Microbial compositions, methods for their preparation and uses

Abstract

The present invention, in some embodiments, relates to methods for producing a composition comprising a co-culture comprising multiple bacteria having a high % similarity and bacterial load to an original sample.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 292,536, entitled "SCREENING SYSTEMS AND ASSAYS USING BACTERIA," filed December 22, 2021, and Israeli Patent Application No. 286748, entitled "MICROBIAL COMPOSITIONS AND METHODS OF PREPARING SAME," filed September 27, 2021, the contents of which are incorporated herein by reference in their entireties.

[0002] FIELD OF THE INVENTION The present invention is in the field of microbiology, and in particular relates to complex microbial compositions and methods for preparing and using the same. [Background technology]

[0003] There is growing evidence advocating the use of probiotics to promote human health, for example by benefiting the immune system and suppressing infection.

[0004] The human body contains bacterial cells, creating a network of bacterial-human cell interactions that profoundly influences health and even behavioral patterns. For example, the gastrointestinal tract harbors a rich and diverse microbial community. This complex system provides an environment, or niche, for many different microbial communities, including diverse bacterial species. Microbial flora also inhabit other body regions, such as the skin, nails, eyes, oral and upper respiratory tracts, and urogenital tract.

[0005] A healthy microbiota includes a balanced community of bacteria colonized to provide multiple benefits to the host, such as resistance to a wide range of pathogens, production and absorption of essential nutrients, and well-regulated systemic immunity. In situations of "dysbiosis" or disrupted symbiosis, the function of the microbiota can be lost or impaired, resulting in increased susceptibility to pathogens, altered metabolic profiles, or the induction of pro-inflammatory signals that can lead to local or systemic inflammation or autoimmunity. Therefore, microbial flora, such as the gut microbiota, are thought to play an important role in the pathogenesis of many diseases and disorders, including, but not limited to, various pathogenic infections of the intestine.

[0006] Current practice for treating intestinal dysbiosis involves fecal transplantation from healthy donors. Fecal transplantation, also known as fecal microbiota transplantation (FMT), is a method for reestablishing and / or modulating the recipient's gastrointestinal microbiota with the goal of preventing, treating, and / or ameliorating a disease or condition. Research has shown that FMT can restore health-related bacteria in the lower intestine, thereby treating, for example, Clostridium difficile (C. diff) infection and preventing its recurrence (recurrent C. diff, rCDI). C. diff is one of the causes of healthcare-associated diarrhea, often due to a severely disrupted microbiome, exacerbated by antibiotic use, for example. According to a systematic search [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 shown a cure rate of over 90% among C. diff patients who received some FMT treatment. Although FMT is rapidly becoming an accepted treatment for many other diseases associated with gut microbiome disturbances [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], concerns remain regarding its safety, poor reproducibility, and scalability of manufacturing.

[0007] There remains a great need for methods to prepare bacterial compositions that are highly similar to the source sample, e.g., a fecal sample, that can be used to treat dysbiosis.

[0008] Advantageously, such methods may eliminate the need for extra source sample collection, reduce safety risks associated with the use of biological samples, and preserve the original microbiota profile while enabling scalability and reproducibility of manufacturing.

[0009] Such compositions can also be used, inter alia, as a system to simulate a subject's in vitro microbial environmental niche, e.g., a subject's gastrointestinal flora, and thus provide insight into the modulation of bacterial populations in light of exposure to at least one compound. Summary of the Invention [Problem to be solved by the invention]

[0010] In some embodiments of the present invention, methods are provided for producing a composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles, wherein the co-culture has i) at least 30% similarity to the source sample, and ii) at least 1 E / g of particles. 4 Contains a bacterial load of

[0011] In some aspects of the invention, compositions produced according to the methods of the invention are provided.

[0012] In some embodiments of the present invention, in vitro methods are provided for assessing the effect of at least one compound on a plurality of bacteria, a plurality of bacteria on at least one compound, or both. [Means for solving the problem]

[0013] In some embodiments, the method includes providing a microorganism comprising a plurality of bacteria from a source sample; contacting the plurality of bacteria with particles to at least partially adhere the plurality of bacteria 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, wherein the culturing includes culturing under anaerobic conditions, whereby i) a similarity to the source sample of at least 30% and ii) a concentration of at least 1 E per gram of particles is achieved. 4 The method produces a composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles comprising a bacterial load of 1000 μg / ml.

[0014] In some embodiments, the method includes providing a plurality of bacteria from a source sample; contacting the plurality of bacteria with particles to at least partially adhere the plurality of bacteria 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, wherein the culturing includes culturing under anaerobic conditions, whereby i) a similarity to the source sample of at least 30% and ii) a density of at least 1 E per gram of particles is achieved. 4 The method produces a composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles comprising a bacterial load of 1000 μg / ml.

[0015] In some embodiments, the plurality of bacteria are characterized by having different growth, cultivation and / or proliferation conditions selected from the group consisting of metabolic requirements, nutritional requirements, pH, temperature, aerobic, obligately anaerobic, facultatively anaerobic, microaerophilic, attached, planktonic, growth medium, fluid, shaking, agitation, stirring, static, moist, low humidity, and any combination thereof.

[0016] In some embodiments, culturing is performed to determine whether the co-culture has a similarity of 30% or more to the source sample and at least 1 E / g of particles. 4 This is carried out until a bacterial load of

[0017] In some embodiments, the culture period ranges from 6 hours to 14 days.

[0018] In some embodiments, the co-culture comprises at least 30% similarity to the source sample, where similarity is determined by a metric that considers the genetic relatedness of bacteria using any one of next-generation sequencing (NGS) technology, whole genome sequencing (WGS), or both.

[0019] In some embodiments, the similarity comprises a quantitative Unifrac similarity of at least 50%. In some embodiments, the co-culture comprises a quantitative Unifrac similarity of at least 50%.

[0020] In some embodiments, the co-culture has at least 70% similarity to the source sample and at least 1 E per gr of particles. 8 Contains a bacterial load of

[0021] In some embodiments, the similarity comprises a quantitative Unifrac similarity of at least 70%, and the co-culture has at least 1 E per gr of particles. 8 Contains a bacterial load of

[0022] In some embodiments, the similarity comprises any one of the following: the presence of bacterial taxonomic classes, genetic relatedness, phylogenetic distance between observed bacteria, bacterial diversity, bacterial abundance, relative abundance, or any combination thereof.

[0023] In some embodiments, the similarity between the co-culture and the source sample comprises the similarity between bacterial populations.

[0024] In some embodiments, the source sample further comprises additional microorganisms, including any one of archaea, viruses, fungi, or any combination thereof.

[0025] In some embodiments, additional microorganisms are contacted with the particles.

[0026] In some embodiments, the additional microorganisms are at least partially attached to the particles.

[0027] In some embodiments, the co-culture further comprises an additional microorganism.

[0028] In some embodiments, the similarity between the co-culture and the source sample further comprises similarity in at least one of archaeal, viral, fungal populations, or any combination thereof.

[0029] In some embodiments, the plurality of bacteria provided belongs to at least five bacterial species and / or at least two bacterial genera.

[0030] In some embodiments, the composition comprises bacteria in a planktonic form and bacteria at least partially attached to particles.

[0031] In some embodiments, the growth medium comprises at least two carbon sources.

[0032] In some embodiments, the growth medium comprises a carbon source from at least two chemical groups selected from the group consisting of monosaccharides, disaccharides, polysaccharides, and any combination thereof.

[0033] In some embodiments, the growth medium comprises at least one monosaccharide, at least one disaccharide, and at least one polysaccharide.

[0034] In some embodiments, the contacting step, the culturing step, or both, occur in a single vessel.

[0035] In some embodiments, the method further comprises separating bacteria not attached to particles from bacteria attached to particles at least one time point selected from the group consisting of 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 particles.

[0036] In some embodiments, the method further comprises mixing composition (i) and composition (ii) in any desired ratio.

[0037] In some embodiments, a plurality of bacteria is provided in a container, and the method further includes adding at least one compound to the container and determining any one of: (i) bacterial diversity, (ii) relative abundance of the bacteria, (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)-(v), thereby assessing the effect of the at least one compound on the plurality of bacteria, the effect of the plurality of bacteria on the at least one compound, or both. In some embodiments, the any change in the at least one compound comprises a chemical modification, a structural modification, or a combination thereof.

[0038] In some embodiments, the source sample is selected from the group consisting of: from at least one source; from at least one subject; a microbiome sample; a skin sample; an oral sample; a fecal sample; a vaginal sample; comprising a gut microbiota; and any combination thereof.

[0039] In some embodiments, the compound provides (i) a similarity level of at least 30% at the end of the culture period, and (ii) at least 1 E per gr of particles at the end of the culture step. 4 of bacterial load, or both (i) and (ii) are altered.

[0040] In some embodiments, modifying comprises increasing or decreasing any one of the similarity level, bacterial load, or both.

[0041] In some embodiments, the method is carried out simultaneously in several single vessels.

[0042] In some embodiments, the bacterial diversity, relative abundance of bacteria, bacterial load, and / or other effect of the at least one compound on the plurality of bacteria is compared to corresponding characteristics in the plurality of bacteria of the source sample, and alterations in such characteristics indicate that the at least one compound has an effect on the plurality of bacteria.

[0043] In some embodiments, the method further includes culturing a control plurality of bacteria in a separate container that has not been exposed to the compound, and comparing the bacterial diversity, relative abundance of bacteria, bacterial load, and / or any other effect of the at least one compound on the plurality of bacteria with corresponding characteristics in the control plurality of bacteria, wherein any alteration in the characteristics indicates that the compound has an effect on the plurality of bacteria.

[0044] In some embodiments, the adding and / or determining is performed using bacteria attached to particles, bacteria not attached to particles, or both.

[0045] In some embodiments, the composition is a pharmaceutical composition for use in modulating microflora in a subject in need thereof.

[0046] In some embodiments, the composition is for use in an in vitro method for assessing the effect of at least one compound on a plurality of bacteria, a plurality of bacteria on at least one compound, or both.

[0047] In some embodiments, the composition further comprises an acceptable carrier or excipient.

[0048] In some embodiments, the method further comprises subjecting the bacteria to at least one compound, thereby generating a desired microbial profile, wherein the desired microbial profile comprises any one of a predetermined bacterial alpha diversity, a predetermined beta diversity, a predetermined bacterial relative abundance, a predetermined bacterial load, or any combination thereof. In some embodiments, subjecting the bacteria to the at least one compound alters, e.g., decreases or increases, the similarity level by at least 30% at the end of the culture period.

[0049] In some embodiments, the co-culture and / or composition is enriched with bacteria from different sources.

[0050] In some embodiments, the method further comprises harvesting the plurality of cultured bacteria. In some embodiments, the harvested plurality of cultured bacteria has a bacterial count per gr, e.g., at least 1 E per gr of particles. 6 In some embodiments, at the end of the culture period, the bacterial load is at least 1 E per gr, e.g., per gr of particles. 6 In some embodiments, the method further comprises harvesting the plurality of co-cultured bacteria, and optionally any additional microorganisms cultured and / or maintained in the culture system, e.g., any additional microorganisms attached and / or unattached to particles (referred to herein as "biological biomass" or "biological cells"). In some embodiments, prior to harvesting the biological biomass, the biological biomass is separated from the growth medium or cell culture broth, e.g., by centrifugation, filtration, and / or by allowing the biological biomass to settle within the culture vessel.

[0051] In another aspect, an in vitro method for assessing the effect of at least one compound on a plurality of bacteria, the plurality of bacteria on the at least one compound, or both is provided, the method comprising: providing a sample comprising the plurality of bacteria contained in a container; contacting the plurality of bacteria with particles to at least partially adhere the plurality of bacteria 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, wherein the culturing comprises culturing under anaerobic conditions, thereby obtaining a co-culture comprising the plurality of bacteria; adding at least one compound to the container; and determining any one of: (i) bacterial diversity, (ii) relative abundance of the bacteria, (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)-(v), thereby assessing the effect of the at least one compound on the plurality of bacteria, the plurality of bacteria on the at least one compound, or both.

[0052] 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 can be used in practicing or testing embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0053] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0054] [Figure 1] Figure 1 includes a nonmetric multidimensional scaling (NMDS) beta-diversity (quantitative Unifrac) plot showing the microbial diversity of particle-attached bacterial fractions (represented as octagons), non-attached bacterial fractions (represented as squares), and planktonic culture samples (represented as triangles), all produced simultaneously from the same sample of origin (fecal samples, represented as pluses). Several samples were taken from the particle-attached bacterial fraction, non-attached fraction, and planktonic culture. Each sample is represented by one dot. Thus, several dots are shown for each system. [Figure 2] Vertical bar graphs showing the quantitative Unifrac similarity of composition and bacterial counts (qPCR measurements) to a reference sample at different time points (T1, T2, and T3) during cultivation in medium with a single carbon source (glucose) compared to cultivation in a multi-carbon source medium (glucose, maltose, trehalose, starch). The original fecal sample is considered as 100% (not shown in the graph). [Figure 3] A column graph shows the effect of supplementing glucose or multi-carbon media with trace elements on the similarity of their composition to a reference sample. The trace elements used were manganese, copper, and iron sources. Samples were taken at three time points during cultivation (T1, T2, and T3) to assess quantitative Unifrac similarity and bacterial counts (based on qPCR measurements). [Figure 4] Graph showing the level of similarity between the composition and the reference origin fecal sample "Origin." The results are shown as a principal coordinate analysis (PCoA, PCA-based metric multidimensional scaling, MDS) plot. Samples containing bacteria were cultured under aerobic and anaerobic conditions in different media (e.g., brain-heart infusion medium (BHI), or pharmaceutical-grade media for growing human gut bacteria). Anaerobic conditions are indicated by a box (anaerobic conditions are indicated as AN). [Figure 5]Included are column graphs showing the similarity level and bacterial count (presented as a line plot, secondary Y-axis, based on quantitative PCR (qPCR) measurements) of bacterial fecal samples cultured under anaerobic conditions using different source medium substrates. Cultivation was carried out in a medium according to an embodiment of the invention containing glucose, yeast extract, peptone extract, sodium chloride, disodium hydrogen phosphate in small-scale production (PG2) or medium-scale production (fermentor / bioreactor PG2) or small-scale culture in BHI medium. Tests were carried out at different time points, and a reference fecal sample is designated as "source." [Figure 6] 1 is a vertical bar graph showing the similarity and bacterial count levels of co-cultures produced according to one embodiment of the present invention using a multi-carbon medium containing glucose, maltose, trehalose, starch, yeast extract, peptone extract, sodium chloride, disodium hydrogen phosphate, a manganese source, a copper source, and an iron source. The results show combined data from three processes using three source samples from the same donor collected on different days. Samples were grown independently at a medium scale (6 L). [Figure 7] 1 shows non-metric multidimensional scaling (NMDS) of the Bray-Curtis similarity index performed at the species level for fungal, viral, and bacterial communities present in compositions produced according to embodiments of the present invention (represented as squares) and a reference fecal sample (represented as diamonds), respectively. [Figure 8] 1 shows non-metric multidimensional scaling (NMDS) of the Bray-Curtis similarity index performed at the species level for fungal, viral, and bacterial communities present in compositions produced according to embodiments of the present invention (represented as squares) and a reference fecal sample (represented as diamonds), respectively. [Figure 9]Figure 1 shows non-metric multidimensional scaling (NMDS) of the Bray-Curtis similarity index performed at the species level for fungal, viral, and bacterial communities present in compositions produced according to embodiments of the present invention (represented as squares) and a reference fecal sample (represented as diamonds). Fecal samples from healthy volunteers were also analyzed for their communities (represented as circles). [Figure 10] 1 shows a scatter plot showing the relative proportions / abundances of metabolic pathways tested in compositions produced according to embodiments of the present invention. The X-axis represents the source sample, and the Y-axis represents the produced composition. Each point represents a metabolic super-pathway, and its location on either side of the gray dashed y=x line means that the pathway is enriched in one of the two compared samples. [Figure 11A] The growth preference of two bacterial families for specific phases (attached or not attached to particles) of the composition is shown. [Figure 11B] The figures show the growth preference of two bacterial families for specific phases (attached or unattached to particles) of the composition. Each plot represents a different test bacterium. Each dot on the graph represents the delta between the relative abundance of the test bacterium in the attached phase and its abundance in the non-attached phase in a sample obtained from the culture vessel at a particular time point. Skew of values ​​to a particular side of the graph represents the preference of the bacterium for a particular phase. Figure 11A shows the preference of the bacterium for the attached fraction. Figure 11B shows the preference of the bacterium for the non-attached fraction. [Figure 12A] Plot A - shows diversity recovery at the genus level, each representing a different β diversity. [Figure 12B]Plot B shows diversity recovery at the species level, with each panel representing a different β diversity. Each graph consists of three panels showing a comparison between two specific phases. The first and second upper panels compare the combined phase with the non-adherent and attached phases, respectively. The third lower panel compares the non-adherent phase with the attached phase. Each dot on the graph represents the delta between a subset of two selected test phases (adherent, non-adherent, combined) in samples obtained from the culture vessel at a specific time point. Higher metric values ​​for a specific phase compared to others are observed by the skewness of the distribution from zero toward that particular side of the graph, with each side representing a different phase. Statistical significance between each of the two tested phases was determined using the Wilcoxon nonparametric signed-rank test and is marked with an asterisk (*). Asterisks are indicated on significantly enriched sides. [Figure 13] A heat map showing the relative abundance of Akkermansia is shown. Data is represented by color, with darker colors indicating a higher relative abundance of the bacteria in the test sample or composition (starting with white—indicating no bacteria present; maximum 1: black—indicating maximum relative abundance). The X-axis represents specific sampling time points of the bacterial fermentation, and the Y-axis represents the origin sample used for cultivation. Single origins Ori1, Ori2, and Ori3. Pooled origins: Ori1 + Ori2 + Ori3; or Ori2 + Ori3. [Figure 14] In some embodiments thereof, by way of non-limiting example, a flow chart illustrating steps of the methods disclosed herein is shown. [Figure 15] In some embodiments thereof, by way of non-limiting example, a flow chart illustrating steps of the methods disclosed herein is shown. [Figure 16] In some embodiments thereof, by way of non-limiting example, a flow chart illustrating steps of the methods disclosed herein is shown. [Figure 17]This figure shows a non-metric multidimensional scaling (NMDS) beta diversity (Bray-Curtis) plot depicting the microbial diversity among four lines generated from four different fecal samples (from four different donors, designated as Donors 1–4). Donor #1 provided samples at three separate time points (designated Experiments 1–3). Multiple lines were generated simultaneously: four lines from four different donors, plus two additional lines generated from Donor #1 in Experiments 1 and 2 (six in vitro culture lines in total). Several samples were taken from the particle-associated bacterial flora. Each sample is represented by a dot on the plot. Thus, several dots are shown for each line / donor. [Figure 18] This figure includes a box-bar graph showing the average effect of Compound A on bacterial counts (based on qPCR measurements) for four different systems generated from four fecal samples, compared to a system based on an untreated control microbiome. Measurements were taken at three different time points during culture (TP1-TP3). Note that the bacterial load results for all systems (four treated systems and four untreated control systems) are shown on the same graph. The x-axis, measuring genome copies per μl, refers to bacterial count measurements by qPCR. This figure provides a non-limiting example of a method for assessing the effect of a compound on multiple bacteria. [Figure 19A] 1 shows a box bar graph depicting the effect of Compound_A on alpha diversity in four different systems generated from four different donors compared to a system based on an untreated control microbiome. [Figure 19B] 1 shows a box bar graph depicting the effect of Compound_A on alpha diversity in four different systems generated from four different donors compared to a system based on an untreated control microbiome. [Figure 19C] 1 shows a box bar graph depicting the effect of Compound_A on alpha diversity in four different systems generated from four different donors compared to a system based on an untreated control microbiome. [Figure 19D]Box-bar graphs show the effect of Compound A on alpha diversity in 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 period from particle-associated bacterial phases. The alpha diversity of the fecal sample before culture is shown in each graph as "origin." This figure provides a non-limiting example of a method for assessing the effect of a compound on multiple bacteria. [Figure 20A] 1 shows a non-metric multidimensional scaling (NMDS) plot showing the effect of Compound_A compared to the control on β-diversity (Bray-Curtis) of the microbial population. [Figure 20B] 1 shows a non-metric multidimensional scaling (NMDS) plot showing the effect of Compound_A compared to the control on β-diversity (Bray-Curtis) of the microbial population. [Figure 20C] 1 shows a non-metric multidimensional scaling (NMDS) plot showing the effect of Compound_A compared to the control on β-diversity (Bray-Curtis) of the microbial population. [Figure 20D] This figure shows a non-metric multidimensional scaling (NMDS) plot showing the effect of Compound_A on the β-diversity (Bray-Curtis) of a microbial population compared to a control. Measurements were taken at three time points during the culture period from particle-attached bacterial phases. Each plot shows the diversity in a different line generated from a different donor at three subsequent time points during the culture period (all time points are shown on the same plot). This figure provides a non-limiting example of a method for assessing the effect of a compound on multiple bacteria. [Figure 21]This is a heat map showing the different bacterial genera affected by exposure of multiple bacteria cultured in an in vitro system to Compound A. Each experiment was performed simultaneously with four treatments (samples 1-4). The darker the color, the greater the change. Patterned colors indicate a decrease in the exposure, while unpatterned colors indicate an increase in the exposure. Analysis was performed relative to an untreated control bacterial population. Color intensity indicates the fold change level (see indicator level in the rectangle on the right). The average color indicator is also shown (Av.). A cross (x) indicates that the genus was not significantly affected in the sample. [Figure 22] 1 is a graph showing the effect of Compound_A and its derivative (Compound_A') on the relative abundance of bacterial populations compared to untreated bacterial populations in a control system. Relative abundance is presented at the genus taxon level. All systems were generated from the same fecal sample. Samples were collected at five time points. This figure provides a non-limiting example of a method for assessing the effect of a compound on multiple bacteria. [Figure 23] Figure 1 shows a graph depicting the relative abundance of nine selected genera in the attached and non-attached bacterial fractions of particles after exposure to Compound D. The top panel shows the treated fraction, and the bottom panel shows the relative abundance in the untreated system. [Figure 24] A graph showing the relative abundance of four selected bacteria (labeled "Bacteria 1-4") within the generated control screening system and after the addition of Compound B is shown. The relative abundance of each bacterium within the total bacterial population was measured in both the particle-attached bacterial ("attached") and planktonic ("planktonic") phases. Measurements were performed at one time point (T1) for the control system and at two time points (labeled T1 and T2) for the treatment system. DETAILED DESCRIPTION OF THE INVENTION

