose, nanocellulose, or methylcellulose, chitin, chitosan, or any combination thereof. In some embodiments, the food-grade particles comprising proteins are selected from: β-lactoglobulin, lactoferrin, lactoferrin-polysaccharide, bovine serum albumin, gelatin, collagen, soy protein isolate, pea protein, zein, or any combination thereof. In some embodiments, the food-grade particles are selected from: flavonoids (tiliroside), waxes, shellac-xanthan gum, or any combination thereof.

[0280] In some embodiments, according to the method of the present invention, the weight / weight ratio between the particles and the sample ranges from 1:2 to 1:10, 1:3 to 1:10, 1:4 to 1:10, 1:5 to 1:10, 1:6 to 1:10, 1:7 to 1:10, 1:8 to 1:10, or 1:9 to 1:10, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0281] In some embodiments, the volume / weight (v / w) ratio of the solution (e.g., buffer or culture medium) used during contact or cultivation to the particles ranges from 1:1 to 200:1, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0282] In some embodiments, the terms "attachment" and "adhesion" with respect to bacteria include adsorption to a surface - e.g., via weak interactions and / or strong interactions, such as by flagella, pili, lipopolysaccharides, exopolysaccharides, collagen-binding adhesin proteins, etc.

[0283] In some embodiments, "additional microorganisms that are at least partially attached to the particles" includes microorganisms other than bacteria that are attached to the particles, particle-attached bacteria, and / or the matrix formed by the attached bacteria, e.g., archaea, viruses, and / or fungi, and refers to at least 0.01% of the attached "additional microorganisms" among the total "additional microorganisms" present in the culture system or at the end of the contact or cultivation step.

[0284] In some embodiments, after the bacteria attach to the particle(s), some or all of the bacteria may detach, and / or other suspended bacteria may attach to the particles, particle-attached / adhered bacteria, and / or the matrix formed by the attached / adhered bacteria. In some embodiments, "bacteria that are at least partially attached to the particles" includes bacteria that are attached to the particle-attached / adhered bacteria and / or the matrix formed by the attached / adhered bacteria. In some embodiments, the phrase "a plurality of bacteria that are at least partially attached to the particles" refers to at least 0.01%, at least 0.05%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% (or any value and range therebetween) of the particle-attached / adhered bacteria among the total bacteria present in the culture system or at the end of the contact or cultivation step. The total bacteria can be determined by a method for determining bacterial load (such as the methods described below). The attached portion and the non-attached portion can be evaluated by separating the two portions, e.g., for evaluating the percentage of attachment and / or for separately analyzing each portion. In some embodiments, the separate analysis includes separating the two portions and determining the bacterial count / load in each portion. In some embodiments, separating these portions or removing the non-attached bacteria is performed by filtration and / or gravitational sedimentation (e.g., centrifugation), or includes filtration and / or gravitational sedimentation (e.g., centrifugation). In some embodiments, the term "filtration" includes all separation techniques and any other process that utilizes a filter capable of separating these portions.

[0285] General Considerations

[0286] All numerical values in this document are assumed to be modified by the term "about". The term "about" generally refers to a numerical range that a person skilled in the art would consider equivalent to the recited value (e.g., having the same function or result). In many cases, when the term "about" precedes a numerical value, the term "about" indicates ±10%.

[0287] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". The term "consisting of" means "including and limited to".

[0288] The term "consisting essentially of" means that a composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel features of the claimed composition, method or structure.

[0289] The term "exemplary" is used herein to mean "serving as an example, instance, or illustration". Any embodiment described as "exemplary" is not necessarily to be construed as preferred over or superior to other embodiments and / or to exclude the incorporation of features of other embodiments.

[0290] The term "optionally" is used herein to mean "provided in some embodiments and not provided in other embodiments". Any particular embodiment of the present invention may include a plurality of "optional" features, unless these features are in conflict.

[0291] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. As used in this specification and the appended claims, the term "or" is generally used in its sense including "and / or" unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0292] Throughout this application, various embodiments of the present invention may be presented in a range format. It should be understood that the range format description is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the present invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as the individual values within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0293] Whenever a numerical range is indicated herein, it is intended to include any recited number (fractional or integral) within the indicated range. The phrases "ranging between" the first recited number and the second recited number and "ranging from" the first recited number "to" the second recited number are used interchangeably herein and are intended to include the first and second recited numbers and all the fractional and integral numbers therebetween.

[0294] As used herein, the term "method" refers to a way, means, technique, and procedure for accomplishing a given task, including but not limited to those ways, means, techniques, and procedures known or readily developed from known ways, means, techniques, and procedures by practitioners in the fields of chemistry, pharmacology, biology, biochemistry, microbiology, and medicine.

[0295] It should be understood that certain features of the present invention that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for the sake of brevity, the various features of the present invention that are described in the context of a single embodiment may also be provided separately or in any suitable sub-combination or suitably provided in any other described embodiment of the present invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment is inoperable without those elements.

[0296] The description of the various embodiments of the present invention has been presented for purposes of illustration, but these descriptions are not intended to be exhaustive or to limit the present invention to the disclosed embodiments. Many changes and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terms chosen herein are intended to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable other ordinary skilled persons in the art to understand the embodiments disclosed herein.

[0297] The various embodiments and aspects of the present invention described above and claimed in the following claims are supported by experiments in the following examples.

[0298] Other terms used herein are intended to be defined by their meanings known in the art.

[0299] After considering the following embodiments, other objects, advantages, and novel features of the present invention will become apparent to those of ordinary skill in the art, and these embodiments are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention described above and claimed in the following claims section is supported by experiments in the following embodiments.

[0300] Embodiments

[0301] Generally, the nomenclature used herein and the laboratory procedures used in this invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are explained in detail in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Volumes 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methodologies described in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J.E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J.E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W.H.Freeman and Co., New York (1980); available immunoassays are generally described in the following patents and scientific literature, see, for example, U.S. Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M.J., ed. (1984); "Nucleic Acid Hybridization" Hames, B.D., and Higgins S.J., eds. (1985); "Transcription and Translation" Hames, B.D., and Higgins S.J., eds. (1984); "Animal Cell Culture" Freshney, R.I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Volumes 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Additional general references are provided throughout this document.

[0302] In the following examples, the culture of multiple bacteria from fecal / stool or saliva samples (obtained from healthy human donors) is used as one embodiment to illustrate the ability of the method according to the present invention to produce a composition (e.g., for pharmaceutical use and / or as an in vitro screening system) comprising a co-culture having a high similarity to the origin sample. To evaluate the effect of a drug on the microbiome sampled from a diseased subject in a screening model, a fecal sample is obtained from a diseased donor. In the examples, "bacterial sample", "stool sample", and "origin fecal sample" are all used to refer to the origin sample used to produce the composition.

