WO2025154074A1 - Fibre-degrading probiotics and uses thereof - Google Patents

Abstract

Fibrolytic bacterial strains are disclosed which carry in their genomes the genes scciC and cttA, Probiotic compositions and cultures comprising same and uses thereof are also disclosed.

Description

FIBRE-DEGRADING PROBIOTICS AND USES THEREOF

RELATED APPLICATION/S

This application claims the benefit of priority of U.S. Patent Application No. 63/621,148 filed January 16, 2024, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING STATEMENT

The XML file, entitled 102465.xml, created on January 16, 2025, comprising 19,223,194 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to probiotic compositions which comprise fibrolytic strains of bacteria. The compositions may be used for a myriad of uses including as weight reducing agents and therapeutics for the treatment of diseases.

Dietary fiber is beneficial to gut microbiome stability and richness, which has strong repercussions on human health. Fermentation of dietary fiber in the human gut was shown to regulate digestive transit, prevent obesity and diabetes, and reduce cardiovascular diseases and cancer. Cellulose is a major part of the chemical composition of fiber and is thus a common component of diets that include plant-based ingredients. The benefits of cellulose on host health have been demonstrated in animals and include the prevention of colon cancer and the reduction of blood sugar levels. The prevalence of cellulose in processed food is very low, however, and in many developed countries there is now a growing preference to decrease the amount of processed food ingredients in favor of a diet with increased fiber levels.

The utilization of dietary fiber, notably cellulose, in the mammalian gut depends exclusively on the enzymatic capabilities of its resident microorganisms. It was long believed that cellulosic material, particularly its crystalline regions, was not digested in the human gut, in contrast to ruminants and other herbivores. Evidence for the degradation of microcrystalline cellulose by human-gut bacteria was first reported in 2003, and the microcrystalline cellulose degrader Ruminococcus champanellensis was isolated a decade later. Subsequently, the presence of cellulosomes - extremely efficient multi-enzymatic complexes produced in bacteria for degradation of plant-fiber polysaccharides - was detected in this bacterium, and biochemical characterization of its interactive cellulosomal proteins and enzymes confirmed its full functionality, expanding the paradigm of cellulosome-dependent fiber breakdown to human- associated microbiota. Despite this discovery cellulose degradation and fermentation in the human gut is scarce and in most humans, it is absent. Nevertheless, the presence of cellulosomes, elaborate and efficient molecular machines across gut ecosystems suggests that they play a unique role in promoting energy release from crystalline cellulosic components of dietary fiber, highlighting the role of their bacterial producers as key specialists in gut ecosystems.

Background art includes Chassard, C., et al. (2012). Int. J. Syst. Evol. Microbiol. 62, 138— 143 and Morais et al., Science, Volume 383, Issue 6688, 15 March 2024, Article number eadj9223.

SUMMARY OF THE INVENTION

According to an aspect of the present invention, there is provided a probiotic composition comprising at least one isolated fibrolytic bacterial strain having a 16S rRNA sequence at least 92 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, wherein the strain carries in its genome the genes scaC and ctA, wherein the composition comprises no more than 150 species of bacteria.

According to an aspect of the present invention, there is provided probiotic composition comprising at least one fibrolytic bacterial strain which comprises a genome having contigs at least 90 % identical to SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937, wherein the composition comprises no more than 150 species of bacteria.

According to an embodiment, the bacterial strain expresses CttA protein which binds to cellulose.

According to an embodiment, the bacteria strain comprises an assembled cellulosome.

According to an embodiment, the bacterial strain expresses the endoglucanase Cel5A which is capable of degrading cellulose.

According to an embodiment, the bacterial strain expresses the glycoside hydrolase GH98 capable of degrading corn arabinoxylan.

According to an embodiment, the isolated fibrolytic bacterial strain has a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5.

According to an embodiment, the isolated fibrolytic bacterial strain has a 16S rRNA sequence at least 99 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5.

According to an embodiment, the composition comprises no more than 100 species of bacteria.

According to an embodiment, the composition comprises no more than 50 species of bacteria.

According to an embodiment, the at least one isolated fibrolytic bacterial strain is capable of residing in the human gut microbiome. According to an aspect of the present invention, there is provided probiotic composition comprising at least one fibrolytic bacterial strain isolated from a human gut, wherein the composition comprises no more than 50 species of bacteria.

According to an embodiment, the at least one bacterial strain has a 16S rRNA sequence at least 92 % identical to SEQ ID NOs: 1, 2, 3, 4, or 5.

According to an embodiment, the fibrolytic bacterial strain comprises a genome having contigs at least 90 % identical to SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391- 461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688- 824; or SEQ ID NOs: 825-937.

According to an embodiment, the composition comprises at least two isolated non-identical bacterial strains, wherein a first strain has a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, and a second strain has a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, wherein both of the bacterial strains carry in their genomes the genes scaC and ctA.

According to an embodiment, the composition comprises no more than 20 species of bacteria.

According to an embodiment, the probiotic composition is lyophilized.

According to an embodiment, the probiotic composition is formulated as a capsule, a tablet, dry powder, a suppository, a food or beverage.

According to an embodiment, the probiotic composition further comprises a preservative that preserves the activity of the bacteria.

According to an embodiment, the probiotic composition further comprises a prebiotic.

According to an embodiment, the prebiotic composition comprises cellulose.

According to an embodiment, the cellulose is crystalline cellulose and/or hemicellulose.

According to an aspect of the present invention, there is provided a bacterial culture comprising at least one bacterial strain having a 16S rRNA sequence at least 92 % identical to SEQ ID NOs: 1, 2, 3, 4, or 5, or comprising a genome having contigs at least 90 % identical to SEQ ID NOs: SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937, wherein the strain expresses the genes scaC and ctA, wherein the culture comprises no more than 150 species of bacteria.

According to an embodiment, the bacterial culture comprises no more than 50 species of bacteria.

According to an embodiment, the bacterial culture is a monoculture. According to an embodiment, the culture is devoid of bacteria isolated from a rumen microbiome.

According to an embodiment, the bacteria of the culture are derived from a human microbiome sample.

According to an aspect of the present invention, there is provided method of maintaining the health of a subject comprising administering to the subject an effective amount of a composition comprising:

(a) at least one isolated fibrolytic bacterial strain:

(i) having a 16S rRNA sequence at least 90 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, or

(ii) comprising a genome having contigs at least 90 % identical to SEQ ID NOs: SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462- 550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937, wherein the bacterial strain expresses the genes scaC and cttA; or

(b) the probiotic composition of any one of claims 1-19, thereby maintaining the health of the subject.

According to an embodiment, the method further comprises administering to the subject a prebiotic composition which enhances the activity or amount of the bacterial strain.

According to an embodiment, the prebiotic composition comprises cellulose /or hemicellulose.

According to an embodiment, the cellulose /or hemicellulose is crystalline cellulose and/or corn arabinoxylan.

According to an embodiment, the subject is a healthy subject.

According to an embodiment, the subject is a non-healthy subject.

According to an aspect of the present invention, there is provided method of treating a disease selected from the group consisting of an inflammatory disease, a metabolic disease, cancer and a cardiovascular disease, the method comprising administering to the subject a therapeutically effective amount of:

(i) at least one isolated fibrolytic bacterial strain having a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4, or 5;

(ii) at least one isolated fibrolytic bacterial strain comprising a genome having contigs at least 90 % identical to SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937, wherein the strain expresses the genes scaC and cttA; or

(iii) the probiotic composition described herein, thereby treating the disease.

According to an embodiment, the metabolic disease is Diabetes or obesity.

According to an embodiment, the cancer is colon cancer.

According to an embodiment, the inflammatory disease is inflammatory bowel disease and/or colitis.

According to an aspect of the present invention, there is provided method of reducing weight of a subject comprising administering to the subject an effective amount of:

(i) at least one isolated fibrolytic bacterial strain having a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4, or 5, wherein the strain expresses the genes scaC and cttA;

(ii) at least one isolated fibrolytic bacterial strain comprising a genome having contigs at least 90 % identical to SEQ ID NOs: SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937, wherein the strain expresses the genes scaC and ctA; or

(iii) the probiotic composition of any one of claims 1-19, thereby reducing weight of the subject.

According to an embodiment, the subject has a BMI greater than 25.

According to an embodiment, the subject is on a weight loss program.

According to an embodiment, the weight loss program comprises an exercise program.

According to an embodiment, the weight loss program comprises a reduced caloric intake.

According to an aspect of the present invention, there is provided a method of identifying probiotic bacteria useful for maintain the health of a human subject comprising analyzing for the presence of a bacteria in a sample of the gut microbiome of a human subject, wherein a bacteria which express both the genes scaC and cttA is indicative that the bacteria is a probiotic useful for maintaining the health of the human subject.

According to an embodiment, the method further comprises isolating the bacteria following the identifying.

According to an embodiment, the sample is a fecal sample.

According to an embodiment, the analyzing comprises sequencing DNA or RNA derived from the sample.

According to an embodiment, the e analyzing comprises determining 16S rRNA sequence of the bacteria. According to an embodiment, the method further comprises storing the sample following the identifying.

According to an embodiment, the method further comprises qualifying the gut microbiome of the subject upon identification of the bacteria which expresses both the genes scaC and cttA.

According to an aspect of the present invention, there is provided an article of manufacture comprising:

(a) at least one isolated fibrolytic bacterial strain having a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4, or 5, wherein the strain expresses the genes scaC and ctA; and

(b) a weight-reducing agent.

According to an embodiment, the at least one isolated fibrolytic bacterial strain and the weight-reducing agent are formulated in a single composition.

According to an embodiment, the at least one isolated fibrolytic bacterial strain and the weight-reducing agent are formulated in separate compositions.

According to an aspect of the present invention, there is provided an isolated fibrolytic bacterial strain having a 16S rRNA sequence at least 92 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, wherein the strain carry in their genomes the genes scaC and ctA.

According to an aspect of the present invention, there is provided an isolated fibrolytic bacterial strain which comprises a genome having contigs at least 90 % identical to SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937.

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 the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGs. 1A-C. Detection of a novel human-gut fiber-degrading ruminococcal species. A. Scheme of cellulosome architecture. The CttA protein, by virtue of its CBMs, mediates the binding of the bacterial cell to the cellulosic substrate, which can be hydrolyzed by dockerin-bearing enzymatic units that are integrated into the cell-surface cellulosome via its cohesin-containing scaffoldin assemblies. B. Phylogenetic tree of 62 selected genomes and MAGs, using the sequence of the ScaC scaffoldin illustrated in Figure 1A as a phylotyping marker ^15,16. The color of the clade indicates the origin of the genomic bin (light blue for human and light green for rumen). Gray circles on the branches represent bootstrap values higher than 90%. The number and composition of cellulosomal elements is indicated as a bar for each genomic bin (the number of dockerincontaining proteins with additional CAZyme elements in dark gray, the number of dockerincontaining proteins with no additional CAZyme elements in medium gray and the number of scaffoldins containing at least one cohesin module in light gray). Brown circles next to the MAG name genomes containing a ctA gene. C. Genomic dissimilarity computed by Mash distance within the novel identified ruminicoccal cellulosomal species and pairwise comparisons to each other as well as to the ruminal R. flavefaciens species and the human species, R. champanellensis

FIGs. 2A-E. The novel strains are abundant in human hunter-gatherer and rural populations, rare in industrialized societies and potentially originate either from the primate or ruminant gut. A. Observed collective prevalence of the MAGs for the novel fiber-degrading strains in various human cohorts. Pie charts represent the observed prevalence for the respective cohorts. B. Worldwide locations of positive human and monkey samples. The locations of the samples in which the human MAGs were detected are denoted on the map as circles: dark blue (industrialized societies), light blue (rural societies and hunter-gatherers) and pink (monkeys). C. Distribution of fibrolytic strains in human and monkey populations. Left panel, stacked bar chart of the distribution of the human cellulosomal strains (R. hominiciens, R. primaciens, R. champanellensis and R. ruminiciens) within each cohort (industrialized societies in dark blue, rural societies in blue, hunter-gatherers in light blue and monkeys in pink). Right panel, heatmap of the distribution of the human cellulosomal strains among the human- and monkey-positive samples. D. A core protein phylogenetic tree illustrating the co-speciation hypothesis (left panel). MAGs are color-coded according to their host origin: green, blue or pink, indicating rumen, human or monkey, respectively. Gray circles on the branches represent bootstrap values higher than 90%. The comparison to the phylogenetic tree of the mammalian host species is given on the right, with red lines indicating proteins that do not follow the host phylogeny. E. A core protein phylogenetic tree illustrating the human-ruminant clade. As in D MAGs are color-coded according to their host origin: green, blue or pink, indicating rumen, human or monkey, respectively. Gray circles on the branches represent bootstrap values higher than 90%. Blue highlight on the right indicates a phylogenetic connection between the human and ruminant clades.