[0055] Methods of preparation and use According to some embodiments, a method is provided for producing a composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles.

[0056] According to some embodiments, a method is provided for producing a composition comprising a co-culture comprising a plurality of bacteria and a particle.

[0057] According to some embodiments, methods are provided for obtaining compositions characterized by or having an increased yield of enteric bacteria.

[0058] According to some embodiments, an in vitro system / model, e.g., an in vitro digestion system, is provided for assessing the effect of at least one compound on a bacterial and / or microbiome population of a subject and / or the effect of a bacterial and / or microbiome population of a subject on at least one compound.

[0059] According to some embodiments, an in vitro system / model is provided for studying and / or modulating the physiology, metabolic activity, prebiotic, probiotic or pathogenic characteristics of the flora of a subject, optionally after the addition of at least one compound.

[0060] According to some embodiments, an in vitro method is provided for assessing the interactive effect of at least one compound and a plurality of bacteria, eg, a bacterial population and / or a microbiome population of a subject.

[0061] According to some embodiments, methods and systems for culturing a bacterial population (eg, a plurality of gut bacteria) are provided.

[0062] According to some embodiments, there are provided compositions obtainable by the methods and / or systems disclosed herein.

[0063] The methods, compositions, and in vitro systems / models according to the invention, in some embodiments, have several advantages, as demonstrated and detailed below. In some embodiments, the methods according to the invention involve co-cultivating distinct bacteria in a single vessel, e.g., under the same culture conditions. In some embodiments, the methods according to the invention allow for the co-cultivation of distinct bacteria using media containing pharmaceutical-grade ingredients, e.g., ingredients suitable for human consumption. In some embodiments, the methods according to the invention allow for the production of complex microbial compositions and / or grow in the presence of particles, containing at least 30% similarity to the source sample, and at least 1 E per gram of particles. 4 , e.g., 1 E per 1g of particles 6This enables the production of compositions comprising co-cultures containing multiple bacteria with bacterial loads of up to 100%. In some embodiments, where the source sample is a fecal sample, developing a controlled industrial process that results in a composition that preserves the gut microbiome profile of, for example, a healthy donor, can advantageously address issues of scalability, reproducibility, and safety in manufacturing for performing fecal microbiota transplantation (FMT). In some embodiments, an in vitro culture system according to the present invention, which includes a culture step using a growth or culture medium in the presence of particles detailed herein, substantially maintains a high degree of similarity (e.g., bacterial diversity and / or relative abundance of bacteria) with the biological sample used to generate the system and can therefore be utilized as an in vitro model to simulate a subject's bacterial and / or microbiome population. In some embodiments, a system according to the present invention simulates or mimics a subject's flora, e.g., the flora of the gastrointestinal tract, and thus is utilized to determine modulation or changes in bacterial populations following exposure, supplementation, or contact with at least one compound / element. In some embodiments, methods and systems according to the present invention can be used as high-throughput screening assays to simultaneously compare the effects of different treatments / compounds (e.g., different bacteria, different drug derivatives, different prebiotics, etc.) on a subject's bacterial and / or microbiome populations. In some embodiments, methods and systems according to the present invention make it possible to identify modulated bacteria (at any taxonomic level) that may be therapeutic targets for increasing the efficacy of a drug, for example, by drug-probiotic or drug-prebiotic combination therapy aimed at restoring / preserving an original or healthier microbiome profile. In some embodiments, methods and systems according to the present invention make it possible to evaluate the effect of a subject's microbiome on at least one compound, for example, any change in the chemical structure of an administered compound.In some embodiments, methods and systems according to the present invention can be used as part of personalized medicine, where the effect of a compound of interest, e.g., a drug, is investigated by generating a system using, e.g., a subject's microbiota or microbiome as the source sample, for example, on a co-culture containing multiple bacteria simulating the subject's flora. In some embodiments, use of a system according to the present methods provides reproducible results, thus accurately determining the effect of a compound on multiple bacteria that commonly colonize environmental niches and / or multiple bacteria that mimic the relevant environmental conditions of the subject's microbiome, regardless of the origin of the biological sample (e.g., subject) used to establish the model. In some embodiments, without wishing to be bound by any theory, systems according to the present invention include both planktonic and adherent morphologies of bacteria, which better represent the natural behavior of bacterial populations within a subject's body, compared to systems containing only planktonic bacteria (cultured in the presence and / or absence of particles) or adherent morphologies. In some embodiments, the system is a stand-alone system and therefore is not affected by the complexity of other physiological and / or metabolic processes present in the human body.

[0064] 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 compositions comprising particle-attached bacteria substantially free of planktonic bacteria, e.g., cultured in the presence of particles; compositions comprising planktonic bacteria cultured in the presence of particles, e.g., with substantially no bacteria attached to the particles; and compositions comprising both particle-attached and planktonic bacteria cultured in the presence of particles. In some embodiments, the method can include a step of separating particle-attached bacteria from the particles. In some embodiments, the composition can be supplemented with bacteria cultured without particles.

[0065] In some embodiments, culturing comprises contacting bacteria with particles. In some embodiments, the bacteria in contact with the particles are at least partially attached to the particles. In some embodiments, the bacteria in contact with the particles are completely attached to the particles. In some embodiments, the bacteria in contact with the particles are not attached to the particles. In some embodiments, the bacteria in contact with the particles but not attached to the particles comprise planktonic bacteria.

[0066] In some embodiments, the term "population(s)" with respect to microorganisms, e.g., bacterial populations or cell communities, is interchangeable with the term "community" and refers to two or more groups, e.g., two or more bacterial or microbial groups, that are in symbiosis.

[0067] In some embodiments, the term "composition" includes terms such as "in vitro system(s) or model(s)," "in vitro culture system(s) / model(s)," in vitro biological system model, etc. In some embodiments, the at least one compound can be added at any step in producing the composition.

[0068] In some embodiments, the phrase "a system that more closely represents the natural behavior of a bacterial population in a subject's body" refers to a system that includes a bacterial population that has 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% similarity to the bacterial population in a subject's body, and any range therebetween.

[0069] In some embodiments, the phrase "a system that more closely represents the natural behavior of a bacterial population within a subject's body" refers to a system that includes a bacterial population within a subject's body that has 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, similarity to bacterial growth morphology, e.g., planktonic and / or adherent. In some embodiments, the term "planktonic" includes bacteria in a non-adherent / attached state.

[0070] In some embodiments, a system or co-culture comprising multiple bacteria simulates the flora of a subject. In some embodiments, the phrase "simulating the flora / microflora of a subject" refers to a community of commensal bacteria and / or commensal microorganisms that mimics the relevant environmental conditions of the subject's microbiome, e.g., exhibits similar in vivo function, physiology, metabolic activity, and / or probiotic properties of the subject's microflora. In some embodiments, the system simulates different regions of the gastrointestinal tract with respect to environmental conditions, e.g., pH level, physiological and / or chemical conditions, enzymes and their concentrations, etc.

[0071] As used herein, the terms "microbiome," "microflora," and "flora" are interchangeable and refer to a collection of microorganisms (including, but not limited to, bacteria, viruses, fungi, e.g., yeast) found, comprising, or known to reside in an environmental niche. In some embodiments, the term "microbiome" also includes structural elements and / or metabolites / signal molecules of the microorganism community and / or the surrounding environmental conditions in the environmental niche. In some embodiments, a plurality of bacteria are co-cultured with a collection of microorganisms found or known to reside in the environmental niche of interest. Non-limiting examples of environmental niches include, but are not limited to, the gut, skin, eyes, bronchi, oral cavity, 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.

[0072] In some embodiments, the plurality of bacteria used in accordance with the present invention is derived from a source sample. As used herein, the term "derived" is interchangeable with the terms "originating" and "obtained" and refers to the source from which the plurality of bacteria is obtained for culturing / fermentation. In some embodiments, the source sample comprises a collection of microorganisms (e.g., bacteria, viruses, fungi, e.g., yeast) known to be found, constitute, or exist in an environmental niche. In some embodiments, the source sample comprises bacteria and further comprises at least one taxon of additional microorganisms, e.g., archaea, viruses, fungi, or any combination thereof. In some embodiments, the plurality of bacteria is isolated from the source / origin prior to culturing, e.g., separated from other microorganisms or microorganisms present in the sample. In some embodiments, other microorganisms or microorganisms are cultured and / or maintained together with the plurality of bacteria in a culture system. In some embodiments, the term "virus" is or includes a bacteriophage.

[0073] In some embodiments, the term "culturing" includes the term "fermentation" and refers to the in vitro maintenance, propagation, and / or growth of microorganisms, such as bacteria, in various types of buffers and / or media under laboratory or industrial conditions. Suitable culture 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 Acids), BHI (Brain Heart Infusion), GAM (Gifu Anaerobic Medium), TSB (Tryptic Soy Broth), TYG (Tryptic Yeast Extract Glucose), FAB (Fastidious Anaerobic Broth), and the like. In some embodiments, the medium contains pharmaceutical-grade ingredients. In some embodiments, the medium contains food-grade ingredients. In some embodiments, the medium contains ingredients 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 involving the conversion of sugar(s) to acids and / or gases, for example, using bacteria.

[0074] In some embodiments, a collection of additional microorganisms or microorganisms present in the source sample are co-cultured with the plurality of bacteria. In some embodiments, the other microorganisms or microorganisms are added separately or mixed into the culture system. In some embodiments, any one of archaea, viruses, and / or fungi is maintained, e.g., preserved and / or present, in the co-culture without changing their quantity and / or the relative abundance of their taxa relative to the source sample. In some embodiments, "preserved and / or present" refers to a population similar to the source sample as defined herein. In some embodiments, the term "virus" includes bacteriophages.

[0075] In some embodiments, the term "co-cultivating" or "co-cultured" as used herein refers to the maintenance, proliferation and / or growth of multiple bacteria, and optionally one or more additional microorganisms or microorganisms, in the culture systems described herein.

[0076] In some embodiments, the source sample containing a plurality of bacteria resembles the microflora of a subject, e.g., a healthy subject or a subject with a condition such as a disease, disorder, or dysbiosis (such a sample is referred to herein as an "analogous sample"). In some embodiments, an "analogous sample" is a sample that contains 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 subject's microflora and / or a plurality of bacteria derived / from the subject (e.g., relative bacterial abundance and / or bacterial load, as measured by bacterial alpha diversity or beta diversity). In some embodiments, an "analogous sample" is generated by mixing bacteria, e.g., single strains and / or multiple bacteria and / or co-cultured bacteria, in an environmental niche of a subject, such as the gut, eye, oral cavity, skin, bronchi, vagina, upper respiratory tract, or genitourinary tract, to name a few, based on a desired microbiome profile. In some embodiments, the "similar sample" further comprises a mixture of other microorganisms, such as archaea, viruses, fungi, or any combination thereof.

[0077] In some embodiments, the source sample contains a predetermined target or desired population of microorganisms, e.g., a bacterial population. In some embodiments, the source sample is used "as is" or subsequently processed as the starting material for the methods described herein. In some embodiments, sample processing includes any one of dilution, such as with a buffer, culture medium, or a combination, homogenization, partial or complete removal of non-flora matter, coarse particulate matter, fiber, or any combination thereof, and / or any other process 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.

[0078] In some embodiments, the source sample is a synthetic sample. In some embodiments, the source sample is a biological sample. In some embodiments, the plurality of bacteria is derived from at least one source. In some embodiments, the plurality of bacteria is derived from a combination of at least two, at least three, at least four, at least five, or more sources / sources. In some embodiments, the plurality of bacteria is sampled or derived from a donor (such as a human subject) that has a desired microbiota population in the sampled environment. In some embodiments, the sample is derived from a healthy subject, an unhealthy subject, and / or a subject with dysbiosis, e.g., in a particular environmental niche. In some embodiments, the sample is derived from two or more bacterial sources. In some embodiments, the plurality of bacteria is derived from at least one environment. In some embodiments, the plurality of bacteria is derived from a sample. In some embodiments, the plurality of bacteria is 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.

[0079] In some embodiments, the term "synthetic sample" refers to a sample containing an artificially created bacterial community, for example, by combining / mixing selected (two or more) bacterial species that can generally colonize a given environmental niche. In some embodiments, a synthetic sample is or contains bacterial groups selected at any taxonomic level based on their relative abundance in desired microbiota populations in different subjects and / or different environmental niches.

[0080] In some embodiments, the sample used in the methods or systems of the present invention is or comprises any one of a stored microbiota, a stored microbiome sample, a plurality of bacterial populations, bacterial colonies, particle-associated bacteria and / or planktonic bacteria, or any combination thereof. In some embodiments, the sample is frozen or lyophilized prior to use in the methods or systems of the present invention. In some embodiments, the sample is stored at a temperature below 8°C, for example, at a temperature in the range of 2-8°C.

[0081] In some embodiments, methods according to the invention include providing a composition comprising a co-culture comprising a plurality of bacteria produced according to the invention, e.g., in frozen or lyophilized form, e.g., for use in an evaluation method and / or as starting material for producing a composition according to the invention.

[0082] In some embodiments, the plurality of bacteria is or comprises at least one bacterial population selected from a fecal bacterial population, a gut bacterial population, an eye bacterial population, an oral bacterial population (e.g., a salivary bacterial population), a skin bacterial population, a bronchial bacterial population, a vaginal bacterial population, an upper respiratory tract bacterial population, a urogenital tract bacterial population, or any combination thereof. In some embodiments, the plurality of bacteria is or comprises at least one bacterial population selected from a soil bacterial population, a groundwater bacterial population, an open water bacterial population, or any combination thereof. In some embodiments, the plurality of bacteria is or comprises a gut microbiota.