[0303] Exemplary origin fecal samples used in the following experiments included 42 - 58 bacterial genera (average 50.1); and 131 - 196 observed bacterial species (average of 168.1). The data were obtained from five different experiments using five different origin samples and are presented as a range, where the minimum and maximum values are the minimum and maximum values obtained in the five different experiments, and the average of the five different experiments is in parentheses.

[0304] Generally, samples from human donors (e.g., fecal samples) include various types of microorganisms, including bacteria, archaea, viruses, and fungi. Within each type of microorganism, there are different populations / communities that require different maintenance, growth, culture, and / or proliferation conditions. Therefore, it is challenging to maintain a high similarity of such different populations in vitro, especially when cultured together in a single vessel.

[0305] In addition, each sample origin has a different microbial composition, which further increases the challenges of maintaining similarity, reproducibility, and process scaleup.

[0306] It has been reported in the literature that most gut microbes are considered unculturable or difficult to culture as an ecosystem [see, e.g., Lagier, J.C., Dubourg, G., Million, M., Cadoret, F., Bilen, M., Fenollar, F., Levasseur, A., Rolain, J.M., Fournier, P.E. and Raoult, D., 2018. Culturing the human microbiota and culturomics. Nature Reviews Microbiology, 16(9), pp.540-550, Liu, Sijia, Christina D. Moon, Nan Zheng, Sharon Huws, Shengguo Zhao, and Jiaqi Wang. "Opportunities and challenges of using metagenomic data to bring uncultured microbes into cultivation." Microbiome 10, no. 1 (2022): 1-14. Mabwi, H.A., Kim, E., Song, D.G., Yoon, H.S., Pan, C.H., Komba, E.V., Ko, G. and Cha, K.H., 2021. Synthetic gut microbiome: advances and challenges. Computational and structural biotechnology journal, 19, pp.363-371].

[0307] The following examples show that, despite the complexity and variability present in the origin samples, the similarity and functionality of various communities with the origin samples (e.g., bacterial populations) are retained during the culturing and expansion processes according to the exemplary embodiments.

[0308] Materials and Methods

[0309] Similarity Parameters

[0310] To obtain the relative abundances of each taxon in the co-culture, similarity indices commonly used in the art were calculated based on 16S next-generation sequencing (NGS) or whole-genome shotgun (WGS) technology.

[0311] "Relative abundance" is the percentage of a specific taxon relative to the total abundance of taxa and is used to evaluate the distribution of taxa within a community in a sample. In the examples, the relative abundances between two test samples are compared.

[0312] "α-diversity" is a measure that takes into account the number of taxa in a sample. In the examples, the α-diversity between communities (or samples) is compared.

[0313] "Recovered OTU" (and / or recovered ASV and / or diversity recovery) is a measure of similarity between two communities (or samples) that takes into account the percentage of OTUs (and / or ASVs) observed in a treated sample (composition) relative to the total OTUs (and / or ASVs) observed in the origin sample. "Recovered OTU" (and / or ASV) refers to the bacterial diversity / richness in a treated and / or cultured sample (composition) compared to the OTUs (and / or ASVs) in the origin sample.

[0314] "β-diversity" in "unweighted Unifrac similarity" ("unweighted Unifrac") or "weighted Unifrac similarity" ("weighted Unifrac") is a measure of similarity between two communities (or samples) that takes into account both: I. the presence / absence data of taxa (e.g., bacterial populations); and II. the phylogenetic relatedness of taxa. Weighted Unifrac similarity also takes into account the relative abundance of taxa in the population.

[0315] "β-diversity" as shown in the Bray-Curtis similarity measure is a measure of similarity between two communities (or samples) that refers to the abundance of taxa in the sample.

[0316] Each point on a β-diversity plot represents the entire microbial / viral / fungal composition profile of a sample. Samples with similar microbial / viral / fungal profiles are shown close to each other, while samples with different profiles are shown far apart.

[0317] The "Chao1 richness index", "Shannon index", "Simpson reciprocal", or any other parameter used in the examples for analyzing population dynamics has meanings known in the art.

[0318] Bacterial count

[0319] In the following examples, the bacterial load of the particles introduced into the culture vessel in dry form per gr is calculated. The weight of the particles is calculated based on the particles used to contact the bacteria.

[0320] The bacterial load was determined using the following known methods: DNA-based quantification (quantitative polymerase chain reaction - qPCR); and colony forming unit (CFU) determination known in the art. Results are presented on a logarithmic scale - per 1 gr of particles in the introduced container in dry form.

[0321] CFU measurement. Serial dilutions of the samples were made and the bacteria were plated on CDC plates, in triplicate. The plates were incubated for 48 - 72 h under anaerobic conditions at 37 °C before CFU counting.

[0322] qPCR measurement. Absolute abundance quantification was performed using quantitative real-time PCR. The standard curve was designed with 10-fold serial dilutions of plasmid DNA containing a single copy of the 16S gene. Total DNA was calculated by normalizing against the mean of the control standard curve and the control assumed genome size.

[0323] In the following examples, the terms "bacterial count" and "bacterial load" are used interchangeably.

[0324] Treatment and culture procedures for origin biological samples

[0325] Unless otherwise stated, all procedures were carried out under anaerobic conditions. Stool samples were collected in disposable stool containers and transferred to anaerobic conditions. Preparation of the inoculum from the fecal samples was carried out under anaerobic conditions. When testing the effect of aerobic culture, the treatment was carried out under anaerobic or aerobic conditions.

[0326] 1 mg to 450 gr of feces was mixed with a sterile anaerobic saline solution (50 ml – 6 L) and blended until the mixture was homogeneous, then filtered. The filtrate containing the bacterial population (hereafter referred to as fecal liquor – FL / "origin" sample; considered 100% in similarity calculations) was transferred to different fermentation containers (50 ml – 6 L). In the presence of particulate MCC:DCP (20:80, e.g., 20 gr MCC; 80 gr DCP), the bacterial population was cultured in a single container, in the medium detailed below, under anaerobic conditions. The contact and culture in the presence of the particles was carried out for a period of less than 14 days.

[0327] To assess the effect of compounds and the bacterial population, 1 mg to 50 gr of feces was mixed with 1 - 150 ml of sterile anaerobic saline solution. In the presence of 10 - 1,200 gr of particulate MCC:DCP (20:80 or 50:50; MCC:DCP), the bacterial samples were cultured in a single container, in the medium (1 - 150 ml) detailed below, under anaerobic conditions. The culture was carried out for a period of less than 14 days.

[0328] Medium exchange and treatment sampling process

[0329] During the entire cultivation process, the culture medium is exchanged several times. Before the culture medium exchange, mixing / stirring is stopped to allow the particles to settle. After the culture medium exchange, mixing is restarted.

[0330] To assess the effect of a compound on a bacterial population or the effect of a bacterial population on a compound, cultivation is carried out under static conditions or under stirring conditions. Sampling is performed at specific time points after the bacterial population has been exposed to the test compound.