FIGs. 3A-D. Intact cellulosome assembly, cellulolytic activity and cellulose adhesion by the novel fiber-degrading strains. A. Summary of interactions between selected cellulosomal cohesin and dockerin modules, derived from an R. primaciens strain (Human_SRR5558136_bin.38), as compared to those of orthologous modules from the R. flavefaciens FD-1 rumen strain ³⁵. Cohesin and dockerin modules are color-coded (red, yellow, green) according to their predicted specificities of interaction. Intensities of the interactions are denoted with -, +, ++ and +++ for no affinity (OD450 lower than 0.15), moderate affinity (OD450 between 0.15 and 0.5), high affinity (OD450 between 0.5 and 1.0) and very high affinity (OD450 between 1.0 and 2.2) (intensities not available in Israeli-Ruimy 2017 study). Eight blue highlight indicates negative interactions, darker blues positive interactions and gray not tested. B. Overview of cellulosomal interactions in R. primaciens compared to those of R. flavefaciens, as deduced from affinity-based ELISA experiments and proposed recognition residues of the dockerin components. C. Comparative cellulolytic activity of ruminococcal GH5 orthologs of either human (R. primaciens) or rumen origin (R. flavefaciens FD-1). Enzyme samples were examined using microcrystalline cellulose (Avicel) as the substrate at 37°C. D. Cellulose binding assay. SDS- PAGE gels loaded with cellulose-bound (B) and -unbound (U) fractions of either R. hominiciens CttA, the CBM3a from the CipA scaffoldin of the Clostridium thermocellum cellulosome as a positive control or green fluorescent protein (GFP) as a negative control (non-binding protein).

FIGs. 4A-E. Functional adaptation of MAGs with their host. In A, C, D and E, MAGs and samples are color-coded according to host origin: green, blue or pink indicating rumen, human or monkey, respectively. A. Principal component analysis (PCA) of the overall predicted ORFs of the MAGs, color-coded by their hosts (see below). Clustering analysis of MAGs gene content according to their hosts was done using PERMANOVA test with 1000 randomizations of the data was performed, and the p-value is indicated. B. Rank distribution of verticality values for core proteins across the 3 types of hosts versus host-specific proteins for a given type of host indicates that specific genes are likely to be transferred via horizontal gene transfer within a given type of host. C. PCA of the fibrolytic system (indicated GH families) of the MAGs, color-coded by their hosts. Clustering analysis of MAGs GH families content according to their hosts was done using PERMANOVA test with 1000 randomizations of the data was performed, and the p-value is indicated. D. PCA of the expression of the fibrolytic system, as examined by transcriptomics analysis of three fecal samples of the three hosts (macaque, human and sheep rumen). E. Left: heatmap of the statistically significant GH families that statistically distinguish the strains associated with the three gut ecosystems as determined by Kruskal-Wallis test p <0.05 after FDR correction. The left bar graph represents the verticality values for each of these orthologous groups of genes. Right: heatmap of the statistically significant GH expression (metatranscripts in FPKM) between the three types of hosts (see Material and Methods Section). Transcriptomic values for GH141-Doc and GH97-Doc, correspond to Rumen_CADBJG01 and Rumen_CACVQO01 MAG sequences³⁷, as these genes are not present in the Rumen_CACVSX01 MAG used for analysis.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to probiotic compositions which comprise fibrolytic strains of bacteria. The compositions may be used for a myriad of uses including as weight reducing agents and therapeutics for the treatment of diseases.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Carbohydrates one of the most important components in human nutrition, include sugars and other molecules used by our bodies to generate energy and support cellular functions. Monosaccharides, such as glucose, are short molecules readily utilized by the human body. In contrast, polysaccharides, such as cellulose or starch, are composed of long chains of monosaccharides, needing breakdown into basic subunits. Amongst these are plant polysaccharides that do not break down in the gastrointestinal tract, known as dietary fibers. Simple carbohydrates are broken down in our digestive system by specific proteins - digestive enzymes. In contrast, more complex carbohydrates like cellulose require the assistance of bacteria residing in our large intestines. The difference in the rate of digestion of different carbohydrates in our body has a significant effect on blood sugar levels and various other processes. Thus, the ecosystem in our large intestines heavily depends on the polysaccharides we consume. Beneficial bacteria need these polysaccharides to thrive. Their regular supply is essential for maintaining a balanced microbiome, which helps prevent harmful bacteria from multiplying and damaging our health. Cellulose-degrading bacteria, such as those found in the intestines of cows and other ruminants, can disassemble the fibers in plant material containing complex sugars and transform them into an available energy source.

The present inventors set to identify novel cellulosome-producing, cellulose-degrading bacteria in the human gut microbiota They analyzed nearly 100,000 genomes assembled from metagenomic data, designed to deepen the understanding of cellulosome-producing bacteria in human gut microbiota. 254 genomes with cellulosomal elements were retrieved using the scaffoldin gene scaC as a phylogenetic marker, and 61 genomes with >90% completion were retained. These were further analyzed for carbohydrate-active enzymes and cellulosomal elements. From the present work, the combination of the scaC and ctA genes emerged as a robust and efficient tool for discriminating close relatives of R. flavefaciens among the cellulolytic and cellulosome-producing bacteria in the human gut microbiome.

The phylogenetic -based strategy used in the present analysis revealed the presence of novel bacteria, referred to herein as R. hominiciens, R. primaciens and R. ruminiciens, in the human- and non-human primate-gut ecosystems, but not in ruminants. Biochemical analysis showed that these strains express intact, fiber-degrading cellulosomal complexes, similar to those of the ruminal R. flavefaciens strains (Figures 3A-D).

Biochemical analysis of multiple cellulosomal components confirmed the presence and proper assembly of the cellulosome in the R. primaciens genome found in the human and monkey gut. Metatranscriptomic analysis of R. hominiciens, R. primaciens, and R. flavefaciens in the gut of humans, monkeys, and ruminants showed high levels of expression of cellulosomal, plant fiber degrading genes and overall gene content that was specific to their respective ecosystems. This suggests that these microorganisms are highly active and play important roles in the functioning of their host ecosystems.

The inventors propose that these bacteria's ability to degrade plant fiber biomass, composed of microcrystalline cellulose and other plant fiber polymers such as hemicellulose, in the human gut enhances plant fiber consumption. This increases the potential for documented health benefits, including improved gut microbiota diversity, enhanced digestion, better regulation of blood sugar levels, reduced cholesterol levels, decreased risk of colonic cancer, reduced gut inflammation, and potential weight management. Furthermore, such microbes could provide a protective effect on the soft tissue of the lower digestive tract from abrasive cellulose fibers as well as other previously thought undigestible plant fibers. These findings also suggest that fiber breakdown and fermentation may impact human health through the production of important short-chain fatty acids (SCFAs). The present examples reveal the ecosystem specificity of the R. hominiciens, R. flavefaciens, R. ruminiciens and R. primaciens genomes to the human, rumen, and monkey ecosystems, at both the whole genome level and within their fibrolytic systems. In particular, the present inventors reveal the specificity and functionality of the GH98 gene, which is coded exclusively by R. hominiciens and R. ruminiciens strains and is present in the human gut ecosystem.

Thus, according to an aspect of the present invention, there is provided a probiotic composition comprising at least one isolated fibrolytic bacterial strain having a 16S rRNA sequence at least 92 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, wherein said strain carries in its genome the genes scaC and ctA, wherein the composition comprises no more than 150 species of bacteria.

The term “probiotic” as used herein refers to a microbial cell preparation or components of microbial cells with a beneficial effect (e.g. weight reducing) on the health or well-being of the host.

The term “fibrolytic bacteria” refers to a bacteria which is able to process complex plant polysaccharides due to its capacity to synthesize cellulolytic and hemicellulolytic enzymes. Fibrolytic enzymes, of which cellulases and hemicellulases (also known as xylanases) are the two main representatives can hydrolyze the P (1 ->4) bonds in plant polysaccharides.

In microbiology, "16S rRNA sequence" refers to the sequence derived by characterizing the nucleotides that comprise the 16S ribosomal RNA gene(s). The bacterial 16S rRNA is approximately 1500 nucleotides in length. In order to reconstruct the evolutionary relationships and sequence identity of one bacterial isolate to another, phylogenetic approaches are used standardly exploiting the 16S rRNA sequence or a portion of the 16S rRNA sequence of the bacteria, although any other sequence or the entire genome of the microorganisms to be analyzed can also be used.

Contemplated bacterial strains include those having a 16S rRNA sequence at least 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.5 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, wherein the strain carries in their genomes the genes scaC and ctA.

Other contemplated bacterial strains include those which comprises a genome having contigs at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.1 %, 99.2 %, 99.3 %, 99.4 %, 99.5 %, 99.6 %, 99.7 %, 99.8 % or 99.9 % identical to SEQ ID NOs: SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937. In one embodiment, the contig identity is measured using Mash distance analysis. As used herein, the term “contig” is an abbreviation for the term “contiguous sequence of DNA” and refers to DNA segments derived from a single source of genetic material. A set of contigs collectively represent the genetic information of an organism.

It will be understood that each set of contigs (e.g. SEQ ID NOs: SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937) are derived from a genome of a single bacterial strain.

Also contemplated are progeny of the bacteria that have a genomic nucleic acid with the above disclosed contig sequences, wherein the progeny has the same fibrolytic activity as that of the parent bacteria. Such progeny may have genomes having a sequence at least 99 % identical, at least 99.1 % identical, at least 99.2 % identical, at least 99.3 % identical, at least 99.4 % identical, at least 99.5 % identical, at least 99.6 % identical, 99.7 % identical, 99.8 % identical 99.9 % identical to the contigs of the parent bacteria.

Also contemplated are progeny of the bacteria that have a 16S rRNA sequence at least 90 %, at least about 91 %, at least about 92 %, at least about 93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, at least about 99.5 %, or more identical (or homologous) to the sequences as set forth in SEQ ID NOs: 1, 2, 3, 4 or 5, wherein the progeny has the same fibrolytic activity as that of the parent bacteria. Such progeny may have genomes having a sequence at least 99 % identical, at least 99.1 % identical, at least 99.2 % identical, at least 99.3 % identical, at least 99.4 % identical, at least 99.5 % identical, at least 99.6 % identical, 99.7 % identical, 99.8 % identical 99.9 % identical to the parent bacteria.

As used herein, the term "progeny of the bacteria" refers to bacteria descending from or derived from the bacterial species identified herein.

Also contemplated are functional homologs of the above disclosed bacteria, wherein the functionally homologous bacteria has a fibrolytic activity.

As used herein “functional homolog” or “functionally homologous” or “variant” or grammatical equivalents as used herein refer to a bacteria with a genomic nucleic acid sequence different than that of the sequenced bacteria (i.e., at least one mutation) resulting in a bacteria that is endowed with substantially the same ensemble of biological activities (+/- 10 %, 20 %, 40 %, 50 %, 60 % when tested under the same conditions) as that of the bacteria having a 16S rRNA sequence as set forth in SEQ ID NOs: 1-5.

According to some embodiments, the 16S rRNA sequence of the bacteria described herein is at least about 90 %, at least about 91 %, at least about 92 %, at least about 93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, at least about 99.5 %, or more identical (or homologous) to the sequences as set forth in SEQ ID NOs: 1-5.

As used herein, “percent homology”, “percent identity”, "sequence identity" or "identity" or grammatical equivalents as used herein in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff JG. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(22): 10915- 9].