[0083] In some embodiments, the source sample containing the plurality of bacteria is derived from a subject, e.g., a fecal sample, an oral sample (e.g., a saliva sample), a skin sample, an eye sample, a bronchial sample, a vaginal sample, or any combination thereof. In some embodiments, the sample containing the plurality of bacteria is derived from at least one, at least two, at least three, at least four, at least five or more sources, e.g., the sample is a pool from several subjects.

[0084] In some embodiments, flora, e.g., bacterial populations, are separated from non-flora matter. In some embodiments where the source is a fecal sample, fecal flora is at least partially separated from non-flora matter, coarse particulate matter, and / or fiber of the fecal material. Separation can be achieved, for example, by homogenization, centrifugation, filtration, and / or any other method known to those skilled in the art. In some embodiments, the fecal sample used in the methods of the present invention contains fiber derived from feces. In some embodiments, the bacterial population is at least partially separated from non-digestible particles or materials.

[0085] In some embodiments, the sample comprises a soil sample.

[0086] In some embodiments, the sample is derived from a plant or plants.

[0087] As used herein, the term "plurality" refers to any integer greater than or equal to two. In some embodiments, the plurality of bacteria comprises at least two distinct bacteria. In other embodiments, 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 distinct bacteria. In some embodiments, the plurality of bacteria is from one or more taxonomic classes.

[0088] In some embodiments, the term "plurality of bacteria" refers to a bacterial population that includes at least two different strains or species of bacteria. In some embodiments, the bacterial population includes from two different types of bacteria, up to 1000 different types of bacteria, or more bacteria.

[0089] In some embodiments, the term "distinct" 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 distinct optimum conditions for growth, culture, and / or proliferation. In some embodiments, the distinct growth, culture, and / or proliferation conditions are at least two optimum conditions, a first condition that allows substantially optimal growth, culture, and / or proliferation of a first bacterium and a second condition that allows substantially optimal growth, culture, and / or proliferation of a second bacterium.

[0090] In some embodiments, the different growth, culture, and / or propagation conditions include any one of metabolic requirements, nutrient requirements, pH, temperature, aerobic, obligately anaerobic, facultative anaerobic, microaerobic, attached morphology, planktonic, growth medium, flowable, shaking, stirring, agitation, static, moist, low humidity, or any combination thereof. In some embodiments, the growth, culture, and / or propagation conditions include at least one obligately anaerobic bacterium and at least one facultative anaerobic bacterium. In some embodiments, the different growth, culture, and / or propagation conditions include at least two bacterial populations selected from the group consisting of obligately aerobic, obligately anaerobic, facultative anaerobic, and microaerobic.

[0091] In some embodiments, the different growth, culture and / or proliferation conditions are biological parameters selected from the group consisting of metabolic requirements, nutrient requirements, growth media, and any combination thereof.

[0092] In some embodiments, the different growth, culture and / or proliferation conditions are metabolic requirements selected from the group consisting of aerobic, obligately anaerobic, facultatively anaerobic, microaerobic, and any combination thereof.

[0093] In some embodiments, the different growth, culture and / or propagation conditions are physical parameters selected from the group consisting of temperature, moisture, low humidity, fluidity, shaking, agitation, stirring, static, and any combination thereof.

[0094] In some embodiments, the different growth, culture and / or proliferation conditions are chemical parameters selected from the group consisting of pH, redox potential, gas composition, dissolved gases, and any combination thereof.

[0095] In some embodiments, the different growth, cultivation and / or propagation conditions are fermentation techniques selected from the group consisting of fed-batch, semi-batch, batch, continuous and any combination thereof.

[0096] In some embodiments, the different growth, culture and / or proliferation conditions are bacterial growth forms selected from the group consisting of planktonic forms, attached forms, and any combination thereof.

[0097] In some embodiments, the term "metabolic requirements" includes any one of carbon and energy sources, basic element sources (e.g., H, O, N), macroelements, trace elements, vitamins, hormones, growth factors, CO2 levels, oxygen levels, light, metabolic precursors or substrates, or any combination thereof.

[0098] In some embodiments, the composition is produced by a method comprising providing a plurality of bacteria from a source sample, contacting the plurality of bacteria with particles to at least partially adhere the plurality of bacteria 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.

[0099] In some embodiments, the composition is produced by a method comprising providing a plurality of bacteria from a source sample and culturing the plurality of bacteria in a growth medium for a period of less than 14 days, wherein culturing comprises culturing under anaerobic conditions.

[0100] In some embodiments, the composition is produced by a method comprising providing a plurality of bacteria from a source sample and culturing the plurality of bacteria in a growth medium for less than 14 days, wherein culturing comprises culturing under aerobic conditions.

[0101] In some embodiments, the growth medium comprises a carbon source and a nitrogen source in a molar / atom / atom ratio ranging from 50:1 to 1:50, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, the ratio between the embodied carbon species in the carbon-to-nitrogen ratio (either w / w or m / m) is based on dry form.

[0102] In some embodiments, the phrases "providing a plurality of bacteria derived from a source sample" and "providing a sample comprising a plurality of bacteria" are interchangeable.

[0103] In some embodiments, a plurality of bacteria or a sample containing same is provided in a container, and the method further includes adding at least one compound to the container and determining any one of: (i) bacterial diversity, (ii) relative abundance of the bacteria, (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, for example, and (vi) any combination of (i)-(v), thereby assessing the effect of the at least one compound on the plurality of bacteria, the effect of the plurality of bacteria on the at least one compound, or both.

[0104] In some embodiments, the in vitro evaluation methods and other uses according to the present invention defined herein can be performed 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 performed in a system with particles, a system without particles, and / or both, e.g., as detailed above and below. In some embodiments, the compound(s) can be added before and / or after the addition of particles to the culture system, e.g., as detailed above and below. The evaluation and / or determination steps can be performed on any of the bacterial fractions / phases produced according to the present invention.

[0105] The phrase "contacting a plurality of bacteria with particles" is used herein in its broadest sense and refers to any type of combinatorial action that brings a plurality of bacteria into close proximity with particles, for example, so that the bacteria can attach / adhere to the particles. In some embodiments, the contacting is performed in a solution. In some embodiments, the contacting includes combining the bacteria, particles, and solution in any order, in any combination and / or partial combination, including any premixing of the two components, before adding a third component. For example, the bacteria are inoculated into a solution, and then the bacteria are cultured in a solution containing particles. In some embodiments, a particle blend is prepared in a solution as a prior step and then combined with the plurality of bacteria.

[0106] In some embodiments, the contacting is performed in a solution selected from saline, phosphate-buffered saline, growth medium, or any combination thereof. In some embodiments, the contacting includes inoculating a plurality of bacteria into a liquid containing the particles (e.g., a buffer solution, a culture medium, or a combination thereof) and incubating the particles with the plurality of bacteria for a sufficient time to allow the plurality of bacteria to at least partially adhere to the particles. In some embodiments, the contacting is or includes culturing the bacteria in the presence of the particles. In some embodiments, the contacting and culturing are performed simultaneously or as subsequent steps.

[0107] In some embodiments, the time sufficient for a plurality of bacteria to at least partially attach or adhere to the particles is between 2 hours and 48 hours. In some embodiments, the time sufficient for a plurality of bacteria to at least partially attach / adhere to the particles is between 2 hours and 12 hours, between 2 hours and 24 hours, between 6 hours and 24 hours, between 10 hours and 24 hours, between 15 hours and 20 hours, between 20 hours and 40 hours, between 6 hours and 40 hours, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0108] In some embodiments, culturing comprises fed-batch or semi-fed-batch culturing, e.g., where some or all of the nutrients are provided to the culture during the culturing process. In some embodiments, fed-batch culturing comprises fixed-volume fed-batch, variable-volume fed-batch, or any combination thereof.

[0109] As used herein, the term "fed-batch culture" refers to a culture method in which components, e.g., nutrients such as a carbon source and / or a nitrogen source, are provided to the culture at least one time point after the start of the culture / culture process. In some embodiments, the culturing is a batch culture, e.g., nutrients are provided at the start of the culture process. In some embodiments, the culturing is a combination of fed-batch and batch culture. For example, one nutrient is provided at the start of the culture and another nutrient is provided during the culture.

[0110] In some embodiments, the growth medium comprises one or more nitrogen sources, hi some embodiments, the nitrogen source is or comprises at least one of yeast extract, peptone, yeast peptone, enriched yeast peptone, casein, guar peptone, synthetic amino acid medium, wheat peptone, potato peptone, ammonium salts (various salts such as ammonium carbonate, ammonium chloride, and ammonium nitrate), or any combination thereof.

[0111] In some embodiments, the culturing is or includes culturing under anaerobic conditions.

[0112] In some embodiments, the term "anaerobic conditions" refers to conditions in which 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 that do not provide free oxygen. In some embodiments, anaerobic conditions include conditions that lack free oxygen.

[0113] In some embodiments, the term "aerobic conditions" refers to conditions that include the presence of molecular oxygen. In some embodiments, the oxygen concentration is greater than 20% (v / v of the total gases present in the culture).

[0114] In some embodiments, methods for producing a composition according to the invention comprise in vitro culturing complex bacterial populations characterized by having different growth, cultivation and / or proliferation conditions, thereby producing a composition comprising a co-culture comprising the complex bacterial populations.

[0115] In some embodiments, the produced composition or co-culture comprises at least 30% similarity, at least 40% similarity, at least 50% similarity, at least 60% similarity, at least 70% similarity, at least 80% similarity, at least 90% similarity, at least 95% similarity, at least 99% similarity, or 100% similarity to the source sample, or any value and range therebetween, with each possibility representing a separate embodiment of the present invention.

[0116] In some embodiments, the bacterial load of the produced composition or co-culture is at least 1 E per gr of particles. 4 ~At least 1 E 9 In some embodiments, the composition or co-culture produced has at least 1 E per gram of particles. 4 , at least 1 E per 1g of particles 5 , at least 1 E per 1g of particles 6 , at least 1 E per 1g of particles 7 , at least 1 E per 1g of particles 8 , at least 1 E per 1g of particles 9 , at least 1 E per 1g of particles 10 , at least 1 E per 1g of particles 11 , at least 1 E per 1g of particles 12 , at least 1 E per 1g of particles 14, at least 1 E per 1g of particles 16 , or at least 1 E per 1g of particles 20 , or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0117] In some embodiments, culturing is performed to determine whether the co-culture has a similarity of 30% or more to the source sample and a similarity of 1 E per gram of particles. 4 ~1 E 9 The procedure is continued until the bacterial load is in the range of 1000 to 10 ...

[0118] In some embodiments, culture periods of less than 14 days include at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most 1 day, at most 5 hours, at most 3 hours, or any value and range therebetween, with each possibility representing a separate embodiment of the present invention.

[0119] In some embodiments, culture periods of less than 14 days include 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, with each possibility representing a separate embodiment of the present invention.

[0120] In some embodiments, the method includes an incubation 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, wherein the co-culture has: i) at least 50% similarity to the source sample; and ii) at least 1 E per gram of particles. 6 Contains a bacterial load of

[0121] In some embodiments, the method comprises culturing in a growth medium comprising at least two carbon sources selected from monosaccharides, disaccharides, and polysaccharides for a period ranging from 12 hours to 5 days, thereby producing a composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles, wherein the co-culture has i) a quantitative Unifrac similarity of at least 70% with the source sample, and ii) a denaturation index of at least 1 E per gram of particles. 8 Contains a bacterial load of

[0122] In some embodiments, culturing includes culturing under any conditions selected from static, fluid, agitated, shaking, stirring, or any combination thereof. In some embodiments, culturing includes agitating the plurality of bacteria, e.g., at 50-750 revolutions per minute (RPM), 50-650 RPM, 100-750 RPM, 100-700 RPM, 150-700 RPM, 200-750 RPM, 130-690 RPM, 90-720 RPM, 70-550 RPM, 110-710 RPM, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0123] In some embodiments, the terms "fluidic," "stirring," "shaking," and "agitating" refer to conditions that result in movement or motion of the liquid phase within the culture vessel. In some embodiments, the movement is axial, radial, mixing, dispersing, or any combination thereof. The movement or movement can be performed using mechanical means, e.g., an impeller, a moving platform, a rocker, a shaker, manually, automatically, or any combination thereof. In some embodiments, the term "static conditions" refers to conditions in which no stirring or any other moving action (manual, automatically, and / or mechanically) is performed on the liquid phase.

[0124] In some embodiments, culturing comprises subjecting the plurality of bacteria to a temperature 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 present invention.

[0125] In some embodiments, culturing comprises subjecting the plurality of bacteria to a pH ranging from 3.0 to 9.0, or from 4.0 to 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, with each possibility representing a separate embodiment of the present invention.

[0126] In some embodiments, culturing comprises exposing the plurality of bacteria to a pH of 6.2-7.2, 6.2-7.1, 6.2-7.0, 6.3-7.2, 6.4-7.2, 6.5-7.1, 6.6-7.2, or 6.8-7.2, or any value and range therebetween, with each possibility representing a separate embodiment of the present invention.

[0127] In some embodiments, prior to culturing the plurality of bacteria, the sample is diluted (e.g., with a buffer, culture medium, or a combination thereof) at a weight / volume (w / v) ratio of 1:1 to 1:300, including, 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. Each possibility represents a separate embodiment of the present invention.

[0128] In some embodiments, the growth medium comprises one carbon source. In some embodiments, the medium comprises at least two carbon sources. In some embodiments, the medium comprises carbon sources from at least two groups selected from 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.

[0129] In some embodiments, the at least one monosaccharide, at least one disaccharide, and at least one polysaccharide are present in the growth medium in a weight / weight (w / w / w) ratio of 1:0:0, 1:1:0, 0:1:1, 0:1:0, 0:0:1, 1:0:1, 1:1:1, or any values ​​and ranges therebetween, with each possibility representing a separate embodiment of the present invention.

[0130] In some embodiments, the at least one monosaccharide, at least one disaccharide, and at least one polysaccharide are present in the growth medium in a weight / weight (w / w / w) ratio of X:Y:Z, where any one of X and Y, X and Z, and / or Y and Z is in the range of 0 to 10, or any value or range therebetween. Each possibility represents a separate embodiment of the present invention.

[0131] In some embodiments, the monosaccharide is selected from glucose (dextrose), fructose (levulose), galactose, or any combination thereof.

[0132] In some embodiments, the disaccharide is selected from sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, isomaltose, nigerose, maltulose, mannobiose, xylobiose, or any combination thereof.

[0133] In some embodiments, the term "polysaccharide" encompasses any polymer of carbohydrate composed of monosaccharide units linked together via glycosidic bonds. In some embodiments, the polysaccharide is selected from alginate, starch, cellulose, pectin, arabinoxylan, glycogen, galactogen, inulin, or any combination thereof. In some embodiments, the polysaccharide comprises a synthetic polysaccharide. In some embodiments, the synthetic polysaccharide encompasses any non-naturally occurring polysaccharide.

[0134] In some embodiments, the growth medium further comprises a trace element selected from an iron source, a zinc source, a copper source, a manganese source, selenium, iodine, a fluorine source, a molybdenum source, a cobalt source, a chromium source, a nickel source, soluble salts thereof, or any combination thereof.

[0135] In some embodiments, the method further comprises substantially separating bacteria not attached to particles from bacteria attached to 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 substantially removing bacteria not attached / non-adherent to the particle(s) from the culture system and / or 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 lack sessile bacteria. In some embodiments, the separation or removal is performed at least once at a time 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.

[0136] In some embodiments, the term "substantially separated" as used herein refers to having 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, of unattached bacteria in a composition comprising particle-attached bacteria, and / or having 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, of particle-attached bacteria in a composition comprising unattached bacteria.

[0137] In some embodiments, the phrase "substantially removes bacteria not attached / adhered to particle(s)" refers to the removal of 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 non-attached / non-adhered bacteria present in the co-culture. Each possibility represents a separate embodiment of the present invention.

[0138] In some embodiments, the evaluation method is carried out in a system according to the present invention using bacteria in a planktonic form. In some embodiments, the evaluation method is first carried out in a system including bacteria in a planktonic form, e.g., before the contacting step and / or before attaching a plurality of bacteria to particles. In some embodiments, after at least partial attachment, e.g., during the culturing step, the method is carried out in a system including either particle-attached bacteria, bacteria in a planktonic form, or both.

[0139] In some embodiments, the method further comprises separating the plurality of bacteria cultured in the presence of the particles into different culture systems to produce i) a culture system containing bacteria in a planktonic form initially cultured in the presence of the particles and ii) a culture system containing bacteria attached to the particles. In some such embodiments, the compound can be added to either system (i), system (ii), or any combination thereof, of the culture systems prior to the separating step. Evaluation can be performed separately for each system to which the compound has been added.

[0140] In some embodiments, after at least partial adherence, the culture containing the plurality of bacteria can be divided into several containers, e.g., for evaluation of different compound(s) / condition(s). In some embodiments, the plurality of bacteria are cultured under the same culture conditions.

[0141] In some embodiments, the contacting step, the culturing step, or both, occurs in a container, hi some embodiments, the contacting step, the culturing step, or both, occurs in a single container, interconnected containers, or communicating containers.

[0142] In some embodiments, the container comprises any compartment adapted or configured to cultivate and / or maintain bacterial growth, proliferation, activity, and / or viability.

[0143] In some embodiments, the term "vessel" refers to any recipient in which bacteria can be cultured by conventional fermentation techniques, such as bioreactor(s), flask(s), test tube(s), microtiter dish(s), well plate(s), multi-well plate assembly, Petri plate(s), etc.

[0144] In some embodiments, the contacting, culturing and / or adding steps are performed sequentially in a single / one vessel.