[0331] Cultivation conditions include: stirring range of 50 - 750 RPM; temperature of 32 - 39 °C; and pH of 4.4 - 8.0.

[0332] Unless otherwise stated, for similarity parameter and bacterial load measurements, sampling is performed from the particle - attached bacterial phase. Phase separation is carried out by allowing the particles to settle, centrifugation, washing, or by filtration.

[0333] Sampling process

[0334] For measuring similarity and bacterial load, sampling is performed at several time points (referred to as: T1 or TP1, T2 or TP2, T3 or TP1) during the cultivation process.

[0335] Culture medium composition

[0336] The culture medium compositions used are detailed in the following examples. Pharmaceutical or food - grade components are used.

[0337] Example 1

[0338] Culturing multiple bacteria in a single container in the presence of particles

[0339] In the following example, the effect of culturing multiple bacteria in the presence of particles on the similarity level is assessed.

[0340] For this purpose, as detailed above in the "Materials and Methods" section, fecal samples are processed and cultured in the presence of particles. As a comparison, the processed fecal samples are cultured under the same conditions in the absence of particles. At the end of the cultivation, the similarity is determined using the weighted Unifrac similarity metric. The similarity of the compositions produced under the two cultivation conditions is compared with a reference fecal sample. At the end of the cultivation cycle, measurements are taken from: (i) the planktonic culture; and (ii) both the planktonic bacterial phase and the particle - attached bacterial phase of the culture carried out in the presence of particles.

[0341] The results show ( Figure 1 ), compared to the distance between the particle - attached bacterial fraction and the non - attached fraction, the planktonic culture samples are further from the origin sample.

[0342] These results indicate that culturing in the presence of particles results in a composition comprising planktonic bacteria and / or attached bacteria that has increased similarity compared to planktonic culturing (e.g., culturing in the absence of particles).

[0343] These results indicate that it is advantageous to culture the plurality of bacteria in the presence of particles to obtain a composition that is highly similar to the origin sample.

[0344] Accordingly, unless otherwise indicated, in all of the following examples, culturing is carried out in the presence of particles.

[0345] Example 2

[0346] Effect of the type of carbon source in the culture medium on similarity to the origin sample and on bacterial load

[0347] In the following examples, the effect of different carbon sources on the ability to grow human bacteria and produce a bacterial composition comprising a high similarity to the origin fecal sample is evaluated. In addition, bacterial load is also tested.

[0348] To this end, as detailed above in the "Materials and Methods" section, the bacterial composition is treated and cultured in a culture medium comprising a single carbon source (e.g., glucose) or a culture medium comprising multiple carbon sources. The multiple carbon sources include: glucose; maltose; trehalose; and starch. Each culture medium further comprises: yeast extract; peptone extract; sodium chloride; and disodium hydrogen phosphate. Culturing is carried out in flasks (small scale).

[0349] Similarity is determined by using the weighted Unifrac similarity metric, and bacterial load is determined by qPCR measurement. Sampling is carried out from the particle-attached bacterial phase at different time points (referred to as T1, T2, and T3).

[0350] The results show ( Figure 2 ) that all test groups have a similarity greater than 50% to the origin sample, and the bacterial load (qPCR) is greater than 1·E 8 / 1 gr of particles. However, compared to the single carbon source culture medium, using a multiple carbon source culture medium results in a higher bacterial load while maintaining a higher similarity.

[0351] Therefore, the current study indicates that it is advantageous to use more than one carbon source in the culture medium, resulting in an increase in similarity to the origin sample.

[0352] Example 3

[0353] Effect of adding trace elements to the culture medium on similarity to the origin sample

[0354] In the following examples, the effect of adding trace elements to a glucose medium or a multi-carbon source medium on the similarity between the composition and the origin sample was investigated. The trace elements used were: a manganese source; a copper source; and an iron source. In addition, the bacterial load was also tested.

[0355] To this end, as detailed above in the "Materials and Methods" section, the bacterial composition was treated and cultured in a medium containing a single carbon source (glucose) or a medium containing multi-carbon sources (glucose; maltose; trehalose; and starch), with or without the addition of trace elements. Each medium further included: yeast extract; peptone extract; sodium chloride; and disodium hydrogen phosphate. The culture was carried out in flasks (on a small scale).

[0356] Samples were taken at three time points (T1, T2, and T3) during the culture, and the weighted Unifrac similarity and the bacterial load (qPCR measurement) were evaluated. The reference sample was labeled as the "origin sample".

[0357] The results showed ( Figure 3 ) that similarities greater than 60% were obtained in all test groups. The addition of trace elements to the multi-carbon medium led to greater similarities - greater than 70%. The results also showed that when using a medium that included both trace elements and multi-carbon sources, there was a tendency for an increase in the bacterial load of the co-cultures obtained.

[0358] Example 4

[0359] Effect of culturing under aerobic conditions vs. anaerobic conditions on the similarity level with the origin fecal sample

[0360] In the following examples, the effect of anaerobic or aerobic conditions on the bacterial similarity level was investigated.

[0361] Under anaerobic or aerobic conditions, bacterial samples were treated and cultured using different culture medium matrices, as described above in the "Materials and Methods" section. The test media were: Brain Heart Infusion medium (BHI) and various food-grade media suitable for the growth of human intestinal bacteria. At the end of the culture, the similarity was evaluated (based on the Bray-Curtis metric) (the origin fecal fluid sample was labeled as "origin"; the anaerobic condition was labeled as AN).

[0362] The results presented as PCoA (Principal Coordinates Analysis, a form of Metric Multidimensional Scaling, MDS) ( Figure 4 ) showed that using anaerobic conditions during the culture was better for maintaining similarity.

[0363] Since it was found that anaerobic conditions were better for obtaining increased similarity, anaerobic conditions were used in all the following examples.

[0364] Example 5

[0365] Similarity with fecal origin samples when using different media and extended fermentation

[0366] In the following examples, the effects of the media used and the expansion process on the level of similarity were evaluated.

[0367] To this end, bacterial samples were processed and cultured using media substrates from different sources, as described above in the "Materials and Methods" section. The culturing was carried out in the presence of granules and under anaerobic conditions in small-scale volumes or medium-scale volumes (50 ml - 6 L). Table 1 below shows the conditions used during culturing.

[0368] To evaluate similarity and bacterial load, representative samples of granule-attached bacteria were collected from each treatment and the weighted similarity level of the bacterial population as well as the bacterial count (based on qPCR measurements) were analyzed.

[0369] Table 1. Treatment groups

[0370]

[0371] *Medium-scale production.

[0372] **Small-scale production.

[0373] ***Standard commercial medium for fecal bacteria growth.

[0374] The results showed ( Figure 5 ), compared to culturing in standard media (e.g., BHI), culturing in PG2 medium simultaneously led to increased bacterial counts and high similarity values. The current results also showed that culturing in PG2 medium favorably maintained a high similarity level over an extended period of time.