Percent identity can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

Other exemplary sequence alignment programs that may be used to determine % homology or identity between two sequences include, but are not limited to, the FASTA package (including rigorous (SSEARCH, LALIGN, GGSEARCH and GLSEARCH) and heuristic (FASTA, FASTX/Y, TFASTX/Y and FASTS/M/F) algorithms, the EMBOSS package (Needle, stretcher, water and matcher), the BEAST programs (including, but not limited to BLASTN, BLASTX, TBLASTX, BLASTP, TBLASTN), megablast and BLAT. In some embodiments, the sequence alignment program is BLASTN. For example, 95% homology refers to 95% sequence identity determined by BLASTN, by combining all non-overlapping alignment segments (BLAST HSPs), summing their numbers of identical matches and dividing this sum with the length of the shorter sequence. In some embodiments, the sequence alignment program is a basic local alignment program, e.g., BLAST. In some embodiments, the sequence alignment program is a pairwise global alignment program. In some embodiments, the pairwise global alignment program is used for protein-protein alignments. In some embodiments, the pairwise global alignment program is Needle. In some embodiments, the sequence alignment program is a multiple alignment program. In some embodiments, the multiple alignment program is MAFFT. In some embodiments, the sequence alignment program is a whole genome alignment program. In some embodiments, the whole genome alignment is performed using BLASTN. In some embodiments, BLASTN is utilized without any changes to the default parameters.

According to some embodiments of the invention, the identity is a global identity, an identity over the entire nucleic acid sequences of the invention and not over portions thereof.

According to an additional or alternative embodiment, a functional homolog is determined as the average nucleotide identity (ANI), which detects the DNA conservation of the core genome (Konstantinidis K and Tiedje J M, 2005, Proc. Natl. Acad. Sci. USA 102: 2567-2592). In some embodiments, the ANI between the functional homolog and the disclosed bacteria is of at least about 95 %, at least about, 96 %, at least about 97 %, at least about 98 %, at least about 99 %, at least about 99.1 %, at least about 99.5 %, at least about 99.6 %, at least about 99.7 %, at least about 99.8 %, at least about 99.9 % or more.

According to an additional or alternative embodiment, a functional homolog is determined by the degree of relatedness between the functional homolog and the disclosed bacteria as determined as the Tetranucleotide Signature Frequency Correlation Coefficient, which is based on oligonucleotide frequencies (Bohlin J. et al. 2008, BMC Genomics, 9:104). In some embodiments, the Tetranucleotide Signature Frequency Correlation coefficient between the variant and the disclosed bacteria is of about 0.99, 0.999 or more.

According to an additional or alternative embodiment, the degree of relatedness between the functional homolog and the disclosed bacteria, is determined as the degree of similarity obtained when analyzing the genomes of the parent and of the variant bacteria by Pulsed-field gel electrophoresis (PFGE) using one or more restriction endonucleases. The degree of similarity obtained by PFGE can be measured by the Dice similarity coefficient. In some embodiments, the Dice similarity coefficient between the variant and the disclosed bacteria is of at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, at least about 99.1 %, at least about 99.5 %, at least about 99.6 %, at least about 99.7 %, at least about 99.8 %, at least about 99.9 % or more. According to an additional or alternative embodiment, the degree of relatedness between the functional homolog and the disclosed bacteria is determined by the Pearson correlation coefficient obtained by comparing the genetic profiles of both phages obtained by repetitive extragenic palindromic element-based PCR (REP-PCR) (see e.g. Chou and Wang, Int J Food Microbiol. 2006, 110: 135-48). In some embodiments, the Pearson correlation coefficient obtained by comparing the REP-PCR profiles of the variant and the above described bacteria is of at least about 0.99, at least about 0.999 or more - see for example bmcmicrobioldotbiomedcentraldotcom/articles/ 10.1186/sl2866-020-01770-2.

According to an additional or alternative embodiment, the degree of relatedness between the functional homolog and the disclosed bacteria is defined by the linkage distance obtained by comparing the genetic profiles of both bacteria obtained by Multi-locus sequence typing (MLST) (see e.g. Maiden, M. C., 1998, Proc. Natl. Acad. Sci. USA 95:3140-3145). In some embodiments, the linkage distance obtained by MLST of the functional homolog and the disclosed bacteria is of at least about 0.99, at least about 0.999 or more.

According to an additional or alternative embodiment, the functional homolog is defined by a comparison of the coding sequence (gene) order.

According to an additional or alternative embodiment, the functional homolog is defined by a comparison of order of non-coding sequences.

According to an additional or alternative embodiment, the functional homolog is defined by a comparison of order of coding and non-coding sequences.

According to some embodiments of the invention, the combined coding region of the functional homolog is such that it maintains the original order of the coding regions as within the genomic sequence of the disclosed bacteria, yet without the non-coding regions.

For example, in case the genomic sequence has the following coding regions, A, B, C, D, E, F, G, each flanked by non-coding sequences (e.g., regulatory elements, and the like), the combined coding region will include a single nucleic acid sequence having the A+B+C+D+E+F+G coding regions combined together while maintaining the original order of their genome, yet without the non-coding sequences.

According to some embodiments of the invention, the combined non-coding region of the functional homolog is such that it maintains the original order of the non-coding regions as within the genomic sequence of the disclosed bacteria, yet without the coding regions as originally present in the original bacteria.

According to some embodiments of the invention, the combined non-coding region and coding region (i.e., the genome) of the functional homolog is such that it maintains the original order of the coding and non-coding regions as within the genomic sequence of the disclosed bacteria.

As used herein “maintains” relate to at least about 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or 100 % of the coding and/or non-coding regions of the functional homolog compared to the genome of the disclosed bacteria.

According to an additional or alternative embodiment, the functional homolog is defined by a comparison of gene content.

According to a specific embodiment, the functional homolog comprises a combined coding region at least about 90 %, at least about 91 %, at least about 92 %, at least about 93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, or more (e.g., 100%) identical to the combined coding region existing in genome of the disclosed bacteria.

As used herein “combined coding region” refers to a nucleic acid sequence including all of the coding regions of the original bacteria yet without the non-coding regions of the original bacteria.

Additional bioinformatics methods that may be used to determine relatedness between two bacterial genomes include Nucmer and Minimap, both of which are alignment based tools; Winzip, Jacard distance and MinHash, each of which are information based tools; and Codon usage similarity, pathway similarity and protein motif similarity.

As mentioned, the novel bacterial strains described herein comprise in their genomes both the genes scaC (e.g. the gene which encode for the protein scaC having the GenBank reference No. CAK18894.1) and cttA (e.g. the gene which encodes the protein CTTA having the Genbank reference No. CAK18897.1).

In one embodiment, the bacterial strain expresses the protein encoded by the scaC gene. Thus, the bacterial strain may express a protein at least 35 % identical, 40 % identical, 45 % identical, 50 % identical, 55 % identical, 60 % identical, 65 % identical, 70 % identical, 75 % identical, 80 % identical, 85 % identical, 90 % identical, 95 % identical, 97 % identical, 99 % identical to one of the sequences as set forth in SEQ ID NOs: 938-945.

In another embodiment, the bacterial strain expresses the protein encoded by the cttA gene. Thus, the bacterial strain may express a protein at least 35 % identical, 40 % identical, 45 % identical, 50 % identical, 55 % identical, 60 % identical, 65 % identical, 70 % identical, 75 % identical, 80 % identical, 85 % identical, 90 % identical, 95 % identical, 97 % identical, 99 % identical to one of the sequences as set forth in SEQ ID NOs: 946-952.

In still another embodiment, the expressed CttA protein is capable of binding to cellulose. An exemplary method of analyzing whether CttA is capable of binding to cellulose is described in the Examples section below.

In further embodiments, the bacterial strains of the present invention express an endoglucanase which is capable of degrading cellulose (e.g. avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl P-D-cellobiose).

The term "endoglucanase" or "EG" refers to a group of cellulase enzymes classified as E.C. 3.2.1.4. These enzymes hydrolyze internal beta- 1,4 glucosidic bonds of cellulose. Endoglucanases include, but are not limited to, enzymes classified in the GH5, GH6, GH7, GH8, GH9, GH12, GH16, GH44, GH45, GH48, GH51, GH61, and GH74 GH families.

In one embodiment, the endoglucanase is Cel5A. Typically, the amino acid sequence of Cel5A is at least 60 %, 70 %, 80 %, or even 90 % identical to the sequence as set forth in SEQ ID NO: 953.

In still further embodiments, the bacterial strain expresses a member of the glycoside hydrolase family which is capable of degrading corn arabinoxylan. Exemplary glycoside hydrolases are those belonging to the GH98 family, those belonging to the GH5 family, those belonging to the GH7 family, those belonging to the GH8 family, those belonging to the GH10 family.

According to a specific embodiment, the bacteria expresses a glycoside hydrolase from the GH98 family.

Exemplary GH98 xylanases are described in PCT International Application No. PCT/US2020/015648 (incorporated herein by reference in its entirety). Optionally, the bacterial strains described herein are capable of residing in the human gut microbiome.

As used herein, the term “microbiome” refers to the totality of microbes (bacteria, fungae, protists), their genetic elements (genomes) in a defined environment.

The term “gut microbiome” microbiota of the digestive track. In one embodiment, the environment is the small intestine. In another embodiment, the environment is the large intestine. The microbiome may be of the lumen or the mucosa of the small intestine or large intestine.

In still another embodiment, the gut microbiome is a fecal microbiome.

In one embodiment, the bacteria may be found to naturally reside in a gut microbiome of a human (without the intervention of artificially providing the bacteria). The human may be a carnivore, a herbivore or a vegetarian.

The bacterial strains described herein may be isolated from a human gut sample using methods known in the art - see for example Wan et al., Microorganisms. 2023 Apr 20; 11(4): 1080. doi:.10.3390/microorganisms 11041080. The term "isolated" as used herein means that the bacterial strain has been removed from its natural environment. "Isolated" thus implies a purification step. However, "isolated" does not necessarily reflect the extent to which the microorganism, more particularly the bacterium has been purified. A bacterial strain of current application is purified at least 2x, at least 5x, at least lOx, at least 50x or at least lOOx from the raw material from which it is isolated, i.e. the human gut flora.

In one embodiment, the bacterial strain is isolated by selective cultivation.

In another embodiment, the bacterial strain is purified from a human gut sample by fluorescent in situ hybridization (FISH). Additionally, or alternatively, fluorescence-activated cell sorting (FACS) may be used.

In still embodiment, the bacteria may be found to be naturally absent in a rumen microbiome of a rumen (e.g. cow) (without the intervention of artificially providing the bacteria).

In another aspect, an enriched culture of one of the disclosed bacterial strains is provided.

The term "culture" as used herein refers to a population of microorganisms that are propagated on or in media of various kinds. An "enriched culture" of one of the bacterial strains of current application refers to a culture of microorganisms, more particular a bacterial culture, wherein the total microbial population of the culture contains more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% of one of the isolated bacterial strains of current application. This is equivalent as saying that a culture of microorganisms, more particularly a bacterial culture, is provided, wherein said culture is enriched with one of the bacterial strains of current application and wherein "enriched" means that the total microbial (or more particularly the total bacterial) population of said culture contains more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% of one of the isolated bacterial strains of current application.

In one embodiment, a biologically pure culture of one of the disclosed bacterial strains is provided.

As used herein, "biologically pure" refers to a culture which contains substantially no other microorganisms than the desired strain of microorganism and thus a culture wherein virtually all of the cells present are of the selected strain. In practice, a culture is defined biologically pure if the culture contains at least more than 96%, at least more than 97%, at least more than 98% or at least more than 99% of one of the bacterial strains of current application. When a biologically pure culture contains 100% of the desired microorganism a monoculture is reached. A monoculture thus only contains cells of the selected strain and is the most extreme form of a biologically pure culture.

In another aspect, a composition is provided comprising the bacterial species or strain herein disclosed and growth medium appropriate for the bacterial strain. Non-limiting examples of said growth medium include those that enrich for bacteria that can digest cellulose. For example, minimal media (without glucose) can be supplemented with carboxymethylcellulose (CMC), Avicel (microcrystalline cellulose), corn glucuronoarabinoxylan, corn-arabinoxylan, glucuronoarabinoxylan and/or Cellobiose.