[0145] In some embodiments, the container comprises an interconnected container or a communicating container. In some embodiments, the terms "interconnected container" and "communicating container" refer to multiple compartments or containers containing a homogenous liquid that are connected well below the top surface of the liquid.

[0146] In some embodiments, the in vitro model simulates different regions within the gastrointestinal (GI) tract. In some such embodiments, several single containers, each containing a stand-alone system, can be arranged in series and interconnected, for example, by tubes or pipes, alternating above the upper liquid surface and below the lower liquid surface. Each container can represent environmental conditions within the GI tract. Simulation of GI motility and / or functionality can be performed, for example, by utilizing mechanical means. In some embodiments, liquids and / or gases are transported through interconnecting tubes or pipes.

[0147] In some embodiments, the plurality of bacteria is not divided into different containers during culture. In some embodiments, the method is carried out in multiple containers. In some embodiments, the plurality of bacteria is divided into different containers before and / or during culture, e.g., to simultaneously evaluate the effects of a compound on multiple bacteria. In some embodiments, the plurality of bacteria is cultured under the same culture conditions in different containers. In some embodiments, the culture conditions are selected from the group consisting of biological parameters (e.g., metabolic requirements, nutrient requirements, growth medium), metabolic requirements (e.g., aerobic, obligately anaerobic, facultatively anaerobic, microaerobic), physical parameters (e.g., temperature, moisture, low humidity, fluidity, 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 morphology (e.g., planktonic, attached morphology), and any combination thereof.

[0148] In some embodiments, the evaluation method includes the following steps: (a) providing a fecal sample from a subject, optionally diluting the fecal sample, for example, 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 from step (a), for example, at a ratio ranging from 1:2 (g / ml) to 1:10 (g / ml), and incubating or culturing the fecal sample-inoculated particles 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: (i) bacterial diversity; (ii) relative abundance of bacteria; (iii) bacterial load; (iv) effect of the at least one compound on the plurality of bacteria; (v) any change, e.g., chemical and / or structural change, in the at least one compound; and (vi) any combination of (i)-(v). In some embodiments, the diluting is performed in a buffer, culture medium, or a combination thereof.

[0149] In some embodiments, pooled samples derived or obtained from two or more sources are used as part of an analytical 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, the effect of a compound of interest can be tested using at least two bacterial populations representing different sources, such as the intestine and the skin, to determine whether the compound of interest is suitable for treating a disease or disorder associated with a first microbial population, provided it does not alter the second microbial population. The following is an exemplary analysis in which a compound identified for not altering a healthy gut microbial population or for modifying a healthy skin microbial population, but not for modifying an altered gut microbial population, is determined to be unsuitable for treating a gut-related disease or disorder. Alternatively, a compound identified for modifying an altered gut microbial population but not a healthy skin microbial population, is determined to be suitable for treating a gut-related disease or disorder.

[0150] In some embodiments, as part of an analytical method or culture system, different source samples (e.g., from different donors, pools of source samples from the same donor but from different niches mixed in different ratios, or any combination thereof) are cultured in different vessels under conditions of the present application, e.g., to compare the effects of compounds on the different source samples.

[0151] See FIG. 14 for a non-limiting example of the setup of the method of the present invention. First, a sample containing a plurality of bacteria (e.g., a microbial population including a sample derived from at least one target or source population) is provided (step 200), after which the sample can be diluted (step 220). The sample may be diluted with any solution, such as PBS, and / or any suitable medium that still maintains the bacteria's natural environment and is suitable for growth. The dilution can be any range suitable for the preservation and / or growth of bacteria, e.g., 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. The sample or diluted sample is then cultured with a particle or particles under conditions that allow the bacteria to at least partially adhere to the particle or particles (step 240). The bacterial population at least partially attached to the particle or particles is then contacted with a compound of interest for a time and under conditions sufficient for the compound of interest to exert its activity against the bacteria (step 260). The effect of the compound of interest on the bacteria is then determined (step 280). The output of the effect of the compound of interest on the bacteria determined in step 280 can be presented as bacterial mass, bacterial diversity, relative bacterial abundance, bacterial abundance, and / or other such as bacterial viability, particle-attached or adherent bacteria to plankton ratio, gene expression profile, toxin production, etc. Additionally or alternatively, the effect of the bacterial population on the compound can be examined by determining any changes in the concentration (e.g., due to consumption and / or degradation of the compound) and / or structure of the compound of interest, for example, by metabolomics, analytical chemistry, biochemical methods, genomics, or any other suitable method known in the art.

[0152] See FIG. 15 for a non-limiting example of the setup of the method of the present invention. First, a source sample containing a plurality of bacteria (e.g., a microbial population including a sample derived from at least one target or source population) is provided (step 200). The sample can then be diluted and contacted with a molecule of interest (steps 220+260). The sample can be diluted with any solution suitable for preserving and / or growing bacteria, as disclosed herein, and subjected to a time and conditions sufficient for the compound of interest to exert its activity across the bacterial population. The sample or diluted sample is then incubated with a particle or particles under conditions that allow the bacteria to at least partially adhere to the particle or particles (step 240). The effect of the compound of interest on the bacteria is then determined (step 280). The output of the effect of the compound of interest on the bacteria determined in step 280 can be presented as bacterial mass, bacterial diversity, relative bacterial abundance, bacterial abundance, and / or other values, such as bacterial survival, particle-attached or adherent bacteria-to-plankton ratio, gene expression profile, toxin production, etc. Additionally or alternatively, the effect of the bacterial population on the compound can be examined by determining any changes in the concentration and / or structure of the compound of interest, for example, by metabolomics, analytical chemistry, biochemical methods, genomics, or any other suitable method known in the art.

[0153] See FIG. 16 for a non-limiting example of the setup of the method of the present invention. First, a sample containing a plurality of bacteria (e.g., a microbial population including a sample derived from at least one target or source population) is provided (step 200), after which the sample can be diluted (step 220). The sample can be diluted with any solution suitable for storage and / or growth, as disclosed herein. The sample or diluted sample is then cultured with one or more particles under conditions that allow the bacteria to at least partially adhere to the particle(s) and substantially simultaneously contacted with a compound of interest. The bacteria and compound can be contacted for a time and under conditions sufficient for the compound of interest to exert its activity across the bacterial population (steps 240+260). The effect of the compound of interest on the bacteria is then determined (step 280). The output of the effect of the compound of interest on the bacteria determined in step 280 can be presented as bacterial mass, bacterial diversity, relative bacterial abundance, bacterial abundance, and / or other values, such as bacterial survival, particle-attached or adherent bacteria-to-plankton ratio, gene expression profile, toxin production, etc. Additionally or alternatively, the effect of the bacterial population on the compound can be examined by determining any changes in the concentration and / or structure of the compound of interest, for example, by metabolomics, analytical chemistry, biochemical methods, genomics, or any other suitable method known in the art.

[0154] In some embodiments, adding the compound at an early stage of the method, e.g., before the bacteria at least partially adhere to the particles, advantageously allows for the effect of the compound on the original bacterial profile, e.g., the original subject bacterial profile, to be determined. In some embodiments, the early stage includes adding the compound before introducing the particles into the culture vessel, contacting the compound with the source sample, e.g., during inoculation, or any combination thereof. In some embodiments, allowing the bacteria to initially adhere to the particles before adding the compound advantageously allows the bacteria to recover and form a more resilient / less susceptible bacterial population before exposure to the compound.

[0155] In some embodiments, methods according to the invention involve contacting a plurality of bacteria with at least one compound of interest, hi some embodiments, methods according to the invention involve contacting a plurality of bacteria simultaneously or sequentially with at least two, at least three, at least four, at least five, or more compounds of interest.

[0156] In some embodiments, the term "at least one compound" includes any factor, condition, and / or treatment, or number or list of factors, conditions, and / or treatments. In some embodiments, the at least one compound is or includes a small molecule, a drug, a chemical, a peptide, a polypeptide, a protein, a carbohydrate, a prebiotic, a bacteriophage, a bacterium, a fungus, a physical parameter, 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.

[0157] In some embodiments, the term "physical parameter" refers to any measurable physical property, or number or list of measurable physical properties, such as, but not limited to, temperature, pH, stress factors, flowability, shaking, agitation, humidity, gases, e.g., O, CO, N, etc.

[0158] In some embodiments, the at least one compound is or comprises a microorganism not present in the source 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 in the subject.

[0159] In some embodiments, the method includes contacting a plurality of bacteria with a drug. In some embodiments, the plurality of bacteria is contacted with the drug at a dose approved for subject consumption. In some embodiments, the plurality of bacteria is contacted with an effective amount of the drug approved for subject consumption, such as, but not limited to, to induce the 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 an approved drug is used. In some embodiments, the approved drug includes any derivative, analog, or salt thereof, or any combination thereof.

[0160] In some embodiments, an "approved dose" is a clinically approved dose. In some embodiments, a clinically approved dose is one that has been clinically approved for consumption or use by human subjects.

[0161] The types of clinically approved drugs and their administration are common and will be apparent to those skilled in the art.

[0162] 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.

[0163] The phrases "adding at least one compound to a container" or "subjecting bacteria to at least one compound" are used herein in their broadest sense and refer to any type of combinatorial action, e.g., exposing multiple bacteria to a compound. Multiple bacteria can be exposed to, subjected to, or contacted with a compound at any step of the method.

[0164] In some embodiments, at least one compound is added to the container before, substantially after, or immediately after adding the sample or diluted sample to the container. The particles can be added before, substantially after, or after adding the compounds. In some embodiments, at least one compound is added to the container after adding the sample, substantially together with, or immediately after adding the particles. In some embodiments, the bacterial population is exposed to at least one compound during the culturing step. In some embodiments, the bacterial population is exposed to at least one compound during contacting the plurality of bacteria with the particle(s), as a preceding step, during the culturing step, or any combination thereof. In some embodiments, the plurality of bacteria is exposed to at least one compound only before attaching the plurality of bacteria to the particles, during the entire culturing period, or both. In some embodiments, the compound can be contacted with a sample containing a plurality of bacteria, a diluted sample, a co-culture containing a plurality of bacteria, particle-attached bacteria, bacteria in a planktonic form, e.g., bacteria grown in the presence of particles, or any combination thereof.

[0165] In some embodiments, the evaluation method comprises the steps of inoculating the particles with a sample (or a diluted sample) and contacting the sample-inoculated particles with at least one compound of interest.

[0166] In some embodiments, determining or evaluating at least one of (i) bacterial diversity, (ii) relative abundance of bacteria, (iii) bacterial load, (iv) any other effect of the at least one compound on the plurality of bacteria, or (v) any change, e.g., chemical and / or structural change, in the at least one compound, is performed at any time throughout the process of this method, e.g., during culturing. In some embodiments, the determining is performed before and / or after contacting the plurality of bacteria with the particles. In some embodiments, determining at least one of (i) bacterial diversity, (ii) relative abundance of bacteria, (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 is performed at any time during culturing, e.g., at 1 hour, 5 hours, 10 hours, 15 hours, 20 hours, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, and / or 14 days of culturing.

[0167] In some embodiments, the method further comprises transferring the plurality of bacteria to a different container at least once at a time selected from: (i) during the contacting step, (ii) during the culturing period, (iii) before the step of adding at least one compound, (iv) after the step of adding at least one compound, or (v) any combination of (i)-(iv).

[0168] In some embodiments, the determining step is carried out using a co-culture comprising a plurality of bacteria, a plurality of bacteria attached to particles, bacteria in a planktonic form, or any combination thereof, hi some embodiments, the effect of the compound is compared between any one of a co-culture comprising a plurality of bacteria, a plurality of bacteria attached to particles, or bacteria in a planktonic form.

[0169] In some embodiments, the bacterial diversity, relative abundance, bacterial load, and / or any other effect of the at least one compound on the plurality of bacteria of the treated bacteria is compared to corresponding characteristics of the plurality of bacteria in the source sample or any culture sample, and a change / alteration in any characteristic indicates that the at least one compound has an effect on the plurality of bacteria.

[0170] In some embodiments, any other effect of the at least one compound on the plurality of bacteria includes planktonic particle adhesion rate, abundance of a particular bacterium / bacteria, gene expression profile, protein and / or metabolite production, toxin production, suitability of the compound to produce a desired / predetermined microbial profile, suitability of the compound to enhance adhesion of a particular bacterium to particles and / or to enhance planktonic growth forms of a particular bacterium within a population of the plurality of bacteria, or combinations thereof.

[0171] In some embodiments, the method is performed simultaneously in several containers. In some embodiments, several containers can be used simultaneously in a high-throughput assay. In some embodiments, the method further includes culturing a "control plurality of bacteria" not exposed to the compound in a separate container. In some embodiments, such a high-throughput assay is performed in a system generated from the same source sample, for example, to determine optimal treatment or effectiveness on a microbial population. In some embodiments, bacterial diversity, relative bacterial abundance, bacterial load, any other effect of at least one compound, and / or any changes in at least one compound in different treatments are compared with each other and / or with corresponding characteristics in the control plurality of bacteria. In some embodiments, any changes or modifications indicate that at least one compound has an effect on the plurality of bacteria. In some embodiments, the effect is determined at several time points during and / or after exposure of the plurality of bacteria to the compound. In some embodiments, comparisons between different characteristics are performed at the same time to determine the effect of the compound on the plurality of bacteria.

[0172] In some embodiments, the term "any change or alteration" includes an increase, decrease, shift, modulation, change and / or deviation in the microbial profile and / or bacterial load compared to a reference sample, e.g., the source sample, a plurality of bacteria in an untreated control, or any combination thereof.

[0173] In some embodiments, at the end of the culture period, the cultured control line comprises at least 30% similarity, at least 40% similarity, at least 50% similarity, at least 60% similarity, at least 70% similarity, at least 75% similarity, at least 80% similarity, at least 85% similarity, at least 90% similarity, at least 95% similarity, at least 99% similarity, or 100% similarity, or any value and range therebetween, with each possibility representing a separate embodiment of the present invention. In some embodiments, the produced control co-culture comprising the plurality of bacteria comprises at least 30% similarity, at least 40% similarity, at least 50% similarity, 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 to the provided source sample, or any value and range therebetween, with each possibility representing a separate embodiment of the present invention.

[0174] In some embodiments, the effect of a compound on a plurality of bacteria is examined by comparing the level of similarity, bacterial load, and / or any other effect between the treated plurality of bacteria with any one of the following: (i) a control / cultured, e.g., untreated, plurality of bacteria, (ii) a source donor sample, (iii) a differently treated plurality of bacteria, (iv) a co-culture comprising a plurality of bacteria, or any combination thereof. The effect can be examined by comparing different bacterial morphologies or fractions / phases, e.g., particle-attached bacteria, non-attached bacteria, or a combination thereof.

[0175] In some embodiments, the bacterial load in the untreated control system (i.e., the production system that was not exposed to the compound(s) or element(s)) is at least 1 E per gr of particles at the end of the incubation period. 4 ~At least 1E 9 In some embodiments, the untreated control system has at least 1 E per gr of particles at the end of the culture period. 4 , at least 1 E per 1g of particles 5 , at least 1 E per 1g of particles 6 , at least 1 E per 1g of particles 7 , at least 1 E per 1g of particles 8 , at least 1 E per 1g of particles 9 , at least 1 E per 1g of particles 10 , at least 1 E per 1g of particles 11 , at least 1 E per 1g of particles 12 , at least 1 E per 1g of particles 14 , at least 1 E per 1g of particles 16 , or at least 1 E per 1g of particles 20 , or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0176] In some embodiments, adding, contacting, or exposing the plurality of bacteria to at least one compound is performed under conditions suitable for the at least one compound to exert its effect on the bacterial population.

[0177] In some embodiments, the suitable conditions are selected from a pH level, humidity, low humidity, or any combination thereof. In some embodiments, the suitable conditions include subjecting the bacteria to either fluidization, shaking, agitation, stirring, static conditions, or any combination thereof.

[0178] The compositions, systems and methods according to the invention can be used in some embodiments to, among other things, analyze metabolites produced by a subject's microbiota, examine how a particular microbial profile population is or can be modulated / modified given exposure to different compound(s) / treatment(s), determine how different compounds are affected by a subject's microbial population, modulate / modify a microbial profile, e.g., identify compounds that induce a desired / targeted microbial profile, identify effective combination treatments that may exert synergistic or additive effects, e.g., drug-probiotic, probiotic-prebiotic, drug-prebiotic therapy, e.g., by adding different compounds to a culture system, and / or as a platform for producing compositions with different desired microbial profiles by subjecting multiple bacteria to different conditions, to evaluate the suitability of compounds / conditions for producing desired microbial profiles, e.g., as a platform for producing compositions enriched for bacteria, e.g., specific bacteria attached to particles, by adding compounds to the system / subjecting multiple bacteria to different conditions that promote or enhance the growth of bacteria attached to particles and / or the adhesion of bacteria to particles, to evaluate the suitability of compounds / conditions for enhancing specific bacterial growth, e.g., adhesion morphology, from specific bacteria within a population of multiple bacteria, to identify bacterial strains that favor adhesion to particles, strains that favor planktonic morphology, and strains that can grow in both morphologies, e.g., under specific conditions. In some embodiments, the conditions in the system can simulate different regions of the gastrointestinal tract to identify bacteria that support specific growth morphologies in a subject.

[0179] In some embodiments, the preferred growth form of a particular bacterium is determined by separating bacteria not attached to particle(s) from particle-attached bacteria, e.g., by filtration, and / or sedimenting the particles, and determining the abundance of the bacteria in each fraction, e.g., based on next-generation sequencing (NGS) technology and / or whole genome sequencing (WGS). In some embodiments, a bacterium is defined as supporting a particular growth form if its abundance is greater in one of the fractions.

[0180] In some embodiments, the term "microbial profile" includes bacterial diversity (e.g., alpha diversity or beta diversity), relative bacterial abundance, and / or bacterial load.

[0181] In some embodiments, the phrase "generating a desired or different microbial profile" refers to forming a plurality of bacteria having a designed / predetermined bacterial diversity (e.g., alpha diversity or beta diversity), relative bacterial abundance, and / or bacterial load (e.g., suitable for treating a disease, disorder, or condition).