[0375] When comparing small-scale vs. medium-scale (fermenter) PG2 medium fermentations, the results showed that the similarity values were higher for fermentations carried out at medium scale. At time point 4, the bacterial counts for medium-scale and small-scale were respectively at 6·E 10 and 1·E 8 and above, indicating the scalability of the process.

[0376] In another experiment, the inventors also examined the number of culturing days required to obtain a composition / coculture with a similarity greater than or equal to 50% increase compared to the origin sample and a bacterial load of at least 1·E 6 / gr. It was found that the culturing period ranged between 6 hours and 6 days, and advantageously both parameters could be obtained (data not shown). When using multiple carbon sources, the culturing period was between 12 hours and 5 days, resulting in a weighted Unifrac similarity greater than or equal to 70% and at least 1·E8 The bacterial load of / gr.

[0377] Thus, the method can achieve scalability and obtain a composition similar to that of fecal biological samples.

[0378] Example 6

[0379] Consistency of the process when using samples of different origins

[0380] The following examples are intended to examine whether the method can still produce a composition highly similar to the origin sample even when using samples of different origins with different populations.

[0381] Figure 6 The results shown in display the combined data from three processes using three origin samples from the same donor collected on different dates. As described in the "Materials and Methods" section above, the samples were independently processed and cultured under mid-scale conditions (6 L) including a multi-carbon source medium (including: glucose; maltose; trehalose; starch; yeast extract; peptone extract; sodium chloride; disodium hydrogen phosphate; manganese source; copper source; and iron source).

[0382] The results show the weighted Unifrac similarity and bacterial load (qPCR & CFU measurements) from three independent cultures at different time points (T1 - T6).

[0383] The results indicate that similarities greater than 71% with the fecal origin sample (labeled "Origin") were obtained at all time points, with a relatively low SD, indicating that the method can consistently obtain a composition highly similar to the origin sample. In addition, the results also show that the similarity level decreases over time while the CFU and bacterial count (qPCR) increase (e.g., comparing T1 with T6).

[0384] This exemplifies the provision of a method according to an embodiment of the present invention to provide a composition with high similarity and increased bacterial count, even when using different origin samples with varying microbiome profiles.

[0385] In another experiment, the reproducibility and repeatability of the process were further evaluated.

[0386] For this purpose, five independent culture processes were carried out as described in the "Materials and Methods", each using a different origin sample. The five independent processes were analyzed by comparing the produced compositions with the corresponding origin samples in terms of the observed species, observed genera, and weighted similarity parameters.

[0387] The number of genera observed in the origin samples ranged from 42 to 58 (average of 50.1), while the compositions contained from 39 to 55 genera (average of 43.3; genus richness was 84.4% compared to the origin samples). The number of species observed in the origin samples ranged from 131 to 196 (average of 168.1), while the compositions contained from 104 to 188 species (average of 138.3; species richness was 82.8% compared to the origin samples).

[0388] The average weighted similarity of the origin samples for the five experiments was 77.1% (each sample compared to its corresponding origin sample).

[0389] The inventors determined the similarity using the Bray-Curtis values for the five experiments and found that it ranged from 19 to 46%, with an average of 35.1%. Thus, when using a similarity index that does not account for phylogenetic relatedness among bacterial populations (such as Bray-Curtis), lower values can be obtained compared to those obtained using other similarity metrics.

[0390] The results indicate that despite using different origins, these compositions maintain a similarity higher than 77% with their origin samples while showing comparable numbers of observed species and genera.

[0391] These results suggest that regardless of the variation in the origin sample communities, the method is capable of advantageously "replicating" the origin samples and obtaining synthetic compositions with high similarity to them.

[0392] Thus, various similarity metrics can be used to determine the similarity analysis of the compositions to the origin samples. However, some methods can result in lower similarity values, for example, as illustrated above.

[0393] The above examples demonstrate the ability of different embodiments of the present invention to obtain high similarity of bacterial populations while obtaining high bacterial counts. The following Examples 7 and 8 demonstrate the ability of the method to further retain other similarity characteristics - for example, similarity of other communities (such as viruses, fungi) in addition to the bacterial community; and similarity of metabolic pathways / paths.

[0394] Example 7

[0395] Similarity of other communities in the compositions produced according to embodiments of the present invention to the origin samples

[0396] In the following examples, the similarity of other communities in the compositions compared to the origin samples was evaluated.

[0397] As described in the "Materials and Methods" section above, fecal origin samples were processed and cultured, and the viral, fungal, and bacterial communities of the obtained compositions were analyzed using WGS. The similarity level was measured using Bray Curtis similarity. The plots are shown as non-metric multi-dimensional scaling (NMDS) of the Bray-Curtis similarity metric performed - between the compositions (squares) and reference fecal samples (diamonds).

[0398] Figures 7 - 9 They represent the similarity of the fungal, viral, and bacterial communities respectively. Similarity was observed by the proximity of the components in the plots to the origin samples.

[0399] The results showed that the compositions produced according to the embodiments of the present invention retained high fungal and viral similarity, and also confirmed the previous results showing high similarity of the obtained bacterial populations. In addition, the compositions were close to dozens of other representative stool samples from healthy individuals. Specifically, the compositions retained 100% of the fungal species, 71.4% of the bacterial viruses (genera), and 93.5% of the bacterial species (diversity recovery) identified in the original stool samples.

[0400] The results showed that the method was advantageously able to produce compositions with microbial communities having high similarity to the origin samples, thus showing high potential for use as pharmaceutical compositions in a manner similar to FMT treatment.

[0401] Example 8

[0402] Maintenance of metabolic pathways in compositions produced according to embodiments of the present invention

[0403] In the following examples, the ability of the method of the present invention to retain the metabolic pathways of the origin fecal samples was evaluated.

[0404] The ability to maintain metabolic pathways / paths was examined using publicly available analysis software based on WGS data, e.g., the potential to produce, synthesize, consume, and / or utilize certain metabolites / organic compounds.

[0405] The results are shown in Figure 10 .

[0406] It can be seen that 104 metabolic super-paths (104 points appear on the graph) out of 108 were retained in the compositions, with an R-squared value of 0.926. The results showed that the relative proportions between these paths were substantially retained.

[0407] This example shows that in addition to the bacterial populations and other microorganisms that may be present in the origin samples, the method also advantageously retains the metabolic potential of the origin samples.

[0408] Since the method according to the invention advantageously enables the production of different compositions, for example, (i) a composition comprising particle-attached bacteria, (ii) a composition comprising planktonic bacteria cultured in the presence of particles, and (iii) a composition comprising two parts - either a mixture of (i) and (ii) in any proportion or combined parts, Examples 9, 10, and 11 below show the advantageous embodiments of the various compositions.