In another aspect, a supernatant is provided wherein said supernatant is obtained from a culture of at least one of the strains of current application and wherein said culture can be an enriched culture of said at least strain or a biologically pure culture of said at least one strain. "Supernatant" refers to the liquid broth remaining when cells grown in broth are removed by centrifugation, filtration, sedimentation or other means well known in the art.

The present inventors contemplate probiotic compositions comprising at least two, at least three, at least four at least five or more of the above disclosed bacteria (i.e. those comprising a 16S rRNA sequence at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % identical to that as set forth in SEQ ID NOs: 1, 2, 3, 4, or 5 and/or those comprising a genome having contigs at least 90 % identical to SEQ ID NOs: SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937.

For any of the compositions or cultures disclosed herein, the number of bacterial species comprised therein is typically not greater than 150, not greater than 120, not greater than 100, not greater than 90, not greater than 80, not greater than 70, not greater than 60, not greater than 50, not greater than 40, not greater than 30, not greater than 20, not greater than 19, not greater than 18, not greater than 17, not greater than 16, not greater than 15, not greater than 14, not greater than 13, not greater than 12, not greater than 11, not greater than 19, not greater than 9, not greater than 8, not greater than 7, not greater than 6, not greater than 3.

The bacterial strains may be provided to a healthy subject and are useful for maintaining the health of the subject.

Alternatively, the bacterial strains may be provided to a non-healthy subject and act as a therapeutic in treating the disease.

In one embodiment, the non-healthy subject is an overweight subject (e.g. obese). The subject may be on an exercise program to enhance weight loss and/or may be on a diet to decrease caloric intake.

Other examples of diseases which may be treated using the bacterial strains disclosed herein include an inflammatory disease, a metabolic disease, cancer and a cardiovascular disease.

The term "inflammation", "inflammatory disorder" or "inflammatory disease" refers to complex but to the skilled person well known biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. However, inflammation is not a synonym for infection. Infection describes the interaction between the action of microbial invasion and the reaction of the body's inflammatory response — the two components are considered together when discussing an infection, and the word is used to imply a microbial invasive cause for the observed inflammatory reaction. Inflammation on the other hand describes purely the body's immunovascular response, whatever the cause may be. Inflammation is a protective response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and to initiate tissue repair. The classical signs of inflammation are heat, pain, redness, swelling, and loss of function. Inflammation is a generic response, and therefore it is considered as a mechanism of innate immunity, as compared to adaptive immunity, which is specific for each pathogen. Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. A series of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation, such as mononuclear cells, and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.

In one particular embodiment, said inflammatory disorder is a gastro-intestinal inflammatory disorder, or alternatively phrased gut inflammation.

The wording "gut inflammation" is equivalent to the wording "microscopic gut inflammation" as used herein and refers to an inflammatory response in the gut as defined above. The inflammation can affect the entire gastrointestinal tract, can be more limited to for example the small intestine or large intestine but can also be limited to specific components or structures such as the bowel walls. In a more particular embodiment, said gut gastro-intestinal inflammatory disorder is selected from the list consisting of inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), irritable bowel syndrome (IBS), celiac disease, pouchitis, postinfection colitis, inflammation associated with gastrointestinal cancer, diarrhoeal disease due to undesirable inflammatory activity, diarrhoeal disease due to an infectious bacterial agent such as E.coli, Clostridium difficile associated diarrhoea, Rotavirus associated diarrhoea, and post- infective diarrhoea. In a most particular embodiment of the fifth aspect, the inflammatory disorder is IBD, more particularly CD and/or UC. Examples of metabolic diseases include Diabetes (type I or type II) and obesity.

Exemplary cancers that may be treated using the bacterial strains disclosed herein include those of the GI tract (for example colon cancer, rectal cancer, stomach cancer etc.) - see for example sciencedirect(dot)com/science/article/pii/S0022316623027372, the contents of which are incorporated herein by reference.

The bacterial strains disclosed herein can be provided to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the bacteria accountable for the biological effect (e.g. weight reducing effect/ health promoting effect).

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Typically, the bacterial compositions described herein are formulated with a preservative which promotes viability of the bacteria.

Methods of preserving bacteria are disclosed in PCT Application No. WO/2019/014338, the contents of which are incorporated herein by reference.

In one embodiment, the bacterial composition comprises a cryoprotectant.

The term "cryoprotectant" means a substance that improves survival during freezing and/or drying and improves storage stability of bacteria. Cryoprotectants as used herein generally comprise a sugar.

Optionally, the bacterial compositions further comprise a prebiotic.

Prebiotics are non-digestible foods which promote the growth of the disclosed bacteria in the gut.

Examples of prebiotics that may be used in the present invention include, but are not limited to, cellulose corn glucuronoarabinoxylan, inulin and inulin-type fructans, oligofructose, xylose, arabinose, arabinoxylan, ribose, galactose, rhamnose, cellobiose, fructose, lactose, salicin, sucrose, glucose, esculin, tween 80, trehalose, maltose, mannose, mellibiose, mucus or mucins, raffinose, fructooligosaccharides, galacto-oligosaccharides, amino acids, alcohols, and any combinations thereof. Other non-limiting examples of prebiotics include water-soluble cellulose derivatives, water-insoluble cellulose derivatives, unprocessed oatmeal, metamucil, all-bran, and any combinations thereof. Examples of water-soluble cellulose derivatives include, but are not limited to, methylcellulose, methyl ethyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, cationic hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, and carboxymethyl cellulose.

Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

In certain embodiments, the probiotic composition is formulated as a food additive. In certain embodiments, the food additive disclosed herein further comprises other materials known in the art for inclusion in food additives, including, but not limited, water or other aqueous solutions, starch, binders, thickeners, colorants, flavorants, odorants, acidulants (e.g., lactic acid or malic acid, among others), vitamins, minerals, and combinations thereof. In certain embodiments, the food additive comprises between about 10³ and about 10⁴ CFU probiotic bacteria per gram of the food additive, between about 10⁴ and about 10⁵ CFU probiotic bacteria per gram of the food additive, between about 10⁵ and about 10⁶ CFU probiotic bacteria per gram of the food additive, between about 10⁶ and about 10⁷ CFU probiotic bacteria per gram of the food additive.

The present disclosure also provides a fortified food comprising the probiotics or probiotic compositions disclosed herein. In certain embodiments, the fortified food disclosed herein further comprises a base food. In certain embodiments, the food additive can be incorporated to a base food to form the fortified food. Any base foods known in the art can be used with the present disclosure. Non-limiting examples of base foods include kefir, yakult, miso, natto, tempeh, kimchee, sauerkraut, water, milk, fruit juices, vegetable juices, yogurt, carbonated soft drinks, noncarbonated soft drinks, coffee, tea, beer, wine, liquor, alcoholic mixed drinks, bread, cakes, cookies, crackers, extruded snacks, soups, frozen desserts, fried foods, pasta products, potato products, rice products, com products, wheat products, dairy products, confectionaries, hard candies, nutritional bars, breakfast cereals, bread dough, bread dough mix, sauces, processed meats, and cheeses. Administration of a probiotic composition comprising probiotic bacteria can be accomplished by any method likely to introduce the bacteria into the desired location. In certain embodiments, the probiotics can be administered to a subject, in the form of a food additive or a fortified food disclosed herein, by oral consumption. In certain embodiments, the probiotic bacteria can be mixed with a carrier and (for easier delivery to the digestive tract) be applied to liquid or solid food, feed, or drinking water. The carrier material should be non-toxic to the bacteria and the subject/patient. In certain embodiments, the carrier contains an ingredient that promotes viability of the bacteria during storage. The formulation can include added ingredients to improve palatability and improve shelf-life.

In certain embodiments, the carrier comprises a diluent, adjuvant, excipient, or vehicle with which probiotic bacteria are administered. In certain embodiments, the carrier can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. In certain embodiments, the carrier can be water or aqueous solution, saline solutions and aqueous dextrose and glycerol solutions. In certain embodiments, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable carriers for therapeutic use are well known in the art and are described, for example, in “Remington’s Pharmaceutical Sciences” by E. W. Martin, and in “Remington: The Science and Practice of Pharmacy.” Lippincott Williams & Wilkins.

The choice of a carrier can be selected based on the intended route of administration and standard practice. In certain embodiments, oral delivery can be used for delivery to the digestive tract. In certain embodiments, oral formulations comprise additional mixtures, such as milk, yogurt, and infant formula.

In certain embodiments, the duration and frequency of administration can vary between a type of malady from which the subject suffers. For example, in one embodiment, the bacteria is formulated (or provided in a dose suitable) for overweight subjects. In another embodiment, the bacteria is formulated (or provided in a dose suitable) for obese subjects.

In certain embodiments, solid dosages in the form of tablets or capsules are used for the delivery of the probiotic bacteria by mixing the probiotic bacteria with one or more components selected from the group consisting of sodium alginate, calcium carbonate, glyceryl monooleate, triethyl citrate, acetylated monoglyceride, and hypromellose acetate succinate (HPMCAS).

In certain embodiments, the probiotic bacteria or probiotic compositions disclosed herein can be administered parenterally. In certain embodiments, the probiotic bacteria or probiotic compositions of the presently disclosed subject matter can be prepared for delivery as a solution, a tablet, a capsule or as a lyophilized culture. Where cultures are lyophilized, the preparation can be rehydrated in, for example, yogurt or water for administration.

In certain embodiments, the probiotic bacteria or probiotic compositions of the presently disclosed subject matter are formulated such that they can survive passage through the acidic environment of the stomach and such that they adjust quickly to the intestinal environment. Such formulation allows the presently described probiotic bacteria and probiotic compositions to have an elongated half-life in the intestines.

In certain embodiments, the probiotics or probiotic compositions disclosed herein are administered to a subject who has a healthy BMI (e.g. 18.5-24.9). In certain embodiments, the probiotics or probiotic compositions disclosed herein are administered to a subject who has an overweight BMI (e.g. 25-29.9). In certain embodiments, the probiotics or probiotic compositions disclosed herein are administered to a subject who have a diagnosed disease, e.g., obesity (e.g. BMI over 30). In certain embodiments, the probiotics or probiotic compositions are administered to the subject in the form of food additives or fortified foods disclosed herein. Dosage of the probiotic bacteria or probiotic composition disclosed herein for the subject (e.g., a subject having a healthy BMI, a subject having an overweight BMI, a subject having an obese BMI, a subject diagnosed with obesity) can vary depending upon the characteristics of the subject (e.g., age, sex, race, weight, height, BMI, body fat percentage, and/or medical history), frequency of administration, manner of administration, clearance of the probiotic bacteria from the subject, and the like.

In certain embodiments, the initial dose can be larger, followed by smaller maintenance doses. In certain embodiments, the dose can be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level. In certain embodiments, a variety of doses are effective to achieve colonization of the gastrointestinal tract with the desired probiotic bacterial, for example and not by way of limitation, about 10⁶ CFU, about 10⁷ CFU, about 10⁸ CFU, about 10⁹ CFU, about IO¹⁰ CFU, about 10¹¹ CFU, about 10¹² CFU, about 10¹³ CFU, about 10¹⁴ CFU, or about 10¹⁴ CFU of probiotic bacteria can be administered in a single dose to a subject. In certain embodiments, lower doses can also be effective, for example and not by way of limitation, about 10⁴ and about 10⁵ CFU of probiotic bacteria. In certain embodiments, the probiotic bacteria are administered to a subject in a dosage of between about 10⁶ and about 10⁷ CFU, between about 10⁷ and about 10⁸ CFU, between about 10⁸ and about 10⁹ CFU, between about 10⁹ and about IO¹⁰ CFU, between about IO¹⁰ and about 10¹¹ CFU, between about 10¹¹ and about 10¹² CFU, between about 10¹² and about 10¹³ CFU, between about 10¹³ and about 10¹⁴ CFU, or between about 10¹⁴ and about 10¹⁵ CFU. In certain embodiments, the probiotic bacteria are administered to a subject in a dosage of about IO¹⁰ CFU of probiotics. In certain embodiments, the probiotic bacteria are administered to a subject in a dosage of up to about 10¹² CFU. In certain embodiments, the subject is a human. In certain embodiments, the subject is a domestic animal, e.g., a canine.