[0182] In some embodiments, the terms "altered" / "altering" or "modulate" / "modulating" include, for example, increasing or decreasing the level of similarity of a microbiome profile compared to an origin or other reference microbial profile.

[0183] In some embodiments, the term "modifying" includes modifying the bacterial load, alpha diversity, relative abundance, and / or beta diversity of the unhealthy microbiota to be 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 similar to that of a healthy subject. Each possibility represents a separate embodiment of the present invention.

[0184] In some embodiments, "modifying" includes changing the bacterial load, alpha diversity, relative abundance, and / or beta diversity of a "non-responding patient" to be 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 similar to that of a "responding patient." Each possibility represents a separate embodiment of the present invention. In some embodiments, a "responding patient" includes a subject who has achieved a response, e.g., a subject who is in remission and / or a subject who is no longer afflicted with a disease, disorder, or condition after treatment. In some embodiments, a "non-responding patient" includes a subject whose disease, disorder, or condition does not decrease or improve after treatment.

[0185] In some embodiments, "reducing" or "reducing" is at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, or 100%, or any value and range therebetween, with each possibility representing a separate embodiment of the present invention.

[0186] In some embodiments, "reducing" or "reducing" is 5-50%, 25-75%, or 10-100%, or any value or range therebetween, with each possibility representing a separate embodiment of the present invention.

[0187] In some embodiments, "increasing" or "increasing" is 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 or range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, "increasing" or "increasing" is 5-50%, 25-75%, 10-100%, 50-350%, 100-400%, 150-550%, 450-785%, or 200-1,000%, or any value or range therebetween. Each possibility represents a separate embodiment of the present invention.

[0188] In some embodiments, the phrase "particle-associated bacteria or bacteria-enriched composition" refers to a composition in which the ratio of particle-associated bacteria to planktonic bacteria is greater than that of a control. In some embodiments, the control comprises a composition cultured under essentially the same conditions without being subjected to a compound that promotes or enhances the growth of bacteria attached to particles and / or bacterial adhesion to particles. In some embodiments, the ratio is at least 1.1-fold or greater, at least 1.5-fold or greater, at least 2-fold or greater, at least 2.5-fold or greater, at least 3-fold or greater, at least 3.5-fold or greater, at least 4-fold or greater, at least 4.5-fold or greater, at least 5-fold or greater, at least 5.5-fold or greater, at least 6-fold or greater, at least 6.5-fold or greater, at least 7-fold or greater, at least 7.5-fold or greater, or at least 8-fold or greater, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0189] In some embodiments, compositions produced according to the methods of the present invention are provided. In some embodiments, the compositions comprise particle-attached bacteria and / or planktonic bacteria, e.g., bacteria cultured / grown in the presence of particles. In some embodiments, the compositions comprise a co-culture comprising multiple bacteria. In some embodiments, the compositions comprise bacteria attached or adhered to particles. In some embodiments, the compositions comprise bacteria in a planktonic form cultured / grown in the presence of particles. In some embodiments, the compositions and / or harvested co-cultures produced according to the present invention are enriched or supplemented with uncultured bacteria, e.g., 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 compositions further comprise at least one additional microorganism, e.g., archaea, viruses, fungi, or any combination thereof. In some embodiments, the compositions comprise at least one bacterial species partially attached to particles and at least one planktonic bacteria.

[0190] 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, a synthetic composition comprises an artificial composition. In some embodiments, a synthetic composition is an artificial composition, for example, but not limited to, a composition created or produced in a laboratory and / or manufacturing site or facility. In some embodiments, a synthetic composition does not comprise a composition isolated or obtained from nature itself. In some embodiments, the composition is frozen, spray-dried, or lyophilized. In some embodiments, the composition is in the form of a dry powder. In some embodiments, the composition comprises a cryoprotectant, a cryoprotectant, an antioxidant, or any combination thereof. In some embodiments, the composition comprises at least one metabolite produced in vitro by at least one bacterium.

[0191] In some embodiments, the composition comprises bacteria in an aggregated form. In some embodiments, the terms "bacteria in an aggregated form" and "bacterial aggregate" are interchangeable and refer to a collection of individual bacterial particles into a single object.

[0192] In some embodiments, the composition comprises bacteria in the form of a biofilm. In some embodiments, the biofilm is in the form of a dry biofilm, e.g., in a solid form, e.g., in a powder form. In some embodiments, the term "biofilm" refers to a community of bacteria embedded in a matrix comprising self-produced exopolysaccharides that are attached to, for example, the surface of particles. In some embodiments, planktonic bacteria are attached, entrapped, fused to, or embedded beneath, on, or within the biofilm.

[0193] In some embodiments, the co-culture comprising a plurality of bacteria comprises particle-associated bacteria. In some embodiments, the co-culture comprises at least one bacterium in a planktonic morphology. In some embodiments, the co-culture comprises or is a mixture of particle-associated bacteria and bacteria in a planktonic morphology.

[0194] In some embodiments, methods according to the invention include removing bacteria not attached to particles or separating bacteria not attached to particles from bacteria attached to particles, thereby producing (i) a composition comprising or enriched in bacteria attached to particles and substantially free of planktonic bacteria cultured in the presence of particles and / or (ii) a composition comprising or enriched in planktonic bacteria cultured in the presence of particles and substantially free of bacteria attached to particles. In some embodiments, the methods include mixing compositions (i) and (ii) in any desired proportions to produce a final composition enriched in, for example, particular bacteria that prefer a particular growth form, e.g., planktonic or attached, and are therefore present in a higher abundance in such separated composition.

[0195] In some embodiments, "a composition comprising or enriched in bacteria attached to particles and that is substantially free of planktonic bacteria cultured in the presence of particles" refers to a composition that contains bacteria in an attached growth form (e.g., greater than 51%, e.g., 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 particles.

[0196] In some embodiments, the phrase "a composition comprising or enriched with planktonic bacteria cultured in the presence of particles and substantially free of bacteria attached to particles" refers to a composition comprising planktonic bacteria cultured in the presence of particles (e.g., greater than 51%, e.g., at least 55%, at least 60%, at least 70%, or 100%, or any value and range therebetween) relative to bacteria in attached growth form.

[0197] In some embodiments, the composition may include bacteria cultured in other growth forms, for example, planktonic cultures.

[0198] In some embodiments, the compositions of the present invention are administered to a subject immediately after culturing. In some embodiments, the compositions are administered after storage, e.g., at room temperature, at a temperature ranging from 2 to 8°C, or at a temperature below -18°C. In some embodiments, the compositions are provided in a solid form, e.g., lyophilized, spray-dried, or frozen. In some embodiments, the solid, e.g., lyophilized or spray-dried, compositions disclosed herein are stable for at least 3 months at room temperature (e.g., a temperature selected from the group consisting of 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 material.

[0199] In some embodiments, the composition is a solid composition.

[0200] In some embodiments, the composition is a spray-dried or freeze-dried composition.

[0201] Typically, the terms "lyophilization" and "freeze-drying" are used interchangeably and refer to the process of freezing a solution and then reducing the water concentration, for example, by sublimation to a level that does not support biological and / or chemical reactions. The resulting lyophilized composition can be stored for long periods of time 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 placed in an appropriate delivery vehicle or reconstituted by the addition of a semi-liquid or liquid solution. The volume added during reconstitution can be similar, less, or more than the initial volume of the solution before the lyophilization process.

[0202] In some embodiments, the composition includes a carrier or excipient. In some embodiments, the carrier is a veterinarily, agriculturally, and / or pharmaceutically acceptable carrier. The terms "carrier" and "excipient" are used interchangeably herein.

[0203] In some embodiments, a pharmaceutical composition is provided comprising a composition disclosed herein and an acceptable carrier or excipient.

[0204] In some embodiments, the term "pharmaceutical composition" includes the terms "dietary composition" and "nutritional composition."

[0205] In some embodiments, the terms "dietary composition," "nutritional composition," and dietary supplement composition refer to compositions suitable for consumption as food supplements, e.g., to supplement a normal diet, correct nutritional deficiencies, maintain adequate intakes of specific nutrients, and / or support specific physiological functions.

[0206] 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 terms "pharmaceutically acceptable" carrier, solvent, diluent, excipient, and vehicle generally refer to a non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material, any type of formulation auxiliary, or simply a sterile aqueous solution, such as physiological 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 which can function as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose, for example, corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate, tragacanth powder, malt, gelatin, talc, excipients such as cocoa butter and suppository waxes and hard fats, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, glycols such as propylene glycol, polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol, esters such as ethyl oleate, ethyl laurate, agar, buffers such as magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and phosphate buffer, and other non-toxic, compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can function as carriers herein include sugar, starch, cellulose and its derivatives, tragacanth powder, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), and other non-toxic, pharmaceutically compatible materials used in other pharmaceutical preparations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as colorants, flavorings, excipients, stabilizers, antioxidants, and preservatives may also be present.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 well known to those skilled in the art, for example, those listed in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001), the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004) and "Inactive Ingredient Guide", US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, all of whose contents are incorporated herein by reference.The examples of pharmaceutically acceptable excipients, carriers and diluents useful in the compositions of the present invention include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution and DMSO. These additional inactive ingredients, as well as effective formulation and administration procedures, are well 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, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated herein by reference in its entirety.The compositions described herein may also be contained in artificially created structures such as liposomes, ISCOMS, sustained-release particles, and other vehicles. Liposomes include emulsions, bubbles, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the peptides described herein are generally formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and sterols, such as cholesterol. Various methods for preparing liposomes are available, as reviewed, for example, in Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York; see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0207] The carriers may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0208] In some embodiments, the composition is for pharmaceutical use, in some embodiments, the composition is for agricultural use, in some embodiments, the composition is for veterinary use.

[0209] In some embodiments, methods are provided for preventing or treating a disease, disorder, or condition (e.g., dysbiosis) and / or regulating the microflora in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition according to the present invention.

[0210] The terms "therapeutically effective amount" and "effective amount" refer to the amount necessary to prevent, ameliorate, and / or treat a disease, disorder, or condition. The effective dose may vary depending on the subject's sex, age, and weight, the disease or condition and its severity, and any other factors that can be recognized by a person skilled in the art.

[0211] In some embodiments, the pharmaceutical compositions are for use in treating or preventing a dysbiosis in a subject in need thereof, hi some embodiments, the compositions disclosed herein are for use in the preparation of a medicament for treating or preventing a dysbiosis in a subject in need thereof.

[0212] In some embodiments, the pharmaceutical composition is for use in modulating the microflora in a subject in need thereof, e.g., for treating or preventing C. diff infection, ulcerative colitis, or atopic dermatitis in a subject in need thereof.

[0213] In some embodiments, the term "modulating microflora" includes reversing established bacteria typically associated with a disease, health condition, or clinical symptom, and / or achieving colonization of beneficial bacteria in any tissue and / or surface of any body part of an organism, including, but not limited to, any external or internal surface of the body, and the deeper layers of the skin, typically associated with a disease, health condition, or clinical symptom.

[0214] As used herein, the term "dysbiosis" is characterized by an alteration, imbalance, dysfunction, and / or dysfunction of the microbiota and / or microbiome in any tissue and / or surface of any body part of an organism, including, but not limited to, any external or internal surface of the body and the deeper layers of the skin, typically associated with a disease, health condition, or clinical symptom. The imbalance can be in any microbial community, including, but not limited to, imbalances in the gastrointestinal tract, skin, oral cavity, bronchus, vagina, or rectum, to name a few. In some embodiments, the dysbiosis is tumor dysbiosis.

[0215] In some embodiments, the term "surface of any body part of an organism" refers to external surfaces of the body that are visible to the naked eye, such as the skin of the face, throat, scalp, chest, back, ears, neck, hands, elbows, knees, and other skin sites, as well as surfaces of internal body parts that are part of the internal anatomy of an individual, such as, but not limited to, the oral cavity, the digestive tract, and the lower reproductive tract, e.g., the vagina.

[0216] In some embodiments, the dysbiosis comprises an imbalance of microbial flora in a substrate or medium, hi some embodiments, the substrate or medium comprises or is soil.

[0217] In some embodiments, an altered microbiota refers to a flora that has diverged from the homeostatic microbiota, such as in an unhealthy subject. In some embodiments, the altered microbiota comprises a different bacterial diversity, relative bacterial abundance, and / or bacterial load compared to a healthy microbiota. As used herein, the term "healthy subject" refers to a subject having a native flora representative of a healthy subject. In some embodiments, the altered microbiota comprises pathogenic microorganisms. In some embodiments, the altered microbiota is associated with a medical condition and / or is detrimental to the health of the subject, e.g., a human subject.

[0218] 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 may be male or female.

[0219] Pharmaceutical compositions can take any physical form necessary for proper administration. The compositions can be administered in any suitable form, including, but not limited to, liquid, gel, semi-liquid (e.g., a liquid, such as a viscous liquid, that contains some solid), semi-solid (a solid that contains some liquid), or solid. The compositions can be provided, for example, in tablet, pessary, cream, suppository, capsule, liquid, food, chewable, non-chewable, buccal, sublingual, sustained-release, non-sustained-release, extended-release, or non-sustained-release form.

[0220] In some embodiments, the composition is formulated for administration by a mode selected from oral, rectal, parenteral, mucosal, vaginal, nasal, local, topical, pulmonary, ocular, oral, buccal administration, or any combination thereof.

[0221] Pharmaceutically acceptable carriers suitable for preparing unit dosage forms of the compositions described herein for oral administration are well known in the art.

[0222] In some embodiments, the similarity comprises at least one feature selected from the group consisting of functionality, e.g., the ability of the bacterium to affect a metabolic process of the subject and / or ameliorate any disease, disorder, or condition of the subject; potential metabolic pathways / pathways, e.g., the potential to produce, synthesize, consume, and / or utilize a particular metabolite / organic compound; relative abundance; alpha diversity; recovered operational taxonomic units (recovered OTUs) or recovered amplicon sequence variants (ASVs); beta diversity; and any combination thereof.

[0223] In some embodiments, the term "similarity" includes the similarity between the bacterial community or population present in the co-culture compared to the bacterial community or population present in the source sample. In some embodiments, the term "similarity" includes the similarity between other microorganisms present in the composition, including at least one of archaea, viruses, e.g., bacteriophages, fungi, or any combination thereof. Similarity can be determined by any method known to those of skill in the art.

[0224] In some embodiments, similarity is determined by an α-diversity index. In some embodiments, the α-diversity index includes any one of observed species, total genes discovered, Shannon, Chao1, Simpson, or any combination thereof. In some embodiments, α-diversity is a measure that considers the diversity or abundance of taxa, e.g., bacterial taxa, in a sample. In some embodiments, α-diversity is compared between two communities (or samples). In some embodiments, when referring to an α-diversity similarity metric, the diversity or abundance of taxa in a composition or co-culture is compared to the diversity or abundance in the source 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.

[0225] In some embodiments, the similarity is determined by a beta diversity index. In some embodiments, the beta diversity index includes any one of Jaccard, qualitative Unifrac similarity, quantitative Unifrac similarity, generalized Unifrac similarity, Bray-Curtis similarity, or any combination thereof. In some embodiments, generalized Unifrac similarity is performed as described in Jun et al., "Associating microbiome composition with environmental covariates using generalized Unifrac distances," Bioinformatics. 2012 Aug 15;28(16):2106-2113.

[0226] In some embodiments, similarity is determined by qualitative Unifrac similarity. In some embodiments, similarity is determined by quantitative Unifrac similarity. In some embodiments, "qualitative Unifrac similarity" ("qualitative Unifrac") and "quantitative Unifrac similarity" ("quantitative Unifrac") refer to a measure of similarity between two communities (or samples) that considers both: I. data with / without data of bacterial populations, and II. phylogenetic relatedness of bacteria. Quantitative Unifrac similarity also considers the relative abundance of bacterial populations.

[0227] In some embodiments, similarity is determined by Bray-Curtis. In some embodiments, the "Bray-Curtis dissimilarity index" is a non-phylogenetic beta diversity similarity measure between two communities (or samples) that takes into account the abundance of bacterial populations within the samples. In other embodiments, the use of non-phylogenetic beta diversity indices may result in lower similarity values ​​compared to other similarity indices that take into account the phylogenetic relatedness of bacteria (e.g., quantitative Unifrac similarity, qualitative Unifrac similarity).

[0228] In some embodiments, when similarity is determined using next-generation sequencing (NGS) technology and / or whole genome sequencing (WGS) using an index that considers the phylogenetic relatedness of bacteria, the co-culture contains at least 30% similarity to the source sample. In some embodiments, the index that considers the phylogenetic relatedness includes any one of diversity recovery, quantitative Unifrac similarity, qualitative Unifrac similarity, generalized Unifrac similarity, or any combination thereof. Analysis can be performed at any taxonomic level. While various similarity indices can be used according to the present invention, it should be understood that some methods, such as using a non-phylogenetic beta diversity index obtained according to one embodiment of the present invention, may result in low similarity values, e.g., less than 30%, compared to other similarity indices tested that consider the phylogenetic relatedness of bacteria.

[0229] In some embodiments, similarity is determined by relative abundance. In some embodiments, the term "relative abundance" refers to the percentage of a particular taxon, e.g., a bacterial taxon, relative to the total abundance of the taxon, and is used herein to compare the distribution of genera and / or any other bacterial taxonomic level among communities, e.g., bacterial communities, within a sample.

[0230] In some embodiments, alpha diversity and / or beta diversity similarity is calculated from recovered OTUs and / or recovered ASVs. In some embodiments, the terms "recovered OTUs," "recovered ASVs," or "diversity recovery" refer to the percentage of taxa observed in a test sample out of all taxa observed in another sample, e.g., a sample of fecal origin.

[0231] In some embodiments, the similarity is compared to a processed source sample, for example, homogenized and / or filtered.

[0232] As will be appreciated by those skilled in the art, the similarity index can be based on next-generation sequencing (NGS) techniques, such as ribosomal 16S RNA or coding DNA, and / or whole genome sequencing (WGS). Similarity can be determined at any taxonomic level, such as phylum, family, genus, species, and / or lineage.