[0409] Example 9

[0410] Different compositions produced according to the invention

[0411] In the following examples, the growth preferences (at the family taxonomic level) of the 10 most abundant bacteria in a test composition sample were examined, i.e., the preference for planktonic or attached growth forms. The analysis was carried out by measuring the difference between the relative abundances of each bacterium in the particle-attached phase and the non-attached phase. For illustrative purposes, two graphs are shown in Figures 11A - 11B as follows.

[0412] The results showed that among the top 10 most abundant bacteria, 3 bacterial families were more abundant in the attached fraction, while 4 bacterial families were more abundant in the non-attached phase. Similar relative abundances of three other bacterial families were also found in both phases. Thus, the inventors demonstrated the potential role of particles in maintaining a bacterial diversity in the composition similar to that of the origin sample.

[0413] Thus, the method of the invention advantageously enables the production of isolated fractions enriched in specific bacteria, which can be used as independent compositions. These two isolated compositions may also be mixed in any desired proportion to obtain a final composition enriched in specific bacteria of interest - for example, having a positive correlation with the improvement, recovery, or alleviation of a certain disease or condition.

[0414] Optionally, by including both the attached and non-attached phases simultaneously, the presence / abundance and relative abundances of most taxonomic groups can be retained after culturing, regardless of the bacterial phase preference, thus maintaining the original ecosystem of the origin sample.

[0415] Example 10

[0416] Similarities of different compositions produced according to the invention

[0417] As is known, an increase in microbial diversity observed in healthy individuals can restore the protective function of the microbiome against Clostridium difficile. Therefore, it is crucial to produce microbial compositions with a high microbial diversity similar to that of fecal samples.

[0418] In the following examples, diversity recovery was analyzed in different compositions produced according to the present invention (i.e., compositions comprising particle-attached bacteria, compositions comprising unattached fractions, and combined compositions comprising both fractions). Bacterial diversity was measured at the genus and species levels. In addition, the bacterial load in each fraction was also measured by qPCR. Furthermore, the weighted similarity of different fractions was also evaluated.

[0419] Figures 12A - 12B δ-diversity recovery (at the genus and species levels, respectively) between two test compositions was shown.

[0420] The results showed that, compared to the unattached fraction, the combined fraction and the particle-attached fraction exhibited increased diversity recovery of genera and species from the origin sample, indicating a potential role of the particles in maintaining a bacterial diversity similar to that of the origin sample in the composition. In addition, the present inventors also found that the combined fraction exhibited an increased bacterial load compared to the use of each fraction alone (data not shown).

[0421] Therefore, combining the two fractions can be advantageous compared to each separate composition - for example, when a composition with increased diversity recovery and increased bacterial load is desired. In addition, it was also found that the combined composition had an increased weighted similarity compared to the use of each fraction alone.

[0422] Example 11

[0423] Relative abundances of different compositions produced according to the present invention

[0424] In another analysis, the proportions (relative abundances) between different taxonomic bacterial groups (at the family level) were compared between different fermentation samples / compositions (particle-attached phase, unattached phase, and combined composition). The proportions obtained in each composition were compared with those in the corresponding origin sample. Data were obtained from multiple experiments using different origin samples.

[0425] In addition, the proportions in samples obtained from planktonic cultures (cultures in the absence of particles) performed under culture conditions similar to those with the presence of particles were also evaluated. The proportions are shown in Table 2 below.

[0426] The selected bacterial groups analyzed belong to the most abundant bacteria present in the test samples. In addition, it has also been reported in the literature that these bacterial groups are potential producers of short-chain fatty acids (SCFAs) and bile acid converters - which are associated with positive treatment outcomes for various diseases or conditions such as Clostridium difficile infection, atopic dermatitis, and ulcerative colitis.

[0427] Table 2. Proportions between different bacterial taxonomic groups in different samples and the origin sample

[0428]

[0429]

[0430] NS – No significant difference

[0431] These results indicate that, in most cases, it is necessary / advantageous to combine these two parts into a final composition in order to better resemble the origin sample and retain the proportions between different bacterial families.

[0432] Importantly, in most cases, the unattached phase (produced according to the method described above) shows less deviation from the test proportions in the origin sample compared to the proportions during planktonic culture (i.e., culture without particles). Thus, culturing in the presence of particles unexpectedly promotes the retention of the test proportions.

[0433] These results also confirm the previous results and indicate that, in order to obtain a composition with high similarity to the origin sample, it is necessary / advantageous to culture the plurality of bacteria in the presence of particles.

[0434] Example 12

[0435] Culturing a pooled origin sample in a container

[0436] In the following examples, the advantages of using a pooled origin sample from more than one origin (e.g., donors) as starting material in a method according to an embodiment of the present invention are investigated.

[0437] For this purpose, several characteristics are evaluated: (i) various alpha-diversity parameters of the pooled origin and the produced composition vs. the isolated samples and their respective produced compositions; and (ii) the ability of the culturing method according to an embodiment of the present invention to retain difficult-to-culture bacteria when using a pooled sample compared to using a single-source origin. Three different origins were sampled for this experiment.

[0438] Table 3 below shows the alpha-diversity parameters for two different origins and the compositions prepared therefrom.

[0439] Surprisingly, it can be seen that the pooled origin sample, which comprises various microbial populations characterized by having different culturing requirements, results in a composition with increased alpha-diversity parameters compared to the composition produced from a single source.

[0440] Table 3. Alpha-diversity parameters in single and pooled origins and in the compositions prepared from such origins

[0441]

[0442]

[0443] * represents the mean of three different origins for the collection of origin samples.

[0444] In addition, the ability of this cultivation method to produce a composition for preserving fastidious bacteria was also investigated. As a representative genus, the relative abundance of the bacterium Akkermansia was evaluated.

[0445] According to the literature, Akkermansia is a promising next-generation probiotic and also a fastidious bacterium in the complex bacterial population (Derrien, M., Vaughan, E.E., Plugge, C.M. and de Vos, W.M., 2004. Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium. International journal of systematic and evolutionary microbiology, 54(5), pp. 1469-1476).

[0446] Figure 13 It was shown that at cultivation time point 1, origin sample 3 contained Akkermansia. However, at time point 2, the test bacteria were not preserved in the composition. It can be seen that at cultivation time point 2, the collection of several origin samples led to the preservation of Akkermansia.

[0447] In addition, the collection samples also improved the preservation of other potentially beneficial genera, such as Roseburia, Lachnoclostridium-Roseburia, Fusicatenibacter, Coprococcus, Ruminiclostridium (data not shown).

[0448] Therefore, advantageously, according to an embodiment of the method of the present invention, using a collection sample as the starting material (vs. using a single origin sample) can provide conditions for achieving the preservation of various bacterial genera.

[0449] Advantageously, the method can produce a composition that has a high similarity to the origin sample and can potentially be used as a pharmaceutical composition as exemplified below. The cultivation system according to the present invention is advantageously similar to the human intestinal microbiota and can in particular be used as an in vitro model for assessing the effect of a compound on a subject (as exemplified below).