In certain embodiments, when administered to a subject having a diagnosed disease (e.g., obesity) or a subject having an obese BMI, the optimal dosage can be empirically determined by treating physicians based on the stage of disease and patient statistics (e.g., age, height, weight, etc.). In certain embodiments, when administered to a subject who has a healthy BMI, or an overweight BMI, the optimal dosage can be empirically determined by the subject or a dietitian based on the subject’s statistics, e.g., age, sex, race, height, weight, BMI, body fat percentage, and/or medical history.

In certain embodiments, a probiotic composition or probiotic bacteria disclosed herein can be delivered every 4, 12, 24, 36, 48, 60, or 72 hours. In certain embodiments, the probiotic composition or the probiotic bacteria can be delivered with at least one second pharmaceutically active ingredient, where the second pharmaceutically active ingredient can be delivered simultaneously or sequentially (e.g., within a 4, 12, 24-hour or 1-week period) with the probiotic composition or the probiotic bacteria. In certain embodiments, the probiotic composition or the probiotic bacteria can be delivered with two, three, four, five, or six second pharmaceutically active ingredients. In certain embodiments, the treatment can last for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 1 year.

In certain embodiments, one or more preparations of different probiotic bacteria can be administered simultaneously (including administering bacteria of the same species or genus, or different species or genus) or sequentially (including administering at different times). Such probiotic bacteria can be prepared from bacteria isolated from microbiota and then grown in a culture using known techniques.

In certain embodiments, the probiotic composition further comprises one or more antiobesity agent selected from the group consisting of an appetite suppressant such as Ozempic™, a metabolic agent that alters a fundamental metabolic processes of the host subject’s body (e.g. increase fat burning, increase in metabolism). It will be appreciated that the anti-obesity agent does not necessarily have to be formulated in a single composition together with the disclosed bacterial strains. Thus, the present invention contemplates articles of manufacture, whereby the bacterial strain is packaged separately from the anti-obesity agent.

In certain embodiments, the second pharmaceutically active ingredient can be an antiobesity agent. Non-limiting examples of anti-obesity pharmaceutical agents include catecholamine release agents, such as amphetamine, phentermine™ and related substituted amphetamines (e.g. bupropion), agents that increase the human body’s metabolism, agents that interfere with the human body’ s ability to absorb specific nutrients in food, for example and not by way of limitation, orlistat® (tetrahydrolip statin), loscaserin, sibutramine, rimonabant, metformin™ (N,N- dimethylbiguanide), exenatide, phentermine, as well as herbal and dietary supplements.

In certain embodiments, the probiotic composition can be administered in combination with at least one anti-obesity agent.

“In combination with,” as used herein, means that the probiotic composition and the one or more anti-obesity agents are administered to a subject as part of a treatment regimen or plan. In certain embodiments, being used in combination does not require that the probiotic composition and the one or more anti-obesity agent are physically combined prior to administration or that they be administered over the same time frame. For example, and not by way of limitation, the probiotic composition and the one or more anti-obesity agent can be administered concurrently to the subject being treated or can be administered at the same time or sequentially in any order or at different points in time.

In certain embodiments, the present disclosure provides probiotic compositions comprising probiotic bacteria at a concentration of between about 1 weight % and about 100 weight % (%w/w) of the probiotic compositions. In certain embodiments, the probiotic bacteria are at a concentration of between about 1 ppm and about 100,000 ppm of the probiotic compositions. In certain embodiments, the probiotic bacteria are at a concentration of about 1 pM of the probiotic compositions.

Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

According to still another aspect of the invention there is provided a method of identifying probiotic bacteria useful for maintain the health of a human subject comprising analyzing for the presence of a bacterium in a sample of the gut microbiome of a human subject, wherein a bacterium which express both the genes scaC and cttA is indicative that the bacterium is a probiotic useful for maintaining the health of the human subject.

Optionally, bacteria of the gut sample (e.g. fecal sample) may be cultured under appropriate conditions so as to enhance the probability of survival of a fibrolytic bacterium (as described herein above) over the survival of a non-fibrolytic bacterium.

Methods of ascertaining whether the bacterium expresses scaC and cttA can be carried out on the protein level or the nucleic acid level. Protein based methods are known in the art and include for example chromatography, electrophoretic methods, immunodetection assays such as ELISA and western blot analysis and immunohistochemistry. Nucleic acid-based methods include for example DNA sequencing, RNA sequencing, RT-PCR, RNase protection, in-situ hybridization, primer extension, Southern blot, Northern Blot and dot blot analysis.

In one embodiment, the 16S rRNA sequence of the bacterium is determined by sequencing.

The bacteria may be further analyzed so as to confirm that it has fibrolytic activity. Cellulase- specific assays that may be used include congo red cellulase assay (plate screening), filter paper assay (quantitative test), DNS assay for reducing sugars. PCR assays may be carried out for identifying specific cellulase genes such as endoglucanases (celA, celB), exoglucanase (cbhA, cbhB), B-glucosidase (bglX, bglB).

The bacteria may be further characterized by one or more of the following methods:

Survival in gastric conditions (test survival at pH2-3);

Bile salt resistance (bacteria may be grown in 0.3 % bile salts);

Adhesion assays (bacteria may be checked to see whether they adhere to human gut epithelial cells (e.g. Caco-2 cells);

Antimicrobial production (test whether the bacteria inhibit pathogenic bacteria).

Once identified in a mixed culture, serial streaking may be carried out (e.g. on selective media) to obtain a single colony. A glycerol stock of the bacteria may be prepared (e.g. 20 % glycerol) and optionally frozen (e.g. at -80 °C).

The bacteria may be lyophilized so as to prepare an industrial formulation.

As used herein the term “about” refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

The term “consisting of means “including and limited to”.

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

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this 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 invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

It is appreciated 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 suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

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.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

MATERIALS AND METHODS

Retrieval and analysis of ruminicoccal genomes containing cellulosomal elements

The ScaC sequence from R. flavefaciens strain FD-1 (accession number CAK18894) was used as a query sequence to retrieve metagenome-assembled genomes (MAGs) of rumen and human origin ^17,18, using local blast ⁵⁰. Hits below E-values of 10’⁴, above 45% of sequence identities and of lengths higher than 250 amino acids were retained. Among these, only associated MAGs with above 90% completeness as determined by CheckM ¹⁹ were analyzed further. ScaC sequences were aligned using MegaX ⁵¹. Annotation of glycoside hydrolases in the selected genomes were performed with dbcan2 ⁵². The presence of the N-terminal sequence of the CttA protein ²¹ (427 amino-acids, accession number CAK18897.1), which corresponds to the cellulose binding component of the cellulosome system, was used as a specific marker for R. flavefaciens strains using tblastx.

Analysis of selected putative R. flavefaciens MAGs

Dockerin and cohesin-containing sequences were retrieved from the predicted proteome (using Prokka ⁵³) by local BLAST ⁵⁰ using known cohesin or dockerin sequences from the Bayer lab databases ⁵⁴. Hits below E-values of 10’⁴, above 30% of sequence identities for cohesins and 45% for dockerins, and of lengths higher than 60 or 130 amino acids for dockerins or cohesins, respectively, were inspected for characteristic dockerin features including Ca²⁺-binding repeats and putative recognition residues ^55,56. Clustal Omega was used for multiple sequence alignments of dockerin modules ⁵⁷ , and annotation of dockerin-containing genes was performed using dbcan2. Mash analysis on the nucleotide level was performed on the R. hominiciens genomes annotated using CttA as a marker ⁵⁸.

Prevalence of selected MAGs in rumen and gut samples

At first, the 30 selected MAGs of rumen, human and monkey origin were aligned to their original sample reads. The number of reads were normalized between samples, and only alignments above 80% completion were retained. A heatmap of MAG abundances in the different samples was created. Then, to examine the prevalence of selected MAGs across gut samples from human and animals, the different MAGs were clustered based on 97% similarity, using the drep algorithm ²⁴. This step resulted in 3 human and 8 rumen MAGs, that were aligned to metagenomes from gut or rumen fecal samples. Samples with coverage of at least 20% for a given MAG at a threshold of 1 were considered as positive. Prevalence for R. champanellensis was calculated similarly by aligning the 18P13 genome to the same fecal samples. PCR on metagenomic DNA, extracted using the phenol-chloroform method ⁵⁹ from a cohort of 39 human feces samples, as well as chimpanzee and gorilla feces samples (collected at the Safari zoo, Israel ⁶⁰), was performed using specific primers for CttA. To ensure amplification of the specific CttA gene, amplicons were sequenced by Sanger sequencing.

Evolutionary analysis of the selected MAGs

Proteinortho ²⁸ was used to group orthologous proteins from human, rumen and monkey genomes. For each of the 315 orthologous groups comprising the core genome shared between the different host-associated strains, the present inventors searched for orthologs in the genome of Clostridium thermobutyricum DSM 4928 to serve as an outgroup. For 197 orthologous groups, outgroup orthologs were retrieved and phylogenetic trees were created using the minimal ancestor deviation (MAD) rooting approach ⁶¹. They then performed an approximately unbiased (AU) analysis ²⁹. To test for potential evolutionary scenarios, the AU test was performed as part of the iqtree2 program package ⁶² while using the ‘-au’ parameter as well as the ‘-zb 10,000’ parameter to indicate the number of RELL ⁶³ replicates to perform several tree topology tests for all 197 core orthologous groups trees. The dendextend R package was used with the cor.dendlist function ³⁰ to identify core protein trees that exhibited similar host clustering as the mammalian host’s evolutionary tree (created using the Timetree database ⁶⁴).

To perform MLSA ⁶⁵, amino-acid sequences of the subunit of RNA polymerase (rpoB), subunit of DNA gyrase (gyrB), translation initiation factor IF-2 (infB), RNA modification GTPase ThdF or TrmE (thdF), chaperonin GroEE (groEL) and sigma 70 (sigma D) factor of RNA polymerase (rpoD) were retrieved from each of the 30 MAGs, aligned [59], concatenated using MegaX and a maximum likelihood phylogenetic tree was generated.

Cloning of cellulosomal modules and enzymes from human ecotypes

Thirteen sequences of dockerins and cohesins were selected from ecotype 1 and synthesized by IDT (Coralville, Lowa USA) with additions of restriction sites at both ends. The synthesized DNA sequences of cohesins and dockerins were inserted into CBM-Coh and Xyn- Doc plasmids cassettes respectively ³¹, using appropriate restriction endonucleases (Thermofisher Scientific). T4 ligase (New England Biolabs) was used for plasmid ligation and Escherichia coli strain DH5 alpha (Bio Lab, Israel) was used for transformation. Plasmids were verified by Sanger sequencing.

The sequences of GH5 and GH48 enzymes from ecotype 1 were also synthesized by IDT and cloned into pET28a, using either restriction or restriction-free cloning. The N-terminal sequence of the GH5 was reconstructed using sequence consensus of highly similar GH5 sequences, recovered by blastp. GH98 was cloned from metagenomic DNA extracted from a human sample using the phenol-chloroform method, in which the CttA gene was detected, cleaved using Ncol and Xhol and inserted into restricted pET28a by ligation. The list of all primers used in this study is provided in Table 1, herein below.

Table 1

The amino-acid sequences of the proteins used in the study are shown in Table 2, herein below.

Table 2 Coh= cohesin, Doc= dockerin

Expression of proteins

E. coli strain BL21 pLysS (DE3) was used as a host for protein expression. The cells were transformed with the desired plasmid and plated on LB-kanamycin plates. 5 ml of LB (Luria- Bertani Broth Lennox) were added to the plate to resuspend cells. The resuspended cells were added to 1 L of LB (or TYG in the case of GH98), supplemented with 50 mg/ml kanamycin (Sigma- Aldrich, Israel) and 2 mM CaCh. The culture was grown at 37°C to an OD6oo=0.8-l. Isopropyl P-D-l -thiogalactopyranoside (IPTG) inducer (Inalco pharmaceuticals) was added to a final concentration of 0.2 mM, and growth was continued at 37°C for 3 h for protein expression. Cells were centrifuged for 15 min at 5000 rpm, the pellet was resuspended in 30 ml binding buffer, consisting of Tris-buffered saline (TBS, 137 mM NaCl, 2.7 mM KC1, 25 mM Tris-HCl, pH 7.4) with 5 mM imidazole (Merck KGaA, Darmstadt, Germany) and protease-inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride (PMSF), 0.4 mM benzamidine (Sigma-Aldrich. St Louis, MO, USA)). Cells were disrupted by sonication, and the sonicate was centrifuged for 30 min at 15000 rpm at 4°C.