[0233] In some embodiments, the source sample comprises a predetermined bacterial population, and the co-culture is characterized by having at least 30% similarity to the predetermined bacterial population. In some embodiments, the term "predetermined" refers to a custom-made source composition comprising multiple bacterial populations selected according to specific needs, e.g., suitable for treating a subject with intestinal dysbiosis. In some embodiments, the term "predetermined" refers to a bacterial population that commonly colonizes an environmental niche. In some embodiments, the source 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 microbial population present in the sample.

[0234] In some embodiments, the plurality of bacteria or the plurality of bacteria provided in the source sample belong to at least 5 to up to 600 species of bacteria as determined by 16S NGS technology, for example, 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, Belonging to at least 180, at least 190, at least 200, or more bacterial species, and / or belonging to at least 2 bacterial genera and up to 250 genera as determined by 16SNGS 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 of bacteria therebetween. Each possibility represents a separate embodiment of the present invention.

[0235] In some embodiments, the plurality of bacteria provided or the plurality of bacteria in the source sample belong to at least 35-80 observed genera and / or at least 120-300 observed species, or any value and range therebetween, as determined by SNGS techniques, with each possibility representing a separate embodiment of the present invention.

[0236] In some embodiments, the compositions or co-cultures have at least 30% similarity in diversity or abundance of multiple bacteria.

[0237] In some embodiments, the co-culture comprising multiple bacteria comprises at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% similarity to the source sample, or any value and range therebetween, with each possibility representing a separate embodiment of the present invention.

[0238] 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-cell-dead staining, propidium monoazide qPCR (PMA-qPCR), microscopy, metabolic assay, spectrophotometry, or any combination thereof.Methods for determining bacterial load as disclosed herein are common and will be apparent to those skilled in the art.

[0239] In some embodiments, the term "bacterial load" is interchangeable with the term "bacterial count." In some embodiments, the co-culture or composition comprises viable bacteria, and the bacterial count is measured by CFU.

[0240] In some embodiments, the amount of bacteria in a composition produced according to the present invention is calculated per gram of dry form particles introduced into the culture medium or container. In some embodiments, the amount of bacteria is calculated per gram of dry form particles used to contact multiple bacteria. In some embodiments, when measuring the amount of bacteria in a co-culture in a composition, the weight of the particles added to the culture system is taken into account. In some embodiments, when measuring the amount of bacteria in a co-culture in a composition, the weight of the particles in the final composition is taken into account.

[0241] In some embodiments, the term "particle" includes a plurality of one or several types of particles and refers to a substance / material adapted, configured, or suitable for the adhesion / attachment and / or growth of at least one bacterium. In some embodiments, a particle comprises a surface to which bacteria can adhere.

[0242] In some embodiments, in the context of particles, 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 and non-porous particles. In some embodiments, the term "porous" refers to void (i.e., "empty") space in a material. In some embodiments, the term "porous" includes having a non-uniform surface area.

[0243] As used herein, the terms "particle(s)," "nanoparticle(s)," "microparticle(s)," "nanosphere(s)," and "microsphere(s)" are used interchangeably.

[0244] In some embodiments, the particles are not derived from the source sample. In some embodiments where the source is a fecal sample, the particles are not undigested fecal fiber. In some embodiments, the source sample lacks particles as disclosed herein.

[0245] In some embodiments, the particles used in accordance with the present invention are water-insoluble active agents. In some embodiments, the term "water-insoluble active agent" refers to particles having a solubility in water of less than 5 mg / ml, preferably less than 1 mg / ml, and most preferably less than 0.1 mg / ml at 25°C.

[0246] In some embodiments, the particles used in accordance with the present invention are water-insoluble at physiological pH, ie, insoluble at a pH in the range of about 1.0 to 8.0.

[0247] In some embodiments, "particles" include particles having diameters ranging from 5 microns to 1 cm in diameter. In some embodiments, particles range from 1 micron to 50 millimeters. In some embodiments, particles are 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 in diameter, or any range or value therebetween. Each possibility represents a separate embodiment of the present invention.

[0248] In some embodiments, the particles have an average diameter in the range of 1 to 1,500 microns. In some embodiments, the particles have an average diameter 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 1,000 microns (including any range therebetween). Each possibility represents a separate embodiment of the present invention.

[0249] In some embodiments, the diameter is an average diameter. In some embodiments, the diameter is a maximum diameter. In some embodiments, the diameter is a minimum diameter.

[0250] In some embodiments, the term "particle" includes any particle of any shape, for example, within the specified size range. In some embodiments, the particle may be round, amorphous, irregular, spherical, ellipsoidal, flower-shaped, cubic, spherical, elongated, rod-shaped, any other shape, or any combination thereof.

[0251] 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 comprise a plurality of one type of particle. In some embodiments, the particles comprise a plurality of particles of multiple types.

[0252] In some embodiments, the particles comprise a combination of calcium and cellulose. In some embodiments, the particles comprise a combination of phosphate and cellulose. In some embodiments, the particles comprise a combination of calcium, phosphate, and cellulose. In some embodiments, the particles comprise or consist of MCC and DCP.

[0253] In some embodiments, the particles are completely submerged in the culture medium, hi some embodiments, the particles are at least partially submerged in the culture medium.

[0254] In some embodiments, the particles are 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), The particles may include MCC and DCP in a weight ratio (w / w) of 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), or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, all w / w ratios herein for particles are based on the anhydrous form.

[0255] In some embodiments, the seeds are selected from cranberries, passion fruit, herbs, oats, or any combination thereof.

[0256] In some embodiments, the particles comprise or consist of food-grade particles. In some embodiments, the food-grade particles comprise or consist of polysaccharides, fat crystals, proteins, or any combination thereof. In some embodiments, the food-grade particles comprising fat crystals are selected from glycerol 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, nano- or methylcellulose, chitin, chitosan, or any combination thereof. In some embodiments, the food-grade particles comprising proteins are selected from β-lactoglobulin, lactoferrin, lactoferrin polysaccharides, 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 (tyrosides), waxes, shellac-xanthan gum, or any combination thereof.

[0257] In some embodiments, the weight / weight ratio of particles to sample according to the methods of the present invention is 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, with each possibility representing a separate embodiment of the present invention.

[0258] In some embodiments, the solution, e.g., buffer or medium, and particles used during contact or culturing are in a volume / weight (v / w) ratio ranging from 1:1 to 200:1, or any value and range therebetween, with each possibility representing a separate embodiment of the present invention.

[0259] In some embodiments, the terms "attachment" and "adhesion" with respect to bacteria include adsorption to a surface via, for example, weak and / or strong interactions, e.g., by flagella, pili, lipopolysaccharides, exopolysaccharides, collagen-binding adhesive proteins, etc.

[0260] In some embodiments, "additional microorganisms at least partially attached to the particles" refers to attached "additional microorganisms," including non-bacterial microorganisms, e.g., archaea, viruses, and / or fungi, attached to the particles, particle-attached bacteria, and / or the matrix formed by the attached bacteria, and refers to at least 0.01% of the total "additional microorganisms" present in the culture system or at the end of the contacting or culturing step.

[0261] In some embodiments, after the bacteria are attached to the particle(s), some or all of the bacteria can be separated and / or other bacteria in the suspension can be attached to the particles, particle-adherent / adherent bacteria, and / or the matrix formed by the attached / adherent bacteria. In some embodiments, "bacteria at least partially attached to particles" includes bacteria attached to particle-adherent / adherent bacteria and / or attached to the matrix formed by the attached / adherent bacteria. In some embodiments, the phrase "a plurality of bacteria at least partially attached to 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% of the total bacteria present in the culture system or at the end of the contacting or culturing step, or any value and range therebetween. Total bacteria can be determined by methods for determining bacterial load, such as those disclosed below. The adherent and non-adherent fractions can be assessed, for example, by separating the two fractions to assess the percentage of adherence and / or to analyze each fraction individually. In some embodiments, the individual analysis involves separating the two fractions and determining the number / amount of bacteria in each fraction. In some embodiments, the separation of fractions or the removal of non-adherent bacteria is performed by or includes filtration and / or gravity sedimentation, e.g., centrifugation. In some embodiments, the term "filtration" includes all separation techniques, as well as any other process that utilizes a filter capable of separating fractions.

[0262] general All numerical values ​​herein are assumed to be modified by the term "about." The term "about" generally refers to a range of numbers that one of ordinary skill 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" is before a numerical value, the term "about" is intended to indicate ±10%.

[0263] The terms "comprise," "comprising," "include," "including," "having," and their conjugations mean "including, but not limited to." The term "consisting of" means "including and limited to."

[0264] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0265] The word "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 or advantageous over other embodiments and / or to exclude the incorporation of features from other embodiments.

[0266] 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 more than one "optional" feature, unless such features are inconsistent.

[0267] As used herein, the singular forms "a," "an," and "the" include plural references 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 content clearly dictates otherwise. For example, the term "compound" or "at least one compound" can include multiple compounds, including mixtures thereof.

[0268] Throughout this application, various embodiments of the present invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0269] Whenever a range of numerical values ​​is given herein, it is meant to include any and all recited numbers (fractional or integer) within the range given. The phrases "ranging between" a first designated number and a second designated number, and "range" from a first designated number to a second designated number, are used interchangeably herein and are meant to include the first designated number and the second designated number and all fractional and integer numbers therebetween.

[0270] As used herein, the term "method" refers to ways, means, techniques, and procedures for accomplishing a given task, and includes, but is not limited to, ways, means, techniques, and procedures known to those of skill in the art of chemistry, pharmacology, biology, biochemistry, microbiology, and medicine, or readily developed from known ways, means, techniques, and procedures by those of skill in the art.

[0271] It is understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination, or as appropriate, in any other described embodiment of the 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.

[0272] The description of various embodiments of the present invention is presented for illustrative purposes, but is not intended to be exhaustive or limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terms used herein are selected to best explain the principles of the embodiments, practical applications, or technical improvements to technology found in the market, or to enable those skilled in the art to understand the embodiments disclosed herein.

[0273] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0274] Other terms used herein are meant to be defined by their known meanings in the art.

[0275] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples. [Example]

[0276] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are fully explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual" by Sambrook et al. (1989); "Current Protocols in Molecular Biology" Volumes I-III by Ausubel, RM, 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", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998), 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, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique," Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology," Volumes I-III, Coligan JE, 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 widely described in the patent and scientific literature. For example, U.S. Patent No. 3,791,932, U.S. Pat. 35,074, 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, MJ, ed. (1984), "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985), “Transcription and Translation” Hames, BD, and Higgins SJ, eds. (1984), “Animal Cell Culture” Freshney, RI, ed. (1986), “Immobilized Cells and Enzymes” IRL Press, (1986), “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 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. Other general references are provided throughout this specification.

[0277] In the following examples, multiple bacterial cultures derived from fecal / stool or saliva samples (obtained from healthy human donors) are used as embodiments to illustrate the ability of the method according to the invention to produce compositions comprising co-cultures with a high degree of similarity to the original sample, e.g., for pharmaceutical applications and / or as an in vitro screening system. A fecal sample was obtained from a diseased donor to evaluate the effect of pharmaceuticals on the microbiome sampled from a diseased subject in a screening model. In the examples, "bacterial sample," "stool sample," and "original fecal sample" are all used to refer to the original sample used to produce the composition.

[0278] Exemplary source fecal samples were used in the following experiments, containing 42-58 bacterial genera (average 50.1) and 131-196 observed bacterial species (average 168.1). Data are obtained from five different experiments using five different source samples and are shown as ranges, where the minimum and maximum values ​​in parentheses are the minimum and maximum values ​​obtained across the five different experiments, and are the average values ​​across the five different experiments.

[0279] Typically, samples derived from human donors, such as fecal samples, contain various types of microorganisms, including bacteria, archaea, viruses, and fungi. Within each type of microorganism, there are distinct populations / communities that require different maintenance, growth, culture, and / or proliferation conditions. Therefore, maintaining a high degree of similarity among such various populations in vitro is difficult, especially when they are cultured together in a single container.

[0280] Additionally, each sample source has a different microbial composition, further increasing the challenges of maintaining similarity, reproducibility, and process scale-up.

[0281] It has been reported in the literature that most gut microbes are considered to be unculturable or difficult to cultivate as an ecosystem [e.g., Lagier, JC, Dubourg, G., Million, M., Cadoret, F., Bilen, M., Fenollar, F., Levasseur, A., Rolain, JM, Fournier, PE 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, HA, Kim, E., Song, DG, Yoon, HS, Pan, CH, Komba, EV, Ko, G. and Cha, KH, 2021. Synthetic gut microbiome: advances and challenges. Computational and structural biotechnology journal, 19, pp. 363-371.

[0282] The following examples demonstrate that despite the complexity and variability of the source samples, the similarity of various communities to the source samples, e.g., bacterial populations, and their functionality were preserved during cultivation and scale-up according to the illustrated embodiments.

[0283] material and method Similarity Parameter To obtain the relative abundance of each taxon in the co-culture, we calculated similarity indices commonly used in the art based on 16S next-generation sequencing (NGS) or whole-genome shotgun (WGS) techniques.

[0284] "Relative abundance" is the percentage of a particular taxon relative to the total abundance of the taxon and is used to assess the distribution of taxa among communities within a sample. In the examples, relative abundance was compared between two test samples.

[0285] "Alpha diversity" is a measure that takes into account the number of taxa in a sample. In the examples, alpha diversity was compared between communities (or samples).

[0286] "Recovered OTUs" (and / or recovered ASVs and / or diversity recovered) is a measure of similarity between two communities (or samples) that considers the proportion (percentage) of OTUs (and / or ASVs) observed in the processed sample (composition) among all OTUs (and / or ASVs) observed in the source sample. "Recovered OTUs" (and / or ASVs) refers to the bacterial diversity / abundance in the processed and / or cultured sample (composition) compared to the OTUs (and / or ASVs) in the source sample.

[0287] "β-diversity," expressed as "qualitative Unifrac similarity" ("Qualitative Unifrac") and "quantitative Unifrac similarity" ("Quantitative Unifrac"), is a measure of similarity between two communities (or samples) that takes into account both: I. data with or without bacterial populations, and II. the phylogenetic relatedness of taxa. Quantitative Unifrac similarity also takes into account the relative abundance of taxa in the populations.

[0288] The "β diversity" presented in the Bray-Curtis similarity index is a measure of similarity between two communities (or samples) that refers to the abundance of taxa within the samples.

[0289] Each dot on the β diversity plot represents the total microbial / viral / fungal composition profile of one sample. Samples with similar microbial / viral / fungal profiles are presented closer to each other, while samples with different profiles are presented at arbitrary distances from each other.

[0290] The "Chao1 richness index," "Shannon index," "Simpson reciprocal index," or any other parameter used in this example to analyze population dynamics has its meaning known in the art.

[0291] bacterial count In the following examples, the amount of bacteria was calculated per gram of dry particles introduced into the culture vessel, the weight of the particles being calculated according to the particles used to contact the bacteria.

[0292] Bacterial load determination was performed using known methods: DNA-based quantification (quantitative polymerase chain reaction - qPCR) and colony-forming unit (CFU) assays known in the art. Results are presented on a logarithmic scale per gram of dry particles introduced into the vessel.

[0293] CFU determination. Samples were serially diluted and bacteria were plated in triplicate on CDC plates. Plates were incubated anaerobically at 37°C for 48–72 hours, after which CFU were counted.

[0294] qPCR measurements. Absolute abundance quantification was performed using quantitative real-time PCR with a standard curve constructed using plasmid DNA containing a single copy of the 16S gene in 10-fold serial dilutions. Total DNA was calculated by normalizing to the standard curve and the average assumed genome size.

[0295] In the examples below, the terms "bacterial count" and "bacterial load" are used interchangeably.

[0296] Processing and culturing procedures for the original biological sample Unless otherwise indicated, all processes were performed under anaerobic conditions. Stool samples were collected in disposable stool containers and transferred to anaerobic conditions. Preparation of inoculum from stool samples was performed under anaerobic conditions. When testing the effect of aerobic incubation, treatments were performed under either anaerobic or aerobic conditions.

[0297] 1 mg to 450 gr of feces was mixed with sterile anaerobic saline (50 ml to 6 L), mixed until homogenous, and filtered. The filtrate containing the bacterial population (referred to herein as fecal liquid - FL / "source" sample, considered 100% for similarity calculations) was transferred to a separate fermentation vessel (50 ml to 6 L). The bacterial population was cultured in a single vessel under anaerobic conditions in the medium detailed below in the presence of particulate MCC:DCP (20:80, e.g., 20 gr MCC; 80 gr DCP). Contacting and culturing in the presence of particulates was performed for a period of less than 14 days.

[0298] To evaluate the effects of compounds and bacterial populations, 1 mg to 50 g of feces was mixed with 1 to 150 ml of sterile anaerobic saline. Bacterial samples were cultured in a single vessel under anaerobic conditions in 1 to 150 ml of the medium detailed herein below in the presence of 10 to 1,200 g of MCC:DCP (20:80 or 50:50; MCC:DCP). Cultures were grown for periods of less than 14 days.

[0299] Medium exchange and treatment sampling process The medium was changed several times throughout the culture procedure. Before the medium change, mixing / agitation was stopped to allow particles to settle. After the medium change, mixing was resumed.

[0300] To assess the effect of compounds on bacterial populations or the effect of bacterial populations on compounds, incubations were performed either under static or agitated conditions, and sampling was performed at specific time points after exposure of the bacterial populations to the test compounds.

[0301] Culture conditions included the following: stirring in the range of 50 to 750 RPM, temperature -32 to 39°C, and pH -4.4 to 8.0.

[0302] Unless otherwise specified, for similarity parameters and bacterial load measurements, sampling was performed from the particle-associated bacterial phase. Phase separation was achieved by settling the particles, by centrifugation, washing, or filtration.