[0450] Example 13

[0451] A co-culture comprising a plurality of bacteria originating from a saliva sample, cultured in a container in the presence of particles and under anaerobic conditions

[0452] In this example, the ability to grow a saliva bacterial population from a saliva sample according to an embodiment of the present invention was examined.

[0453] As described in the "Materials and Methods" section above, saliva was cultured in a flask using a medium comprising: a multi-carbon source, yeast extract, peptone extract, sodium chloride, disodium hydrogen phosphate, trace elements, and particles.

[0454] The flasks were incubated in an anaerobic chamber and sampled throughout the fermentation process. The CFU / ml reached was approximately 1·E 8 .

[0455] The results showed a weighted similarity higher than 50%, indicating that the method according to the present invention is suitable for culturing a plurality of bacteria originating from a saliva sample (data not shown).

[0456] Non-limiting examples of systems / compositions according to embodiments of the present invention

[0457] The following experiments illustrate that an in vitro system / model generated according to an embodiment of the present invention can be effectively used as, for example, a gastrointestinal model to determine the effect of a compound on the microbiome of a subject; and that different small biota samples can be used to establish the model. In addition, these experiments illustrate that the system can be used to generate different microbial profiles.

[0458] As an example of the effectiveness and reproducibility of the system in evaluating the effect of a compound on a plurality of bacteria, different compounds or known drugs were examined.

[0459] Unless otherwise indicated, the compound was added to the in vitro model system during inoculation. The effect of the compound on the plurality of bacteria was examined in the particle-attached bacterial phase and the non-attached bacterial phase. Compound_A, Compound_A', and Compound_D were added at therapeutic use concentrations and increasing concentrations.

[0460] In all of the following experiments, the culture was carried out in a multi-carbon source medium (glucose; maltose; trehalose; starch) further comprising a combination of trace elements (manganese source; copper source; iron source); yeast extract; peptone extract; sodium chloride; and disodium hydrogen phosphate.

[0461] Example 14

[0462] Microbiome profiles of different samples

[0463] In the following examples, the ability to generate different systems / models comprising similarities to different origin samples with different small biota population profiles was examined.

[0464] In addition, this embodiment also demonstrates: verification of the ability of the system to maintain similarity with the initial sample under small-scale conditions; and investigation of the sensitivity of the system to identify changes in the object microbiome profile by culturing samples obtained from each donor on three different dates (i.e., by obtaining 3 samples from donor 1 for 3 independent experiments).

[0465] For the above purposes, small biota populations including multiple bacteria were obtained from four different donors and 4 systems were generated ( Figure 17 ); donor 1 provided origin samples at three different independent time points; that is, several systems were generated simultaneously: 4 systems from 4 different donors; and another 2 systems were generated from experiment 1 and experiment 2 of donor 1 (a total of 6 in vitro culture systems). The particulate-attached bacterial phase was sampled several times.

[0466] The β-diversity (Bray-Curtis) of different samples obtained from 6 different culture systems was investigated and presented by non-metric multidimensional scaling (NMDS).

[0467] Results ( Figure 17 ) showed that each of the 4 donors was located at a certain distance from the other donors on the graph, and different samples collected from the same donor at different time points during the entire culture cycle were located in the same region on the graph. These results indicate that different systems can be generated according to the unique initial microbial patterns of the donors, and the systems can maintain the unique initial microbial patterns for a long time.

[0468] When investigating the β-diversity of the microbiome populations in the systems generated from three different origin samples from the same donor for three consecutive days, all samples were located in the same region on the graph, with slight variation from each other. This indicates that the system is capable of identifying slight / subtle changes in the microbiome profile.

[0469] Example 15

[0470] Effect of Compound_A on the bacterial load of different culture systems generated from different samples from different donors

[0471] The previous examples showed that different systems generated from different origin samples and different donors led to different small biota population profiles.

[0472] To investigate whether the system can accurately determine the effect of a compound on multiple bacteria (regardless of the microbiome profile), Compound_A was contacted with 4 fecal samples from 4 donors. After the fecal samples were contacted with the particles, the compound was added to different culture systems and incubated with the bacterial population during the culture cycle.

[0473] The effect of the compound on bacterial counts (based on qPCT) was examined at three time points (designated as TP1; TP2 and TP3) in different generated systems and is shown in Figure 18 as follows.

[0474] Figure 18 It is shown that the addition of compound_A resulted in an increase in bacterial counts at the three test time points in all 4 generated systems (all shown on the same graph) compared to the untreated control system, indicating that the system is capable of identifying the common effect of the compound on the microbial population regardless of the initial bacterial profile of the system.

[0475] Example 16

[0476] Effect of compound_A on the microbial profiles of different culture systems generated from different samples

[0477] To better define the ability of the system to examine the effect of the compound on the microbiome profile and to identify changes in the microbiome community, compound_A was added to 4 different systems (generated from 4 fecal samples obtained from 4 different donors) and α- and β-diversity were examined. As pointed out in the previous examples, the compound was added during system cultivation and sampling was performed at three different time points (TP1, TP2 and TP3).

[0478] Figure 19 A- Figure 19 D shows the α-diversity in each system. The graphs show the α-diversity of samples before cultivation and at 3 time points during the cultivation period in the untreated control system and the system treated with compound_A. At each time point, the mean values of TP1, TP2 and TP3 were calculated and used to evaluate the effect of the compound.

[0479] The results showed that the addition of compound_A to each system resulted in a significant increase in α-diversity compared to the untreated control bacterial population - regardless of the origin sample.

[0480] Figure 20 A- Figure 20 D shows the β-diversity of each system. The graphs show the diversity at 3 time points during the cultivation period in the untreated control system and the system treated with compound_A (all time points are shown on the same graph).

[0481] The results showed that the bacterial population was modulated / altered when exposed to the test compound compared to the untreated control bacterial population.

[0482] These results indicate that, according to the embodiments of the present invention, the systems illustrated herein are capable of identifying an increase in the number of species and any change in the level of similarity as a result of exposure to the compound.

[0483] Example 17

[0484] Modulatory effect of Compound_A on specific bacterial genera

[0485] The previous examples showed that the culture system according to an embodiment of the present invention is an effective tool capable of identifying the combined effects or trends of compounds on bacterial load and / or on the microbiome profile - regardless of the initial microbial profile of the origin sample / donor.

[0486] The purpose of the following examples is to use the data extracted from the system (e.g., bacterial load, α-diversity, and / or β-diversity) and further explore and identify the bacteria that are modulated upon exposure of the bacterial population to the test compound. As illustrated above, bacterial modulation can be seen through the changes in the positioning and / or distribution of the points of the treated system vs. the untreated system on the NMDS plot.