Purification of Xyn-Doc fusion proteins and GH-containing dockerins

The sonicated supernatant fluids were loaded onto a column of Ni-NTA beads (EMD, MERCK-Millipore) equilibrated with binding buffer and purified using gravity. The column was washed with 14 ml of binding buffer, followed by a similar volume of binding buffer supplemented with 15 mM imidazole (washing buffer). The recombinant protein was eluted from the column using 100 to 250 mM of imidazole solutions in TBS, 2-ml fractions were collected and analyzed by SDS-PAGE, using a protein molecular weight marker (Gene Direx). The fractions containing pure recombinant protein were pooled. Protein samples were stored in 50% (v/v) glycerol (Mallinckrodt Baker- Av antor) at -20 °C.

Purification of CBM-Coh fusion proteins

15 ml of amorphous cellulose (PASC) at 7.5 g/1 were added to the sonicated supernatant fluids and incubated for 1 h on a rotator at 4°C. The amorphous cellulose was pelleted by centrifugation (5 min, 4000 rpm, 4°C). The pellet was washed three times with 45 ml TBS, containing 1 M NaCl and three times with 45 ml TBS. The protein was eluted from the pellet with 3 volumes (5 ml each) of 1% (v/v) trimethylamine (Sigma- Aldrich), and the pH of the eluted fractions were neutralized using acetic acid (Bio lab, Israel). Purity was assessed by SDS-PAGE, and proteins were stored in 50% (v/v) glycerol at -20 °C.

Affinity-based ELISA analysis of cohesins using immobilized dockerins

The concentration of all purified proteins was estimated by absorbance (280 nm) based on the known amino acid composition of the desired protein and by using the Protparam tool ⁶⁶. IMMUNO MaxiSorp ELISA plates (Greiner Bio one Inc.) were coated overnight at 4°C with 10 pg/ml solution of the desired Xyn-Doc in 0.1 M sodium carbonate (pH 9) (100 pl/well). The following steps were performed at room temperature with all reagents at a volume of 100 pl/well. The coating solution was discarded, and blocking buffer (TBS, 10 mM CaCh, 0.05% Tween 20 (Sigma-Aldrich), 2% BSA (MP Biomedicals)) was added for 1-h incubation. The blocking buffer was discarded, and incremental concentrations of the desired CBM-Coh fusion proteins, diluted in blocking buffer, were added. After a 1-h incubation period, the plates were washed three times with wash buffer (blocking buffer without BSA), and primary antibody (rabbit anti-CBM3a antibody, diluted 1 : 10,000 in blocking buffer, was added. Following another 1-h incubation period, the plates were washed three times with wash buffer, and the secondary antibody (HRP- anti-rabbit antibody (Jackson ImmunoResearch Laboratories Inc. West Grove, PA, USA), diluted 1:10,000 in blocking buffer, was added. After another 1-h incubation, the plates were again washed (four times) with wash buffer, and 100 pl/well TMB Substrate-Chromogen (Dako Corp.) were added. Color formation was terminated after 3 min, upon addition of 1 M H2SO4 (50 pl/well), and the absorbance was measured at 450 nm using a microplate reader. Absorbance was plotted as a function of CBM-Coh concentration, usually resulting in a sigmoidal (dose-response) curve.

Cohesins and dockerins from R. champanellensis 18P13 and R. flavefaciens FD-1 for cross-species interactions were cloned and produced as described earlier ^10,35.

Enzymatic activity assay

Cellulolytic activity was tested with 0.5 pM of either GH5 or GH48 from Human_SRR5558136_bin.38 or from R. flavefaciens FD-1 on 1 % Avicel (FMC, Delaware USA) at pH 5 (50 mM acetate buffer, final concentration) for 24, 48 and 72 h at 37°C. After incubation, the tubes were centrifuged for 2 min at 14,000 rpm at room temperature, and 100 pL of supernatant fluids were added to 150 pL dinitrosalicylic acid (DNS) solution ⁶⁷ , boiled for 10 min, and the absorbance at 540 nm was measured. Released sugar concentrations were determined using a glucose standard curve.

Glucuronoarabinoxylanase activity was tested by incubating 0.2% com arabinoxylan ³⁹ with 20 pL of either purified GH98, double-distilled water (ddw) or the lysate of a R. flavefaciens strain 17 culture, grown in M2 medium in 20 mM citrate buffer (pH 6), supplemented with 0.2% cellobiose, incubated overnight at 37°C ⁶⁸. Two ml of the reactions were spotted on TLC Silica gel 60F (Merck), and chromatography was carried out for 1.5 h, using butanol: acetic acid: water 3:1:1 as a developing solvent. After drying the plate, spots were visualized by orcinol stain (5 g orcinol dissolved in 376.65 ml ethanol, 107 ml DDW and 16.15 ml sulphuric acid), and the silica plate was heated for 10 min at 70 °C.

Cellulose binding assay

Binding ability of CttA to cellulose was tested by the cellulose binding assay as described earlier ⁶⁹. The CBM and cohesin-CBM3a from the CipA scaffodin of Clostridium thermocellum ⁶⁹ were used as a positive control, and the GFP protein as a negative control for binding abilities 70

Comparative genomics of selected human, rumen and monkey genomes

Among the 5958 gene clusters obtained by Proteinortho, the 315 clusters common to the three groups were analyzed for verticality as well as clusters specific to one or two hosts. For verticality mapping, sequences were compared to the verticality values calculated by Nagies et al. ³⁶. This was done by blasting all sequences in the database, which formed the basis for the clustering used in the latter report, against each sequence of interest. Results were filtered by an E-value of IO ¹⁰, and sequences of interest were then mapped to the cluster with the highest number of hits. If the mapped cluster had a calculated verticality value, this value was then mapped to the sequence of interest.

The presence-absence of the overall 5958 gene clusters, or number of annotated glycoside hydrolases (with and without dockerin modules) obtained using dbcan2, were compared among the three groups of selected genomes (human, rumen and monkeys) using PCA plot in R with phyloseq ⁷¹ and ggplot2 ⁷², followed by the PERMANOVA test using 1000 randomizations of the data and the vegan package ⁷³. To highlight statistically different groups of GH, a Kruskal-Wallis test was performed, followed by false-discovery rate correction. Abundance heatmaps were created for genes or transcripts.

Expression ofR. hominiciens genes in human samples

RNA was extracted in 4 positive fecal samples, using the Qiagen AllPrep PowerFecal DNA/RNA Kit, and samples which yielded high-quality RNA were sequenced by NovaSeq SP 2xl50nt (Roy J. Carver Biotechnology Center, Illinois). Reads from sample 50466110 from project PRJNA354235, which was found positive in the MAG alignments, were also used. Reads from the metatranscriptomics of three macaque fecal samples ⁷⁴ and three sheep rumen samples ³⁷ were retrieved from the ENA database (macaque project SRX3517701-SRX3517724, samples SRR6425354, SRR6425396 and SRR6425408 and cow project PRJNA202380, samples SRR1206249, SRR1138694 and SRR1138697). Reads were subsampled to 1,000,000 reads, and transcripts were quantified using RSEM ⁷⁵ against their respective MAGs Human_SRR6028624_bin.l6, Rumen_CACVSX01 and Monkey_bin.22. The transcripts of the annotated glycoside hydrolases (with and without dockerin modules) obtained with dbcan2, were compared among the three groups of selected genomes (human, rumen and monkeys) using PCA plot in R with phyloseq ⁷¹ and ggplot2 ⁷², followed by the PERMANOVA test using 1000 randomizations of the data and the vegan package ⁷³. RESULTS

Detection of novel fiber-degrading species in the human gut microbiome

Cellulosomes are one of the most efficient enzymatic machines for microbial fiber degradation in nature and are exclusively coded in gut systems by the Ruminococcus genus. By identifying known cellulosomal components in genomes of this genus, the present inventors proposed that they should be able to identify the breadth of the diversity of human gut cellulosome- producing species. Cellulosome complexes are heterogeneous modular assemblies of structural proteins (scaffoldins) and enzyme arrays that target the different recalcitrant plant fiber components (Figure 1A). The cellulosome complex is composed of multiple scaffoldins that contain cohesin modules, each of which interacts with a complementary dockerin module located on each of the cellulosomal enzyme components (Figure 1A).

In order to retrieve and analyze cellulo some-producing ruminococcal genomes in the present work, the definitive scaC gene was used that codes for one of the scaffoldin proteins of the cellulosome and is unique to the Ruminococcus genus ^15,16 (Figure 1A). Using this approach, the present inventors searched for ScaC sequences in 4,941 rumen metagenome-assembled genomes and 92,143 human metagenome-assembled genomes (MAGs) ^17,18. In doing so, they identified 251 ruminococcal genomes that code for ScaC and therefore potentially contain cellulosomal elements. After filtering these genomes that exhibit at least 90% completion as determined by CheckM ¹⁹, they obtained 25 and 22 genomes of rumen and human origin, respectively. Maximum likelihood phylogenetic analysis of their ScaC sequences revealed a clustering pattern that almost entirely separates clades of the ruminococcal genomes to human or rumen origin, respectively. This analysis was augmented by ScaC sequences of 12 sequenced genomes of R. flavefaciens isolates from the rumen environment and 3 sequenced genomes from isolates of their close relative from the human gut, R. champanellensis.

To deepen the phylogenetic analysis, the fibrolytic potential of these 62 genomes was further examined by searching for the presence of cellulosomal elements and CAZymes (i.e., carbohydrate-active enzymes that act on glycosidic bonds ²⁰), with an emphasis on enzyme components that integrate into cellulosome complexes, which would be detected by the presence of a dockerin module on the enzyme. A total of 3687 dockerin-containing proteins was identified, among which 1853 also contained a CAZy module (Figure IB), which group into glycoside hydrolases (GH), carbohydrate esterases, polysaccharide lyases and carbohydrate-binding modules (CBMs) from various families. In addition, a total of 308 scaffoldins (structural backbone proteins that serve as a framework of the cellulosome complex) were recovered. The phylogenetic clusters of the tree corresponded to the distribution of the functional cellulosomal components of the identified MAGs. The human-associated MAGs were separated into four distinct clades (bootstrap values higher than 90%) (Figure IB), two clades with low numbers of cellulosomal elements, designed here as Ruminococcus sp. 1 and Ruminococcus sp. 2 and two clades with a high number of cellulosomal elements that were further examined below, one of which composed of sequences from R. champanellensis, previously isolated from the human gut microbiome.

Interestingly, the ScaC sequences of the members of the second human- associated clade with high numbers of cellulosomal elements were phylogenetically closer to the ScaC sequences of R. flavefaciens rumen isolates genomes, (bootstrap value of 60%). Like the R. flavefaciens scaffoldin gene cluster, they also contained the cttA gene marker. CttA is a cellulosomal protein whose role is to bind the bacterium to cellulose (Figure 1A) ²¹. The gene for this cellulosome component represents a marker unique to R. flavefaciens and is lacking in the genome of its relative from the human gut, R. champanellensis. The cttA gene can therefore be used to specifically distinguish between the two closely related, cellulosome-producing species. Members of this clade that encode the cttA gene and occur in the human gut potentially represent novel human-gut fiberdegrading cellulosomal strains. The present inventors analyzed the similarity of the genomes of this clade and obtained an average of >99% similarity to each other but only 78% to the genomes of isolates and MAGs affiliated with rumen R. flavefaciens (Figure 1C) ²². In addition, the 16S- rRNA sequence of 4 out of the 6 MAGs exhibited an average of 95.8% and 92.7% identity to the rumen R. flavefaciens and human R. champanellensis strains, respectively, while 100% identity to each other, thus further corroborating their potential association as a novel distinct ruminococcal species, which are designated herein as Ruminococcus hominiciens (homini.ciens, from Latin homini and “ciens” for similarity to R. flavefaciens').