[0303] Sampling Process To measure similarity and bacterial load, sampling was performed at several time points (denoted as T1 or TP1, T2 or TP2, T3 or TP1) during the cultivation procedure.

[0304] Medium composition The media compositions used are detailed in the Examples below. Pharmaceutical or food grade ingredients were used. [Example]

[0305] Multiple bacteria are cultured in a single container in the presence of particles. In the following examples, the effect of culturing multiple bacteria in the presence of particles on the level of similarity was evaluated.

[0306] For this purpose, fecal samples were treated and cultured in the presence of particles as detailed in the Materials and Methods section above. As a comparison, treated fecal samples were cultured under the same conditions in the absence of particles. Similarity was determined at the end of the culture period using the quantitative Unifrac similarity index. The similarity of the compositions produced under both culture conditions was compared to a reference fecal sample. Measurements were taken at the end of the culture period from: (i) planktonic cultures; (ii) both the planktonic and particle-associated bacterial phases of cultures performed in the presence of particles.

[0307] The results show that the planktonic culture samples are at arbitrary distances from the source sample compared to the distances of the particle-attached bacterial fraction and the non-attached fraction (Figure 1).

[0308] These results indicate that culturing in the presence of particles results in compositions containing either planktonic and / or attached bacteria that have a high degree of similarity compared to planktonic cultures (e.g., culturing in the absence of particles).

[0309] These results indicate that it is advantageous to culture multiple bacteria in the presence of particles to obtain compositions with a high degree of similarity to the source sample.

[0310] Therefore, in all the following examples, the incubation was carried out in the presence of particles unless otherwise indicated. [Example]

[0311] Effect of the type of carbon source in the medium on the similarity to the source sample and bacterial abundance In the following examples, the effect of different carbon sources on the ability to grow human-derived bacteria and produce bacteria-containing compositions with a high degree of similarity to the source fecal sample was evaluated. Additionally, bacterial load was tested.

[0312] For this purpose, bacteria-containing compositions were processed and cultured in either a medium containing a single carbon source (e.g., glucose) or a medium containing multiple carbon sources, as detailed above in the Materials and Methods section. The multiple carbon sources included glucose, maltose, trehalose, and starch. Each medium further contained yeast extract, peptone extract, sodium chloride, and disodium hydrogen phosphate. Cultivation was performed in flasks (small scale).

[0313] Similarity was determined by using the quantitative Unifrac similarity index, and bacterial load was determined by qPCR measurements. Samples were taken from the particle-associated bacterial phase at different time points (designated T1, T2, and T3).

[0314] These results show that all test groups had a similarity of more than 50% to the source sample and 1E per gram of particles. 8The results show that the bacterial abundance (qPCR) exceeded 100% (Figure 2). However, the use of multi-carbon source medium resulted in a higher bacterial abundance while maintaining a high similarity compared to the single-carbon source medium.

[0315] Thus, current studies have shown that the use of two or more carbon sources in the culture medium advantageously increases the degree of similarity to the source sample. [Example]

[0316] Effect of trace element addition in the culture medium on the similarity to the source sample In the following examples, the effect of adding trace elements to glucose or multi-carbon source media on the similarity of the composition to the original sample was investigated. The trace elements used were manganese, copper, and iron sources. In addition, the bacterial load was examined.

[0317] For this purpose, compositions containing bacteria were treated and cultured as detailed in the Materials and Methods section above. Cultures were performed in either a medium containing a single carbon source (glucose) or a medium containing multiple carbon sources (glucose, maltose, trehalose, and starch) with or without trace element supplementation. Each medium additionally contained yeast extract, peptone extract, sodium chloride, and disodium hydrogen phosphate. Cultures were performed in flasks (small scale).

[0318] Sampling was performed at three time points during the culture (T1, T2, and T3) to assess quantitative Unifrac similarity and bacterial load (qPCR measurement). The reference sample is designated as the "source sample."

[0319] These results (Figure 3) show that similarities of over 60% were obtained in all test groups. High similarities of over 70% were obtained by adding trace elements to the high-carbon medium. These results also showed a tendency for the resulting co-cultures to have higher bacterial abundances when media containing both trace elements and a high-carbon source were used. [Example]

[0320] Effect of incubation under aerobic versus anaerobic conditions on the level of similarity to the source fecal sample In the following examples, the effect of anaerobic or aerobic conditions on bacterial similarity levels was investigated.

[0321] Bacterial samples were processed and cultured under anaerobic or aerobic conditions using different media substrates as described above in the Materials and Methods section. Test media included brain-heart infusion medium (BHI) and various food-grade media compositions suitable for growing human gut bacteria. At the end of the culture, similarity (based on the Bray-Curtis assay) was assessed (the source fecal sample is labeled "Source"; anaerobic conditions are labeled "AN").

[0322] These results are shown as a PCoA (metric multidimensional scaling, MDS) plot (Figure 4), which shows that using anaerobic conditions during cultivation was superior for maintaining similarity.

[0323] Anaerobic conditions were found to be superior in obtaining high similarity scores, and therefore anaerobic conditions were used in all of the following examples. [Example]

[0324] Similarity to samples of fecal origin using different media and scale-up fermentations In the following examples, the effect of the media and scale-up process used on the level of similarity was evaluated.

[0325] For this purpose, bacterial samples were processed and cultured as described above in the Materials and Methods section using different source media substrates. Cultivation was performed in either small or medium volumes (50 ml to 6 L) in the presence of particles and under anaerobic conditions. Table 1 below lists the conditions used during cultivation.

[0326] To assess similarity and bacterial abundance, representative samples of particle-attached bacteria were collected from each treatment, quantitative similarity levels of bacterial populations were collected, and bacterial counts (based on qPCR measurements) were analyzed. [Table 1]

[0327] These results indicate that culturing in PG2 medium resulted in both increased bacterial numbers and higher similarity values ​​compared to culturing in standard media, such as BHI (Figure 5). The results also show that culturing in PG2 medium advantageously maintained high similarity levels over long periods of time.

[0328] When comparing small-scale and medium-scale (fermentor) fermentations using PG2 medium, the results show a higher similarity value in the medium-scale fermentation. The bacterial counts at the four time points in the medium and small scale fermentations were 6 E 10 and 1 E 8 This exceeds the scalability of the process.

[0329] In another experiment, we found that the fermentation rate was higher than 50% and the fermentation rate was at least 1 E per 1g. 6 The number of days of cultivation necessary to obtain a composition / co-culture with a bacterial load of 1000 kJ / g was investigated. It was found that cultivation for a period ranging from 6 h to 6 days advantageously allows obtaining both parameters (data not shown). When using multiple carbon sources, a cultivation period of 12 h to 5 days resulted in a quantitative Unifrac similarity of 70% or more and a bacterial load of at least 1 E per gr. 8 The bacterial load was .

[0330] This method therefore allows for scalability and the ability to obtain compositions similar to fecal biological samples. [Example]

[0331] Process consistency using samples from different sources The following examples are aimed at investigating whether the method can use different source samples with different populations and still produce compositions with a high degree of similarity to the source sample.

[0332] The results shown in Figure 6 represent combined data from three processes using three origin samples from the same donor collected on different days. The samples were processed and cultured independently in medium-scale conditions (6 L) containing a multi-carbon source medium containing glucose, maltose, trehalose, starch, yeast extract, peptone extract, sodium chloride, disodium hydrogen phosphate, a manganese source, a copper source, and an iron source, as described above in the Materials and Methods section.

[0333] These results show quantitative Unifrac similarity and bacterial load (qPCR and CFU measurement) at different time points (T1–T6) from three independent cultures.

[0334] These results show that all time points were greater than 71% similar to the fecal origin sample (labeled "Origin") with a relatively low SD, demonstrating the method's ability to consistently obtain compositions with high similarity to the origin sample. These results also show that the level of similarity decreased over time, while CFU and bacterial counts (qPCR) increased (e.g., comparing T1 to T6).

[0335] This illustrates that providing a method according to one embodiment of the present invention results in compositions with high similarity and increased bacterial counts, even when using distinct source samples with different microbiome profiles.

[0336] In a separate experiment, the reproducibility and repeatability of the process was further evaluated.

[0337] To this end, five independent cultivation processes were performed, each using a different source sample, as described in Materials and Methods. The five independent processes were analyzed by comparing the produced composition with each source sample in terms of observed species, observed genus, and quantitative similarity parameter indices.

[0338] The genera observed in the source samples ranged from 42 to 58 (mean 50.1), whereas the compositions contained 39 to 55 genera (mean 43.3, 84.4% greater genus richness compared to the source samples). The species observed in the source samples ranged from 131 to 196 (mean 168.1), whereas the compositions contained 104 to 188 species (mean 138.3, 82.8% greater species richness compared to the source samples).

[0339] The average quantitative similarity to the source sample of the five experiments was 77.1% (each sample was compared to its corresponding source sample).

[0340] We determined similarity using the Bray-Curtis values ​​of five experiments and found that this ranged from 19 to 46%, with an average of 35.1%. Therefore, using a similarity index that does not consider the phylogenetic relatedness between bacterial populations, such as Bray-Curtis, may yield lower values ​​compared to those obtained when using other similarity indices.

[0341] These results show that although different origins were used, the compositions maintained a similarity of over 77% to their source samples, with comparable numbers of observed species and genera.

[0342] These results demonstrate that, despite variations in the source sample community, the present method advantageously makes it possible to "replicate" the source sample and obtain a synthetic composition that has a high degree of similarity to it.

[0343] Thus, the similarity analysis of a composition to the source sample can be determined using various similarity indices, although some methods may result in lower similarity values, for example, as exemplified above.

[0344] The above examples demonstrate the ability of different embodiments of the present invention to obtain high bacterial population similarity while obtaining high bacterial counts. Examples 7 and 8 below demonstrate the ability of the method to further preserve other similarity features, such as similarity of other communities in addition to bacterial communities, such as viruses, fungi, and at least one metabolic pathway. [Example]

[0345] Similarity of other communities in compositions produced according to embodiments of the present invention to the source sample In the following examples, the similarity of other communities in the composition was assessed compared to the source sample.

[0346] Fecal samples were processed and cultured as described above in the Materials and Methods section, and the resulting compositions were analyzed for their viral, fungal, and bacterial communities using WGS. Similarity levels were measured using Bray-Curtis similarity. Graphs are presented as nonmetric multidimensional scaling (NMDS) of the Bray-Curtis similarity index between the compositions (squares) and reference fecal samples (diamonds).

[0347] Figures 7-9 show the similarity of fungal, viral, and bacterial communities, respectively. Similarity is observed by the proximity of the composition to the source sample in the plot.

[0348] These results demonstrate that compositions produced according to embodiments of the present invention preserved high fungal and viral similarity, supporting previous results demonstrating high similarity of bacterial populations. Furthermore, the compositions closely match dozens of other representative stool samples from healthy individuals. Specifically, the compositions preserved 100% of the fungal species, 71.4% of the bacterial virus genera, and 93.5% of the bacterial species identified in the source stool samples (diversity recovery).

[0349] These results demonstrate that the present method advantageously allows for the production of compositions having microbial communities with a high degree of similarity to the source sample, thereby demonstrating their potential for use as pharmaceutical compositions in a manner similar to that used to perform FMT procedures. [Example]

[0350] Maintenance of metabolic pathways in compositions produced according to embodiments of the present invention In the following example, the ability of the method of the present invention to preserve the metabolic pathways of the source fecal sample was evaluated.

[0351] The ability to maintain metabolic pathways / pathways, e.g., the possibility to produce, synthesize, consume and / or utilize specific metabolites / organic compounds, was investigated by publicly available analytical software based on WGS data.

[0352] The results are shown in Figure 10.

[0353] It can be seen that 104 (out of 108) metabolic superpathways are conserved in the composition (104 dots are shown on the graph), with an R-squared value of 0.926. These results indicate that the relative proportions between pathways are substantially conserved.

[0354] This example demonstrates that this method advantageously preserved the metabolic capacity of the source sample, in addition to the bacterial population and other microorganisms that may be present in the source sample.

[0355] Since the method according to the invention can advantageously produce different compositions, for example (i) compositions comprising particle-attached bacteria, (ii) compositions comprising planktonic bacteria cultivated in the presence of particles, and (iii) compositions comprising both fractions, either as a mixture of (i) and (ii) in any ratio or as a combined fraction, Examples 9, 10 and 11 below demonstrate the advantages of various composition embodiments. [Example]

[0356] Different compositions produced according to the present invention In the following examples, the growth preference (at the family taxonomic level) of the 10 most abundant bacteria in the tested composition samples, i.e., preference for planktonic or attached growth forms, was examined. Analysis was performed by measuring the delta between the relative abundance of each bacterium in the particle-attached and non-attached phases. For illustrative purposes, two graphs are shown in Figures 11A-11B.

[0357] These results show that among the top 10 most abundant bacteria, three bacterial families were more abundant in the attached fraction, while four bacterial families were more abundant in the non-attached phase. Three additional bacterial families were found at similar relative abundances in both phases. Thus, we demonstrated the potential role of particles in maintaining bacterial diversity in compositions similar to those of the source sample.

[0358] Thus, the methods of the present invention can advantageously produce isolated fractions enriched for specific bacteria that can be used as stand-alone compositions. Two isolated compositions can also be mixed in any desired ratio to obtain a final composition enriched for a specific bacterium of interest that positively correlates, for example, with the improvement, recovery, or remission of a particular disease or condition.

[0359] Alternatively, by including both attached and non-attached phases, the presence / abundance and relative abundance of most taxonomic groups can be preserved after cultivation regardless of bacterial flora dominance, thereby maintaining the original ecosystem of the source sample. [Example]

[0360] Similarity of different compositions produced according to the present invention The increased microbial diversity observed in healthy individuals is known to restore the protective function of the microbiome against C. difficile, and therefore it is of great importance to produce a microbial composition with a microbial diversity highly similar to that of a fecal sample.

[0361] In the following examples, we analyzed the diversity recovery in different compositions produced according to the present invention (i.e., a composition containing particle-attached bacteria, a composition containing a non-attached fraction, and a combined composition containing both fractions). Bacterial diversity was measured at the genus and species level. We also measured the bacterial load in each fraction by qPCR. Furthermore, we assessed the quantitative similarity of the different fractions.

[0362] 12A-12B show the delta diversity recovery (at the genus and species level, respectively) between the two test compositions.

[0363] These results indicate that the combined and particle-attached fractions recovered increased genus and species diversity from the source sample compared to the non-attached fraction, demonstrating the potential role of particles in maintaining bacterial diversity in a composition similar to that of the source sample. Furthermore, we found that the combined fraction exhibited increased bacterial load compared to each fraction alone (data not shown).

[0364] Therefore, combining both fractions can be advantageous compared to each separated composition, for example, when a composition with increased diversity recovery and increased bacterial load is desired. The combined composition was also found to have increased quantitative similarity compared to each fraction alone. [Example]

[0365] Relative abundance of different compositions produced according to the present invention In another analysis, the proportions (relative abundance) of different taxonomic bacterial groups (at the family level) were compared between different fermentation samples / compositions (particle-attached phase, non-attached phase, and composite composition). The resulting proportions in each composition were compared with the proportions in the respective source samples. Data were obtained from multiple experiments using different source samples.

[0366] The above percentages were also evaluated in samples obtained from planktonic cultures (cultures in the absence of particles) conducted under the same culture conditions as the cultures in the presence of particles. These percentages are shown in Table 2 below.

[0367] The bacterial groups selected for analysis were among the most abundant bacteria present in the test samples. Furthermore, these bacterial groups have been reported in the literature as potential producers of short-chain fatty acids (SCFAs) and bile acid transformants, which correlate with positive treatment outcomes in various diseases or conditions, such as C. diff infection, atopic dermatitis, and ulcerative colitis. [Table 2]

[0368] These results show that in most cases it is necessary / advantageous to combine the two fractions into the final composition in order to better resemble the source sample and to maintain the ratio between the different bacterial families.

[0369] Importantly, in most cases, the non-attached phase (produced according to the method described above) showed less deviation from the tested proportions in the source sample compared to the proportions in planktonic cultures (i.e., cultures without particles). Thus, culturing in the presence of particles surprisingly facilitated the preservation of the tested proportions.

[0370] These results also confirm previous findings and show that it is necessary / advantageous to culture multiple bacteria in the presence of particles to obtain compositions with a high degree of similarity to the source sample. [Example]

[0371] Pooled source samples are cultured in a single vessel In the following examples, the advantages of using pooled source samples from more than one source, for example donors, as starting material in methods according to embodiments of the present invention were investigated.

[0372] To this end, several features were evaluated: (i) various alpha diversity parameters in both pooled origins and produced compositions versus separated samples and their respective produced compositions, and (ii) the ability of cultivation methods according to embodiments of the invention to preserve hard-to-cultivate bacteria when using pooled samples compared to when using single-source origins. Three different origins were collected in this experiment.

[0373] Table 3 below shows the alpha diversity parameters for two different sources and compositions prepared therefrom.

[0374] Surprisingly, it can be seen that pooled source samples containing various microbial populations characterized by different culture requirements resulted in compositions with increased alpha diversity parameters compared to compositions produced from a single source. [Table 3]

[0375] We also investigated the ability of this culture method to produce a composition that preserves difficult-to-culture bacteria.The relative abundance of the bacteria Akkermansia was evaluated as a representative genus.

[0376] According to the literature, Akkermansia is a promising next-generation probiotic and difficult-to-culture bacterium in complex bacterial populations (Derrien, M., Vaughan, EE, Plugge, CM and de Vos, WM, 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).

[0377] As can be seen from Figure 13, source sample 3 contained Akkermansia at the time of culture 1, but the test bacteria were not preserved in the composition at the time of culture 2. When several source samples were pooled, it was confirmed that Akkermansia was preserved at the time of culture 2.

[0378] Furthermore, pooled samples also improved the preservation of other potentially beneficial genera such as Roseburia, Lachnoclostridium-Roseburia, Fusicatenibacter, Coprococcus, and Ruminiclostridium (data not shown).