[0487] Figure 21 Heatmap showing different bacterial genera affected by exposure of the bacterial population to Compound_A. Each experiment was performed in 4 treated systems simultaneously (Samples 1 - 4). Analysis was performed in comparison to the untreated control bacterial population.

[0488] In these experiments, a change at the genus level was considered significant if the same trend (increase or decrease) was observed in 3 out of 4 test samples (i.e., 75% of the samples showed the same trend). The results showed that a total of 9 genera increased and 3 genera decreased. Similar trends were observed when comparing the biomarkers identified by the culture system with those identified in clinical trials using the same compound.

[0489] Advantageously, examining the effect of a compound on the microbiome population in a culture system according to an embodiment of the present invention - simulating the microbiota of a subject - can provide insights into the modulation of the bacterial population - in view of exposure to such a compound. Identifying the modulated bacterial genera (or modulation at any other taxonomic level) can be a therapeutic target for improving the efficacy of a drug (e.g., by administering to the subject a drug - probiotic or drug - prebiotic combination therapy aimed at restoring / reserving the original or a healthier microbiome profile).

[0490] Example 18

[0491] Use of the system according to an embodiment of the present invention as a high - throughput screening assay and as a platform for generating different microbiome profiles

[0492] In the following examples, the effects of two compounds, namely Compound_A and its derivative (Compound_A’), on the relative abundances were compared.

[0493] Under the same conditions, two compounds were added simultaneously to two different systems, and samples were taken at five time points throughout the culture period for relative abundance measurement. The results were compared with untreated control systems sampled at the same time points (a total of three systems were generated from the same original sample / donor).

[0494] Results ( Figure 22 ) showed that the relative abundances of genera at all five time points changed after treatment with Compound_A or Compound_A’ relative to the untreated system. It was also seen that the two treatment groups exhibited different microbial profiles.

[0495] These results highlight the sensitivity and ability of the systems and methods used simultaneously in several individual containers as high-throughput screening assays - for comparing the effects of different treatments (e.g., different drug derivatives) on the microbiome population of an object - and also show that the systems can be used to generate different microbial profiles.

[0496] Example 19

[0497] Sensitivity of the culture system to different compounds

[0498] In another experiment, the ability and sensitivity of the system to identify changes in the microbiome profile and bacterial count - in response to different compounds, namely Compound_D and Compound_J - were evaluated.

[0499] For this purpose, each compound was added to separate systems generated from the same origin sample, and similarity parameters and bacterial counts were evaluated. Sampling was done at different time points during different stages detailed below.

[0500] Table 4 shows the effects of the compounds on alpha diversity and bacterial count.

[0501] Table 4. Effects of compounds on richness and bacterial count

[0502]

[0503] *Percentage change compared to the value of the relatively untreated particle-attached bacterial fraction.

[0504] **Fold change compared to the count of the untreated combined fraction. Bacterial counts in each sample were measured by CFU.

[0505] Exposure of bacteria to Compound_D resulted in a significant reduction in bacterial count, which then gradually increased to a level close to that of the untreated fraction. In contrast, exposure of bacteria to Compound_J resulted in a 7.450-fold increase in bacterial count, which then decreased.

[0506] In response to exposure to Compound_D, α-diversity decreased, while exposure to Compound_J led to a slight increase in richness.

[0507] Notably, for both test parameters, the effects observed after exposure to Compound_D were similar to those known in the art.

[0508] Thus, these results confirm previous results and demonstrate the effectiveness and sensitivity of the system.

[0509] Secondly, the ability to evaluate the effects of compounds using the particle-attached and non-attached fractions was investigated. To this end, after contact of the bacteria with Compound_D, the relative abundances of nine specific genera in both fractions were measured. The results are presented in Figure 23 .

[0510] Overall, the results showed that the microbial profiles of the fractions were differentially affected after exposure to the compounds. For example, at TP3, the relative abundance of Genus 1 - in response to Compound_D - resulted in 36.25% and 87.70% for particle-attached and non-attached bacteria, respectively (see Figure 23 ; upper figure).

[0511] The results demonstrate the importance of evaluating the effects of added compounds on microbial populations in the two combined and / or separated fractions. In addition, the results also confirm our previous results, showing a trend for genera to adhere to particles and / or prefer to grow in the planktonic form. Notably, the tendency of specific bacteria to adhere to particles may imply the possibility of translocation to the target site.

[0512] While performing the sensitivity experiments, the ability of the "stable" or "initial" system to show changes in the microbial profile and bacterial count was evaluated by adding Compound_D, Compound_F, and Compound_J at two different time points. "Initial" - at the time of contact of the compounds with the plurality of bacteria during the inoculation phase before addition of particles to the system; and "stable" - at the time of addition of the compounds after contact of the plurality of bacteria with the particles and addition of the growth medium. These conditions may represent different physiological conditions of the subject, e.g., a more stable healthy microbiome vs. a dysbiotic microbiome. The α-diversity and bacterial count results are presented in Figure 5 .

[0513] Table 5. Effects of various compounds on richness and bacterial load

[0514]

[0515] * Percentage change compared to the value of the relatively untreated particle-attached bacterial fraction.

[0516] Fold change compared to the unprocessed combined fraction count. Bacterial counts in each sample were determined by the CFU method.

[0517] The results showed that compared to bacteria being exposed to the "stable system", bacteria being exposed to Compound_D and Compound_F at the "initial" stage significantly reduced the α-diversity similarity level. However, bacteria being exposed to Compound_D had a similar impact at these two stages.

[0518] The α- and β-diversity parameters after exposure to several other compounds under "initial" or "stable" conditions confirmed these results and indicated that the "stable system" was less sensitive to changes in the bacterial population compared to the "initial system".

[0519] These results suggest that it is advantageous to evaluate the impact of compounds at different stages of the method.

[0520] Example 20

[0521] Use of the system according to an embodiment of the invention for producing a composition enriched in bacteria attached to particles by adding a compound

[0522] In the following examples, the use of the system for enhancing specific bacterial growth forms, namely bacteria attached to particles or in planktonic growth form, was investigated.

[0523] For this purpose, Compound_B was added to the culture system prepared as described above and incubated with the bacterial population during the culture period (vessel 2). The compound was added after the plurality of bacteria had contacted the particles. The impact of the compound on the growth preference of four selected bacteria in the entire bacterial population, both in the attached phase and the planktonic phase, was investigated. The results were compared with an untreated control system (vessel 1).

[0524] Figure 24 It was shown that in view of the exposure to Compound_B, the relative abundance of Bacterium 1 increased, while no difference was observed in the preferred growth form (planktonic growth trend). For Bacterium 2 and Bacterium 4, the addition of Compound_B led to a change in the growth form. Bacterium 2 showed a preference shift from the attached form to the planktonic form, while Bacterium 4 showed the opposite trend. For Bacterium 3, the addition of Compound_B led to a preference for the attached growth form, while in the untreated system, substantially similar relative abundances were observed in both parts.