Two MAGs of human origin that also encode both the cttA gene marker and numerous cellulosomal elements were not located within the R. hominiciens clade. Genome similarity data and marker genes (specified below) suggest that these MAGs may also represent distinct cellulosome-producing bacterial species occupying similar niches as R. hominiciens (Figure IB). One MAG was positioned within the rumen-associated MAG clade and the second appears as a single isolated branch of the phylogenetic tree. The 16S-rRNA sequence of the former MAG exhibited low average genome similarities to the R. hominiciens (80%) and R. flavefaciens genomes (75.6%) (Figure 1C, green background). The latter MAG also exhibited low genome similarity to the R. hominiciens and R. flavefaciens strains, 71 and 77.3%, respectively (Figure 1C, orange background), and its 16S-rRNA sequence exhibited relatively low identity to both the rumen R. flavefaciens and human R. hominiciens strains (91,3% and 90.6%, respectively). The 16S-rRNA and genome similarity of these two strains to the other examined species suggest that they are new distinct species, and they were thus given new species names as well. The human- associated MAG that was positioned within the rumen clade was named Ruminococcus ruminiciens (rumini.ciens, from Latin rumini and “ciens” for similarity to R. flavefaciens), and the other human-associated MAG that appeared as a single branch on the phylogenetic tree was named Ruminococcus primaciens (prima.ciens, from Latin prima and “ciens” for similarity to R. flavefaciens). In addition, Protologger analysis ²³ of the R. ruminiciens, R. primaciens and R. hominiciens genomes suggested that these are new species with potential for cellulose and starch utilization and acetate, propionate, L-glutamate production, similar to that of R. flavefaciens (strain FD-1), which further emphasizes the similar functionality of these strains.

Novel fiber-degrading bacterial species exhibit higher prevalence in hunter-gatherer and non-industrialized human societies, suggesting a possible microbiome extinction process

The prevalence of the novel genomes of the fiber-degrading species was analyzed compared to that of the known ruminococcal species, R. flavefaciens, across gut samples of humans and 17 animal species from various localities around the world. For this analysis, the different MAGs were clustered based on 97% similarity, using the drep algorithm ²⁴. This step resulted in the three new human species (R. primaciens, R. hominiciens and R. ruminiciens) and eight rumen MAGs representing various strains of R. flavefaciens . The prevalence of these MAGs across various gut metagenomic samples from divergent localities around the world was estimated through read alignment (Figure 2A). The samples originated from ruminant animals (wild and domesticated), human cohorts and different species of monkeys. This revealed that the human- associated genotypes were specific to humans and several primate species (macaques, baboons, gorillas and chimpanzees) but absent from the ruminant samples tested. In addition, the rumen MAGs were specific to ruminants but absent from both the various human and monkey cohorts tested. The rumen MAGs appeared more abundant in their source samples compared to the human and monkey MAGs in their respective samples, suggesting that the rumen environment, which contains a large proportion of plant cell wall material, is a more favorable habitat for such cellulosome-producing species.

These findings support that the R. primaciens, R. hominiciens and R. ruminiciens species comprise novel cellulosome-producing species, which exhibit host association in humans, apes and several species of old-world monkeys (prevalence of 80, 53, 40 and 10,4% in macaques, baboons, gorillas and chimpanzees, respectively). This raises the question of whether an evolutionary process might be driving specific adaptation and potential diversification of these strains within different gut ecosystems. The prevalence of R. primaciens, R. hominiciens and R. ruminiciens varied significantly across the human cohorts. In industrialized countries (Denmark, China, Sweden, Israel and the US), their prevalence was collectively 2.7%, while in hunter gatherers and non-industrialized populations their collective prevalence was 15.8% and 33.3%, respectively (i.e., Hadza huntergatherer and Burkina Faso rural societies). These discrepancies in prevalence could be connected to the difference between diets of individuals in industrialized versus those of non-industrialized countries (processed food versus more raw natural plant fiber) ^25,26. The novel strains were detected in many different geographical locations (Figure 2B), suggesting that although these strains exist in small portions of the human populations, they are widely scattered around the world, which could potentially be connected to dietary habits.

Examination of the prevalence of R. champanellensis genomes across the samples revealed a similar pattern of 2.8% in samples of industrialized countries, 34.6% in samples from huntergatherer and 7.8 % non-industrialized populations. The similar overall prevalence of the novel fiber-degrading species and R. champanellensis strains in human gut samples led the present inventors to investigate the potential exclusion or cooperation processes that would drive the distribution of these species and strains. Analysis of the strain distribution of R. primaciens, R. hominiciens, R. ruminiciens and R. champanellensis revealed that in most of the samples originating from humans of industrial countries, the presence of only a single fibrolytic strain was detected, i.e., either the R. hominiciens, R. champanellensis strain or R. primaciens, suggesting potential competitive exclusion between these species (Figure 2C, right panel). In contrast, human samples from either hunter-gatherer societies or non-industrialized countries, as well as monkey samples, exhibited various combinations of two or more strains of R. hominiciens, R. primaciens and R. champanellensis, which indicates reduced competition that could result from increased availability of fiber-rich diets and/or potentially higher niche availability that results in a higher diversity of fibrolytic strains in these samples (Figure 2C, right panel). Evidence was found for preference and association of the different strains to specific hosts, where R. primaciens was associated with monkeys compared to human samples (Fisher exact test p value <0.00001) and R. hominiciens was significantly associated with humans compared to monkey samples (Fisher exact test p value < 0.0263, Figure 2C, left panel). In addition, R. ruminiciens, which exhibits high similarity to rumen strains (see sections below), was rare in all of the samples (Figure 2C, right panel). Cellulosomes of the ruminococcal strains display intact assembly, fiber-degradation activity and cellulose adhesion

Next, the inventors sought to determine whether the R. primaciens strain, that either is the ancestor or at the same evolutionary scale of the human strains, can degrade crystalline cellulose and carry active assembled cellulosomes, and if so, whether they resemble previously characterized cellulosomes from rumen- and human-associated ruminococcal strains. The analysis revealed that this strain can indeed carry out all of the latter functions. To this end, cardinal structural cellulosomal elements (scaffoldins) and enzymes were identified, that were shared among the R. primaciens, R. hominiciens, R. ruminiciens and R. flavefaciens strains. The binding abilities of 7 cohesin and 6 dockerin modules was measured (Table 2), using the matching fusionprotein approach developed by Barak et al ³¹. A total of 10 positive interactions were identified out of 36 interactions tested that enabled the inventors to estimate the cellulosomal assembly of these modules (Figure 3A). The proposed recognition residues of the ruminococcal dockerins of the different cellulosomal components and the predicted specificities of their interaction with the cellulosomal cohesins lend credence to the proposed scheme of the R. primaciens cellulosome (Figure 3B).

Indeed, the present findings show that the overall organization of the R. primaciens cellulosome, as represented in Figure 3B, resembles the known R. flavefaciens cellulosomal organization in strains isolated from ruminants ³². In both R. primaciens and R. flavefaciens strains, the scaffoldin proteins show a similar interaction pattern, where the dockerins of the ScaA and ScaC scaffoldins interact with the cohesins of ScaB via divergent cohesin-dockerin interactions (see Figure 3A). The cellulosome is attached to the microbial cell wall via the selective cohesin- dockerin interaction between ScaB and ScaE. Furthermore, the dockerin-containing enzymes interact with their cohesin counterparts of ScaA, ScaB and ScaC with divergent specificities. Finally, like ScaB, CttA is integrated onto the bacterial cell wall via a similar type of cohesin- dockerin interaction with ScaE. The ability of cellulosomal components from the two species as well as from R. champanellensis to interact with each other was measured. Cross-species interactions of the cellulosomal components of R. primaciens with representative cohesin-dockerin combinations from R. champanellensis 18P13 and R. flavefaciens FD-1 were found, suggesting an evolutionary conservation of the interaction residues and a certain degree of promiscuity among these components. These findings highlight the similarity and conservation between the functional and assembly potential of these species with regard to the cardinal cellulosomal modules, thus corroborating the notion that human strains produce functional cellulosomes. One of the GH5 cellulase enzymes was selected for biochemical characterization of its cellulolytic activity, since this GH5 gene was common to 25 MAGs of the 30 MAGs used in the present analyses. The GH5 enzyme was found to exhibit cellulolytic activity on microcrystalline cellulose as a substrate (Figure 3C), and its enzymatic activity was in a range similar to that of the R. flavefaciens FD-1 ortholog (68% sequence identity). The CttA protein was purified from R. hominiciens and its ability to bind to cellulose was measured. Robust binding ability to microcrystalline cellulose (Figure 3D) was uncovered, thus indicating that the bacterial cells would bind to cellulose, owing to its interaction with the cell-wall-anchored ScaE (see below and Figure 3B) ³³’³⁴.

R. hominiciens, R. primaciens and R. ruminiciens strains reveal clear functional adaptation to their respective host gut systems

The phylogenetic clustering of R. hominiciens, R. primaciens and R. ruminiciens strains (Figure IB and Figure 2D and E) and the prevalence of these strains across hosts suggest that these strains display host association, raising the question of whether the host association is reflected in the coding capacity of the different strains. To address this, the overall set of R. flavefaciens, R. hominiciens, R. primaciens and R. ruminiciens genomes from the different host ecosystems (14 rumen, 8 human and 8 monkey MAGs) was analyzed. They showed clear host specificity in their gene content and expression pattern, which is also associated with the host’s dietary preferences.

Discussion

Here, the present inventors set to identify novel cellulosome-producing, cellulosedegrading bacteria in the human gut microbiota and address the question of whether such bacteria are ancestral and inherent members of the human gut microbial community. They analyzed nearly 100,000 genomes assembled from metagenomic data ^{17 18}, designed to deepen the understanding of cellulosome-producing bacteria in human gut microbiota. 254 genomes with cellulosomal elements were retrrieved using the scaffoldin gene scaC as a phylogenetic marker ^15,16, and 61 genomes with >90% completion were retained. These were further analyzed for carbohydrateactive enzymes and cellulosomal elements. These findings agree with a previous report ¹⁰ for the presence of a bacterial contig in a human fecal sample that resembled the genome of cellulosome- producing ruminal bacterial species, R. flavefaciens. From the present work, the combination of the scaC and ctA genes emerge as a robust and efficient tool for discriminating close relatives of R. flavefaciens among the cellulolytic and cellulosome-producing bacteria in the human gut microbiome. The cbm37 reporter sequence was utilized to investigate the potential presence of Ruminococcus albus ⁴⁰ in the human gut samples, as this rumen microbe is closely related to R. flavefaciens strains that do not produce cellulosomes. No R. albus cbm37 sequences were found in the samples. This could indicate that microbes that produce cellulosomes are more likely to thrive in the recalcitrant fiber polymer niche within the human gut, potentially due to increased efficiency.

The phylogenetic -based strategy used in the present analysis revealed the presence of novel bacteria, i.e., R. hominiciens, R. primaciens and R. ruminiciens, in the human- and monkey-gut ecosystems, but not in ruminants. This evolutionary analysis suggests that the human strains likely originated from an inhabitant of the primate or ruminant gut. Biochemical analysis showed that these strains express intact, fiber-degrading cellulosomal complexes, similar to those of the ruminal R. flavefaciens strains (Figures 3A-D). The prevalence of the human- and rumen- associated genomes was analyzed in large cohorts of humans, primates, and wild ruminants. While the novel genomes occur in only 2.7% of the human populations in industrialized countries, (i.e. in populations that consume diets rich in processed food), these novel ruminococcal strains were significantly more prevalent in rural human populations and were widespread in old-world monkeys, which consume raw natural plant fiber. Therefore, it is possible that dietary fiber underlies that pattern. R. primaciens was found in more than 80% of the macaque cohort, meeting the criteria of a core microbe ⁴¹. This difference in bacterial prevalence may be due to microbial diversity loss in the human gut stemming from diets rich in processed food and augmented by common antibiotic use, consistent with the disappearing microbiome theory ^{42 45} and urbanization effect ⁴⁶. Human-associated R. hominiciens and R. primaciens genomes were detected in human and monkey fecal samples from various geographic locations, indicating a wide distribution of these bacteria. This is consistent with a recent study that reported a higher distribution of R. champanellensis in ancient human and non-industrialized gut microbiomes compared to industrialized human societies, suggesting that industrialized diets and lifestyles impair the presence and survival of such microorganisms in the human gut ecosystem ⁴⁷. These findings therefore suggest that microbial cellulose degradation was an integral part of the normal, ancestral human microbiome, and that cellulose-degrading bacteria, such as those identified in this study, may be in the process of extinction from the gut microbiomes of industrialized human populations.