[0379] Therefore, advantageously, pooled samples can be used as starting material according to one embodiment of the method of the present invention (versus the use of single-source samples) to provide conditions that allow for the preservation of a variety of bacterial genera.

[0380] Advantageously, the method makes it possible to produce compositions with a high degree of similarity to the original sample, which can potentially be used as pharmaceutical compositions as exemplified hereinbelow. The culture system according to the invention advantageously resembles the human gut microbiota and can be used, inter alia, as an in vitro model for assessing the effect of compounds on a subject, as exemplified hereinbelow. [Example]

[0381] Co-cultures containing multiple bacteria derived from saliva samples grown in a single vessel in the presence of particles and under anaerobic conditions. In this example, the ability to grow saliva bacterial populations according to embodiments of the present invention from saliva samples was tested.

[0382] Saliva was grown in small flasks using a medium containing multiple carbon sources, yeast extract, peptone extract, sodium chloride, disodium hydrogen phosphate, trace elements, and particles as described above in the Materials and Methods section.

[0383] The flasks were incubated in an anaerobic chamber and sampled throughout the fermentation process. The resulting CFU / ml was approximately 1 E 8 It was.

[0384] These results showed that the quantitative similarity was higher than 50%, indicating that the method according to the present invention is suitable for culturing multiple bacteria from saliva samples (data not shown).

[0385] Non-limiting examples of utilizing systems / compositions according to one embodiment of the present invention The following experiments illustrate that in vitro systems / models generated in accordance with embodiments of the present invention can be effectively used, for example, as gastrointestinal models to determine the effect of compounds on a subject's microbiome, and that different microbiota samples can be used to establish the models. These experiments also illustrate that the systems can be used to generate different microbial profiles.

[0386] As an example of the effectiveness and reproducibility of the system in assessing the effects of compounds on multiple bacteria, different compounds or known drugs were examined.

[0387] Unless otherwise indicated, compounds were added to the in vitro model system during inoculation. The effects of compounds on multiple bacteria were examined in particle-attached and non-attached bacterial phases. Compounds A, A', and D were added at increasing concentrations at therapeutic concentrations.

[0388] In all the following experiments, cultivation was carried out in a multi-carbon source medium (glucose, maltose, trehalose, starch) further containing a combination of trace elements (manganese source, copper source, iron source), yeast extract, peptone extract, sodium chloride, and disodium hydrogen phosphate. [Example]

[0389] Microbiome profiles of different samples In the following examples, the ability to generate different systems / models containing similarities with different source samples having different microbiota population profiles was investigated.

[0390] Furthermore, this example demonstrates the system's ability to maintain similarity to the initial sample even under small-scale conditions. It also examines the system's sensitivity to identifying changes in the microbiome profile of interest by culturing samples from each donor on three different days (i.e., three independent experiments conducted by obtaining three samples from donor 1).

[0391] For the above purposes, microbiota populations containing multiple bacteria were obtained from four different donors to generate four lines (Figure 17). Donor #1 provided the source sample at three different independent time points. That is, several lines were generated simultaneously: four lines from four different donors, and two additional lines generated from Donor #1 in Experiments 1 and 2 (a total of six in vitro culture lines). Several samples were taken from the particle-associated bacterial phase.

[0392] The β-diversity (Bray-Curtis) of different samples obtained from six different culture systems was examined and is shown in a non-metric multidimensional scaling (NMDS) plot.

[0393] These results (Figure 17) show that each of the four donors was located at a random distance from the other donors on the plot, and that different samples taken from the same donor at different times throughout the culture period were located in the same area of ​​the plot. These results demonstrate that different systems can be generated according to the donor's unique initial microbial pattern, and that these systems can maintain the unique initial microbial pattern over time.

[0394] When examining the beta diversity of microbiome populations in a system generated from three different origin samples from the same donor on three consecutive days, all samples were located in the same region of the plot and varied slightly from each other, demonstrating the system's ability to identify subtle / subtle changes in the microbiome profile. [Example]

[0395] Effect of Compound A on bacterial load in different culture systems produced from different samples derived from different donors The above examples show that different lines generated from different source samples and different donors yield different microbiota population profiles.

[0396] To test the system's ability to accurately determine the effects of compounds on multiple bacteria, regardless of their different microbiome profiles, Compound A was contacted with four fecal samples from four donors. After contact between the fecal samples and the particles, the compound was added to the different culture systems and allowed to incubate with the bacterial populations for the duration of the culture period.

[0397] The effect of the compounds on bacterial numbers (based on qPCR measurements) was investigated in the different production lines at three time points (labeled TP1, TP2 and TP3) and is shown in FIG.

[0398] FIG. 18 shows that addition of Compound A resulted in increased bacterial counts at all three test time points in all four generated systems compared to the untreated control system (all presented on the same plot), demonstrating the system's ability to identify common effects of compounds on microbial populations regardless of the system's initial bacterial profile. [Example]

[0399] Effect of Compound A on microbial profiles of different culture systems produced from different samples To investigate the effects of compounds on microbiome profiles and better characterize the system's ability to identify changes in the microbiome community, Compound A was added to four different systems (generated from four fecal samples obtained from four different donors) to examine alpha and beta diversity. The compounds were added during system culture as described in the previous examples, and samples were taken at three different time points (TP1, TP2, and TP3).

[0400] The α-diversity in each system is shown in Figures 19A to 19D. Each figure shows the α-diversity of samples taken before culture and at three time points during the culture period for the untreated control system and the system treated with Compound A. At each time point, the average values ​​of TP1, TP2, and TP3 were calculated and used to evaluate the effect of the compound.

[0401] These results indicate that the addition of Compound_A to each of the systems significantly increased α-diversity compared to the untreated control bacterial population, regardless of the source sample.

[0402] The β-diversity of each line is shown in Figures 20A-20D. Each figure shows the diversity of the untreated control line and the line treated with Compound A at three time points during the culture period (all time points are shown on the same plot).

[0403] These results indicate that exposure of the bacterial population to the test compound modulated / altered the bacterial population when compared to the bacterial population of the untreated control.

[0404] These results demonstrate that the system exemplified herein according to one embodiment of the present invention is capable of identifying an increase in the number of species and any changes in similarity levels as a result of exposure to a compound. [Example]

[0405] Effect of Compound A on the Regulation of Certain Bacterial Genera The foregoing examples demonstrate that culture systems according to embodiments of the present invention are an efficient means by which common effects or trends of compounds on bacterial load and / or microbiome profile can be identified, regardless of the initial microbial profile of the source sample / donor.

[0406] The goal of the following examples is to use data extracted from the system (e.g., bacterial load, alpha diversity, and / or beta diversity) and further investigate and identify bacteria that are modulated after exposing the bacterial population to a test compound. As illustrated above, bacterial modulation can be confirmed by a change in the position and / or distribution of dots for treated systems relative to untreated systems on an NMDS plot.

[0407] A heat map showing the different bacterial genera affected by exposure of bacterial populations to Compound A is shown in Figure 21. Each experiment was performed simultaneously with four treatment series (samples 1-4). Analysis was performed in comparison with an untreated control bacterial population.

[0408] In these experiments, genus-level changes were considered significant if the same trend (increase or decrease) was observed in three of the four test samples (i.e., 75% of the samples showed the same trend). These results show that nine genera increased and three genera decreased. Similar trends were observed when comparing biomarkers identified by the culture system with those identified in clinical trials conducted using the same compounds.

[0409] Advantageously, studying the effect of a compound on the microbiome population in a culture system according to one embodiment of the present invention, simulating the subject's flora, can provide insight into the modulation of bacterial populations in light of exposure to such compounds. Identifying modulated bacterial genera (or modulation at any other taxonomic level) can be therapeutically targeted to increase the efficacy of a drug, for example, by administering a drug-probiotic or drug-prebiotic combination treatment to a subject with the aim of restoring / preserving an original or healthier microbiome profile. [Example]

[0410] Use of a system according to one embodiment of the present invention as a high-throughput screening assay and as a platform for generating different microbial profiles In the following example, the effects of two compounds, Compound_A and its derivative (Compound_A'), on relative abundance were compared.

[0411] The two compounds were added simultaneously under the same conditions to two different lines, and samples were taken at five time points throughout the culture period to measure relative abundance. Results were compared to an untreated control line sampled at the same time points (three lines generated from the same original sample / donor).

[0412] These results (Figure 22) show that the relative abundance of genera at all five time points was altered after treatment with either Compound A or Compound A' compared to the untreated system. It can also be seen that the two treatment groups exhibited different microbial profiles.

[0413] These results highlight the sensitivity and power of the system and method to be used simultaneously in several single vessels as a high-throughput screening assay to compare the effects of different treatments, such as different drug derivatives, on a subject's microbiome population. The system also demonstrates that it can be used to generate distinct microbial profiles. [Example]

[0414] Sensitivity of the culture system to different compounds In a separate experiment, the ability and sensitivity of the system to identify changes in microbiome profile and bacterial counts in response to different chemical compounds, Compound_D and Compound_J, was evaluated.

[0415] To this end, each compound was added to separate systems, both generated from the same source sample, and similarity parameters and bacterial counts were assessed. Samples were taken at different stages and at different time points, as detailed below.

[0416] Table 4 shows the effect of compounds on alpha diversity and bacterial numbers. [Table 4]

[0417] Exposure of bacteria to Compound D resulted in a significant decrease in bacterial numbers, followed by a gradual increase to levels close to those of the untreated controls. In comparison, exposure of bacteria to Compound J resulted in a 7,450-fold increase in bacterial numbers, followed by a subsequent decrease.

[0418] Alpha diversity decreased in response to exposure to compound D, whereas exposure to compound J slightly increased richness.

[0419] Notably, in both parameters tested, the effects observed after exposure to Compound_D are similar to those known in the art.

[0420] Thus, these results confirm previous findings and clearly demonstrate the effectiveness and sensitivity of the system.

[0421] Next, we evaluated the ability to use particle-attached and non-attached fractions to assess the effects of compounds. To this end, the relative abundance of nine specific genera was measured in both fractions after contacting bacteria with Compound D. These results are shown in Figure 23.

[0422] In general, these results indicate that the microbial profiles of each fraction were differentially affected after exposure to the compounds. For example, the relative abundance of genus 1 in response to compound D in TP3 was 36.25% and 87.70% in particle-attached and non-attached bacteria, respectively (Figure 23, top panel).

[0423] These results demonstrate the importance of evaluating the effects of added compounds on the microbial populations of both the combined and / or separated fractions. Furthermore, these results support our previous findings, showing the tendency of genera to adhere to particles and / or prefer planktonic growth. In particular, the tendency of certain bacteria to adhere to particles may suggest their ability to colonize at the target site.

[0424] In parallel with the susceptibility experiments, we evaluated the ability of the "stabilized" or "initial" systems to exhibit changes in microbial profile and bacterial counts by adding Compound D, Compound F, and Compound J at two different time points: "initial"—where the compounds were exposed to multiple bacteria during the inoculation phase before particles were added to the system; and "stabilized"—where multiple bacteria were exposed to particles and the compounds were added after the addition of growth medium. These conditions may represent different physiological conditions of interest, such as a more stable, healthy microbiome versus a dysbiotic microbiome. The alpha diversity and bacterial count results are shown in Table 5. [Table 5]

[0425] These results indicate that exposure of bacteria to compounds D and F in the "initial" stage significantly reduced the α-diversity similarity level compared to exposure of bacteria to the "stabilized system," but exposure of bacteria to compound D had a similar effect in both stages.

[0426] The α- and β-diversity parameters after exposure to several additional compounds in either the “initial” or “stabilized” conditions confirmed the results, showing that the “stabilized system” was less susceptible to changes in the bacterial population compared to the “initial system.”

[0427] These results demonstrate the advantage of assessing the effect of compounds during different steps in the method. [Example]

[0428] Use of a system according to an embodiment of the present invention to produce a composition enriched in bacteria attached to particles by the addition of a compound The following examples explore the use of the system to enhance specific bacterial growth forms, namely particle-attached or planktonic growth forms.

[0429] To this end, Compound B was added to the culture system prepared as described above and allowed to incubate with the bacterial population for the duration of the culture period (Container 2). After contacting the bacteria with the particles, the compound was added. The effect of the compound on the growth preference of four selected bacteria was investigated by measuring the relative abundance of the entire bacterial population in both the sessile and planktonic phases. These results were compared with those of the untreated control system (Container 1).

[0430] Figure 24 shows that the relative abundance of Bacterium 1 increased upon exposure to Compound B, but no difference in preferred growth form (tendency toward planktonic growth) was observed. For Bacteria 2 and 4, addition of Compound B resulted in a change in growth form. Bacterium 2 showed a preferential conversion from an attached to a planktonic form, while Bacterium 4 showed the opposite trend. For Bacterium 3, addition of Compound B favored an attached growth form, whereas in the untreated system, substantially similar relative abundances were observed in both fractions.

[0431] These results suggest that the system according to the present invention can be used to identify compounds capable of enhancing specific bacterial growth forms of specific bacteria within a population of bacteria, as well as to produce compositions enriched for specific bacteria in attached and / or non-attached forms.

[0432] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. 1. A method for producing a composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles, said co-culture having i) at least 30% similarity to the source sample, and ii) at least 1 E per gr of particles. ４ wherein the method comprises: providing a microorganism comprising a plurality of bacteria from a source sample; contacting the plurality of bacteria with particles to at least partially adhere the plurality of bacteria to the particles; culturing the plurality of bacteria at least partially attached to the particles in a growth medium for a period of less than 14 days; the culturing includes culturing under anaerobic conditions; This ensures that the sample has i) at least 30% similarity to the source sample, and ii) at least 1 E per gr of particles. ４ and producing said composition comprising a co-culture comprising a plurality of bacteria grown in the presence of particles comprising a bacterial load of said composition.

2. 2. The method of claim 1, wherein the plurality of bacteria are characterized by having different growth, cultivation and / or proliferation conditions selected from the group consisting of metabolic requirements, nutritional requirements, pH, temperature, aerobic, obligately anaerobic, facultatively anaerobic, microaerophilic, attached form, planktonic, growth medium, fluid, shaking, stirring, agitation, static, moist, low humidity, and any combination thereof, and optionally, the culturing is carried out until the co-culture reaches a similarity of 30% or more to the original sample and a bacterial load of at least 1 E4 per gr of particles.

3. 2. The method of claim 1, wherein the incubation period ranges from 6 hours to 14 days.

4. 2. The method of claim 1, wherein the co-culture comprises at least 30% similarity to the source sample, where the similarity is determined by a metric that takes into account the genetic relatedness of the bacteria using any one of next-generation sequencing (NGS) technology, whole genome sequencing (WGS), or both.

5. The method of claim 1 , wherein the similarity comprises a quantitative Unifrac similarity of at least 50%.

6. The similarity comprises a quantitative Unifrac similarity of at least 70%, and the co-culture has at least 1 E per gr of particles. ８ The method of claim 1, wherein the bacterial load is

7. 10. The method of claim 1, wherein the similarity between the co-culture and the source sample comprises a similarity between bacterial populations.

8. 2. The method of claim 1, wherein the source sample further comprises additional microorganisms, including any one of archaea, viruses, fungi, or any combination thereof; and optionally, the co-culture further comprises the additional microorganisms; and optionally, the similarity between the co-culture and the source sample further comprises similarity of at least one of archaea, viruses, fungi populations, or any combination thereof.

9. 2. The method of claim 1, wherein the provided plurality of bacteria belongs to at least five bacterial species and / or at least two bacterial genera.

10. The method of claim 1 , wherein the composition comprises bacteria in a planktonic form and bacteria at least partially attached to the particles.

11. 10. The method of claim 1, wherein the growth medium comprises at least two carbon sources.

12. 10. The method of claim 1, wherein the growth medium comprises a carbon source from at least two chemical groups selected from the group consisting of monosaccharides, disaccharides, polysaccharides, and any combination thereof.

13. 10. The method of claim 1, wherein the growth medium comprises at least one monosaccharide, at least one disaccharide, and at least one polysaccharide.

14. 10. The method of claim 1, wherein either the contacting step, the culturing step, or both, is performed in a single vessel.

15. 2. The method of claim 1, 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 the group consisting of 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.

16. The plurality of bacteria is provided in a container, and the method comprises the steps of adding at least one compound to the container and determining (i) bacterial diversity, (ii) relative abundance of bacteria, (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 characteristic selected from any combination of (i)-(v).

10. The method of claim 1, further comprising the step of evaluating the effect of the at least one compound on the plurality of bacteria, the effect of the plurality of bacteria on the at least one compound, or both.

17. 2. The method of claim 1, wherein the source sample is selected from the group consisting of a microbiome sample, a skin sample, an oral sample, a fecal sample, a vaginal sample, a gut microbiota sample, a microbiome ...

18. The compound provides (i) a similarity level of at least 30% at the end of the incubation period, and (ii) at least 1 E per gr of particles at the end of the incubation period. ４ 17. The method of claim 16, wherein (i) or (ii), or both (i) and (ii), are modified, and optionally, modifying comprises increasing or decreasing any one of the similarity level, the bacterial load, or both.

19. The method of claim 16, wherein (i) the method is carried out in several single containers simultaneously, and the bacterial diversity, the relative abundance of the bacteria, the bacterial load, and / or the other effect of the at least one compound on the plurality of bacteria are compared with corresponding characteristics in the plurality of bacteria of the source sample, and any alteration in the characteristics indicates that the at least one compound has an effect on the plurality of bacteria; (ii) the method further comprises culturing in a separate container a control plurality of bacteria not exposed to the compound, and the bacterial diversity, the relative abundance of the bacteria, the bacterial load, and / or the other effect of the at least one compound on the plurality of bacteria are compared with corresponding characteristics in the control plurality of bacteria, and any alteration in the characteristics indicates that the compound has an effect on the plurality of bacteria; (iii) the adding and / or determining is carried out using bacteria attached to the particles, bacteria not attached to the particles, or both; and (iv) any one of any combinations of (i) to (iii).

20. 10. A composition produced according to the method of claim 1, optionally further comprising an acceptable carrier or excipient.

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