[0525] These results indicate that the system according to the invention can be used to identify compounds capable of enhancing specific bacterial growth forms of specific bacteria in a plurality of bacterial populations, and can be used to produce a composition enriched in specific bacteria in the attached form and / or the unattached form.

[0526] Although the present invention has been described in connection with specific embodiments thereof, it is evident that numerous alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. A method for producing a composition, the composition comprising a co-culture, the co-culture comprising a plurality of bacteria grown in the presence of particles, the co-culture comprising: i) A similarity of at least 30% to the origin sample, and ii) at least 1·E 4 / 1gr particle bacterial load, the method comprising: Providing a microorganism comprising a plurality of bacteria derived from an origin sample; Contacting the plurality of bacteria with a particle and causing at least a portion of the plurality of bacteria to attach to the particle; and Culturing the plurality of bacteria that are at least partially attached to the particle in a growth medium for a period of less than 14 days, wherein the culturing comprises culturing under anaerobic conditions, Thereby producing the composition, the composition comprising a co-culture, the co-culture comprising a plurality of bacteria grown in the presence of particles, the co-culture comprising: i) at least 30% similarity to the origin sample; and ii) at least 1·E 4 bacterial load per 1 gr of particles.

2. The method according to claim 1, wherein the plurality of bacteria are characterized by having different growth, culturing, and / or proliferation conditions selected from the following: metabolic requirements, nutritional requirements, pH, temperature, aerobic, obligate anaerobic, facultative anaerobic, microaerophilic, attachment form, planktonic, growth medium, flow, shaking, stirring, agitation, static, moisture, low humidity, and any combination thereof.

3. The method according to claim 1 or 2, wherein the cultivation is carried out until the co-culture reaches a similarity with the origin sample of greater than or equal to 30%, and the bacterial load is at least 1·E 4 / 1 gr granule.

4. The method according to any one of claims 1 to 3, wherein the culturing period ranges from 6 hours to 14 days.

5. The method according to any one of claims 1 to 4, wherein when using any one of the following: next-generation sequencing (NGS) technology, whole-genome sequencing (WGS), or both, and determining the similarity by a measure considering the genetic relatedness of the bacteria, the co-culture comprises at least 30% similarity to the origin sample.

6. The method according to any one of claims 1 to 5, wherein the similarity comprises at least 50% weighted Unifrac similarity.

7. The method according to any one of claims 1 to 6, wherein the similarity comprises at least 70% weighted Unifrac similarity, and the co-culture comprises at least 1·E 8 / 1 gr particle bacterial load.

8. The method according to any one of claims 1 to 7, wherein the similarity between the co-culture and the origin sample comprises similarity between bacterial populations.

9. The method according to any one of claims 1 to 8, wherein the origin sample further comprises additional microorganisms, and the additional microorganisms comprise any one of archaea, viruses, fungi, or any combination thereof.

10. The method according to claim 9, wherein the co-culture further comprises the additional microorganisms.

11. The method according to claim 10, wherein the similarity between the co-culture and the origin sample further comprises similarity of at least one of archaea, virus, fungus populations, or any combination thereof.

12. The method according to any one of claims 1 to 11, wherein the plurality of bacteria provided belong to at least 5 bacterial species and / or at least 2 bacterial genera.

13. The method according to any one of claims 1 to 12, wherein the composition comprises bacteria in planktonic form and bacteria that are at least partially attached to the particle.

14. The method according to any one of claims 1 to 13, wherein the growth medium comprises at least two carbon sources.

15. The method according to any one of claims 1 to 14, wherein the growth medium comprises a carbon source that is from at least two chemical groups selected from monosaccharides, disaccharides, polysaccharides, and any combination thereof.

16. The method according to any one of claims 1 to 15, wherein the growth medium comprises at least one monosaccharide, at least one disaccharide, and at least one polysaccharide.

17. The method according to any one of claims 1 to 16, wherein the contacting step, the culturing step, or any one of the two is carried out in a single container.

18. The method according to any one of claims 1 to 17, further comprising the step of separating bacteria not attached to the particles from bacteria attached to the particles at at least one time point selected from: before, during, after the culturing step, and any combination thereof, thereby producing: (i) a composition comprising bacteria in a planktonic form; and / or (ii) a composition comprising bacteria attached to the particles.

19. The method according to any one of claims 1 to 18, wherein the plurality of bacteria are provided in a container, and wherein the method further comprises the following steps: adding at least one compound to the container; and determining any feature selected from: (i) bacterial diversity; (ii) relative bacterial abundance; (iii) bacterial load; (iv) any other effect of the at least one compound on the plurality of bacteria; (v) any change in the at least one compound; and (vi) any combination of (i) to (v); thereby evaluating: the effect of at least one compound on a plurality of bacteria; the effect of a plurality of bacteria on at least one compound; or both.

20. The method according to any one of claims 1 to 19, wherein the origin sample is selected from: derived from at least one origin; derived from at least one subject; is a microbiome sample, a skin sample, an oral sample, a fecal sample, a vaginal sample; comprises intestinal microbiota; and any combination thereof.

21. The method according to claim 19 or 20, wherein the compound changes: (i) at least 30% of the similarity level at the end of the culture period, (ii) the bacterial load of the 4 / 1 gr granule at the end of the culturing step, or both (i) and (ii). 4 ​ 22. The method according to claim 21, wherein the change comprises increasing or decreasing any one of: the similarity level, the bacterial load, or both.

23. The method according to any one of claims 19 to 22, wherein the method is carried out simultaneously in a plurality of single containers.

24. The bacterial diversity; the relative bacterial abundance; the bacterial load; and / or the other effect of the at least one compound on the plurality of bacteria are compared with the corresponding features in the plurality of bacteria of the origin sample, and wherein a change in the feature indicates that the at least one compound has an effect on the plurality of bacteria.

25. The method according to any one of claims 19 to 24, further comprising culturing a control plurality of bacteria not exposed to the compound in a separate container, and wherein the bacterial diversity; the relative bacterial abundance; the bacterial load; and / or the other effect of the at least one compound on the plurality of bacteria are compared with the corresponding features in the control plurality of bacteria, and wherein any change in the feature indicates that the compound has an effect on the plurality of bacteria.

26. The method according to any one of claims 19 to 25, wherein the addition and / or determination is carried out using bacteria attached to the particles, bacteria not attached to the particles, or both.

27. A composition produced by the method according to any one of claims 1 to 18 and optionally further comprising an acceptable carrier or excipient.

28. The composition according to claim 27, which is a pharmaceutical composition for modulating the microbiota of a subject in need thereof.

29. The composition according to claim 27, which is used in an in vitro method to evaluate: the effect of at least one compound on a plurality of bacteria; the effect of a plurality of bacteria on at least one compound; or both.

30. The composition according to claim 29, wherein the in vitro method is the method according to any one of claims 19 to 26.

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