Biochemical analysis of multiple cellulosomal components confirmed the presence and proper assembly of the cellulosome in the R. primaciens genome found in the human and monkey gut. Metatranscriptomic analysis of R. hominiciens, R. primaciens, and R. flavefaciens in the gut of humans, monkeys, and ruminants showed high levels of expression of cellulosomal, plant fiber degrading genes and overall gene content that was specific to their respective ecosystems. This suggests that these microorganisms are highly active and play important roles in the functioning of their host ecosystems. Overall, these findings provide insight into the ecological roles of these microorganisms and the importance of studying their activity in different host ecosystems. The ability of these bacteria to degrade microcrystalline cellulose in the human gut may provide a protective effect on the soft tissue of the lower digestive tract from the abrasive cellulose fibers found in fruits and vegetables. These findings also suggest a role of the cellulosome itself as possibly the key mediator of human fiber breakdown, with impact on human health.

The present examples reveals the ecosystem specificity of the R. hominiciens, R. flavefaciens, R. ruminiciens and R. primaciens genomes to the human, rumen, and monkey ecosystems, at both the whole genome level and within their fibrolytic systems. In particular, the present inventors reveal the specificity and functionality of the GH98 gene, which is coded exclusively by R. hominiciens and R. ruminiciens strains and is present in the human gut ecosystem. Most of the R. hominiciens MAGs harbor two copies of the GH98 gene, one cellulosomal and one non-cellulosomal, suggesting its importance in the adaptation to the human gut ecosystem, as it is used in both enzymatic strategies of free and cellulosome- anchored enzymes. The GH98 gene was characterized herein as an enzyme capable of cleaving the backbone of corn arabinoxylan - a prerequisite for the utilization of this polysaccharide, which is a major dietary component of humans ³⁹. The presence of this gene highlights the importance of fiberdegrading bacteria in the human gut microbiota, particularly in the digestion of plant fibers that are commonly consumed in the human diet and may be indicative of the domestication of plants by human populations and the adaptation of these bacteria to the host's lifestyle and diet. A recent report describes the influence of widespread consumption of dietary xanthan gum in industrialized countries on the increased prevalence of an uncultured bacterium from the Ruminococcaceae family of the human gut microbiome ⁴⁸. The present findings involve a very different biochemistry but suggest a similar evolutionary trajectory. The apparent genome adaptation of R. hominiciens, R. flavefaciens and R. primaciens to their host lifestyle is attributed to standard gene acquisition from their surrounding microbiome members. This is consistent with the suggestion that ruminococcal species in the human gut owe their different substrate preferences to acquisition of genes from microbial inhabitants in their environment⁴⁹.

The findings suggest that ruminants and monkeys served, and serve, as a source-reservoir for important cellulosome-producing ruminococcal strains, which continue to invade and adapt to the human gut ecosystem. This is highlighted by the human-assembled MAGs of R. primaciens and R. ruminiciens, which seem to be in the transitional stages of transfer from the monkey and rumen gut ecosystems to the human intestine, respectively. The example sheds light on the widespread prevalence of cellulosome-producing, fiber-degrading ruminococcal strains across plant fiber-utilizing mammalian gut systems, their ecosystem specificity and their potential adaptation to the host's lifestyle and diet. The findings bear potential implications for human health and probiotics, as they suggest that although these strains are disappearing from the human gut, they could potentially be reintroduced or enriched through targeted diets and specific probiotics once the strains are available in pure cultures.

Although the 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. It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.

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Claims

WHAT IS CLAIMED IS:

1. A probiotic composition comprising at least one isolated fibrolytic bacterial strain having a 16S rRNA sequence at least 92 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, wherein said strain carries in its genome the genes scaC and ctA, wherein the composition comprises no more than 150 species of bacteria.

2. A probiotic composition comprising at least one fibrolytic bacterial strain which comprises a genome having contigs at least 90 % identical to SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937, wherein the composition comprises no more than 150 species of bacteria.

3. The probiotic composition of claims 1 or 2, wherein said bacterial strain expresses CttA protein which binds to cellulose.

4. The probiotic composition of claims 1 or 2, wherein said bacteria strain comprises an assembled cellulosome.

5. The probiotic composition of claims 1 or 2, wherein said bacterial strain expresses the endoglucanase Cel5A which is capable of degrading cellulose.

6. The probiotic composition of claims 1 or 2, wherein said bacterial strain expresses the glycoside hydrolase GH98 capable of degrading corn arabinoxylan.

7. The probiotic composition of claim 1, wherein the isolated fibrolytic bacterial strain has a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5.

8. The probiotic composition of claim 1, wherein the isolated fibrolytic bacterial strain has a 16S rRNA sequence at least 99 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5.

9. The probiotic composition of any one of claims 1-8, wherein the composition comprises no more than 100 species of bacteria.

10. The probiotic composition of any one of claims 1-9, wherein the composition comprises no more than 50 species of bacteria.

11. The probiotic composition of any one of claims 1-10, wherein said at least one isolated fibrolytic bacterial strain is capable of residing in the human gut microbiome.

12. A probiotic composition comprising at least one fibrolytic bacterial strain isolated from a human gut, wherein the composition comprises no more than 50 species of bacteria.

13. The probiotic composition of claim 12, wherein said at least one bacterial strain has a 16S rRNA sequence at least 92 % identical to SEQ ID NOs: 1, 2, 3, 4, or 5.

14. The probiotic composition of claim 12, wherein said fibrolytic bacterial strain comprises a genome having contigs at least 90 % identical to SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937.

15. The probiotic composition of any one of claims 1-13, wherein the composition comprises at least two isolated non-identical bacterial strains, wherein a first strain has a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, and a second strain has a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, wherein both of said bacterial strains carry in their genomes the genes scaC and ctA.

16. The probiotic composition of any one of claims 1-15, comprising no more than 20 species of bacteria.

17. The probiotic composition of any one of claims 1-16, being lyophilized.

18. The probiotic composition of any one of claims 1-16, formulated as a capsule, a tablet, dry powder, a suppository, a food or beverage.

19. The probiotic composition of any one of claims 1-18, further comprising a preservative that preserves the activity of the bacteria.

20. The probiotic composition of any one of claims 1-19, further comprising a prebio tic.

21. The probiotic composition of claim 20, wherein said prebiotic composition comprises cellulose.

22. The probiotic composition of claim 21, wherein said cellulose is crystalline cellulose and/or hemicellulose.

23. A bacterial culture comprising at least one bacterial strain having a 16S rRNA sequence at least 92 % identical to SEQ ID NOs: 1, 2, 3, 4, or 5, or comprising a genome having contigs at least 90 % identical to SEQ ID NOs: SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937, wherein said strain expresses the genes scaC and ctA, wherein the culture comprises no more than 150 species of bacteria.

24. The bacterial culture of claim 23, comprising no more than 50 species of bacteria.

25. The bacterial culture of claim 23, being a monoculture.

26. The bacterial culture of any one of claims 23-24, wherein the culture is devoid of bacteria isolated from a rumen microbiome.

27. The bacterial culture of any one of claims 23-26, wherein the bacteria of the culture are derived from a human microbiome sample.

28. A method of maintaining the health of a subject comprising administering to the subject an effective amount of a composition comprising:

(a) at least one isolated fibrolytic bacterial strain:

(i) having a 16S rRNA sequence at least 90 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, or

(ii) comprising a genome having contigs at least 90 % identical to SEQ ID NOs: SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462- 550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937, wherein said bacterial strain expresses the genes scaC and cttA; or

(b) the probiotic composition of any one of claims 1-19, thereby maintaining the health of the subject.

29. The method of claim 28, further comprising administering to the subject a prebiotic composition which enhances the activity or amount of said bacterial strain.

30. The method of claim 28, wherein said prebiotic composition comprises cellulose /or hemicellulose.

31. The method of claim 30, wherein said cellulose /or hemicellulose is crystalline cellulose and/or com arabinoxylan.

32. The method of claim 28, wherein the subject is a healthy subject.

33. The method of claim 28, wherein the subject is a non-healthy subject.

34. A method of treating a disease selected from the group consisting of an inflammatory disease, a metabolic disease, cancer and a cardiovascular disease, the method comprising administering to the subject a therapeutically effective amount of:

(i) at least one isolated fibrolytic bacterial strain having a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4, or 5;

(ii) at least one isolated fibrolytic bacterial strain comprising a genome having contigs at least 90 % identical to SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937, wherein said strain expresses the genes scaC and cttA; or

(iii) the probiotic composition of any one of claims 1-19, thereby treating the disease.

35. The method of claim 34, wherein said metabolic disease is Diabetes or obesity.

36. The method of claim 34, wherein said cancer is colon cancer.

37. The method of claim 34, wherein said inflammatory disease is inflammatory bowel disease and/or colitis.

38. A method of reducing weight of a subject comprising administering to the subject an effective amount of:

(i) at least one isolated fibrolytic bacterial strain having a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4, or 5, wherein said strain expresses the genes scaC and ctA

(ii) at least one isolated fibrolytic bacterial strain comprising a genome having contigs at least 90 % identical to SEQ ID NOs: SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937, wherein said strain expresses the genes scaC and cttA; or

(iii) the probiotic composition of any one of claims 1-19, thereby reducing weight of the subject.

39. The method of claim 38, wherein the subject has a BMI greater than 25.

40. The method of claim 38 or 39, wherein the subject is on a weight loss program.

41. The method of claim 40, wherein said weight loss program comprises an exercise program.

42. The method of claim 40, wherein said weight loss program comprises a reduced caloric intake.

43. A method of identifying probiotic bacteria useful for maintain the health of a human subject comprising analyzing for the presence of a bacteria in a sample of the gut microbiome of a human subject, wherein a bacteria which express both the genes scaC and cttA is indicative that the bacteria is a probiotic useful for maintaining the health of the human subject.

44. The method of claim 43, further comprising isolating the bacteria following the identifying.

45. The method of claim 43, wherein the sample is a fecal sample.

46. The method of claim 45, wherein the analyzing comprises sequencing DNA or RNA derived from the sample.

47. The method of claim 43, wherein the analyzing comprises determining 16S rRNA sequence of the bacteria.

48. The method of claim 43, further comprising storing the sample following the identifying.

49. The method of any one of claims 43-48, further comprising qualifying the gut microbiome of the subject upon identification of the bacteria which expresses both the genes scaC and cttA.

50. An article of manufacture comprising:

(a) at least one isolated fibrolytic bacterial strain having a 16S rRNA sequence at least 95 % identical to SEQ ID NOs: 1, 2, 3, 4, or 5, wherein said strain expresses the genes scaC and ctA; and

(b) a weight-reducing agent.

51. The article of manufacture of claim 50, wherein said at least one isolated fibrolytic bacterial strain and said weight-reducing agent are formulated in a single composition.

52. The article of manufacture of claim 50, wherein said at least one isolated fibrolytic bacterial strain and said weight-reducing agent are formulated in separate compositions.

53. An isolated fibrolytic bacterial strain having a 16S rRNA sequence at least 92 % identical to SEQ ID NOs: 1, 2, 3, 4 or 5, wherein said strain carry in their genomes the genes scaC and ctA.

54. An isolated fibrolytic bacterial strain which comprises a genome having contigs at least 90 % identical to SEQ ID NOs: 30-262, SEQ ID NOs: 263-390; SEQ ID NOs: 391-461; SEQ ID NOs: 462-550, SEQ ID NOs: 551-637; SEQ ID NOs: 638-687; SEQ ID NOs: 688-824; or SEQ ID NOs: 825-937.