Functional compositions and methods of use thereof in eradication and / or decolonization of antibiotic-resistant bacteria and restoration of healthy microbiota

Abstract

The present disclosure relates to bacterial compositions having specific genotypes that are useful in antibiotic-resistant bacteria (ARB) decolonization, eradication, and colonization prevention via establishing a "colonization resistance" mechanism and (but not exclusively) simultaneously restoration of a healthy microbiome to prevent antibiotic-resistant bacteria and other (non-resistant and non-bacterial) invasive infections. More specifically, the bacterial compositions provided herein have been designed to exhibit specific properties (functions) useful in the clinical settings described above.

Description

[0001] FUNCTIONAL COMPOSITIONS AND METHODS OF USE THEREOF IN ERADICATION AND / OR DECOLONIZATION OF ANTIBIOTIC-RESISTANT BACTERIA AND RESTORATION

[0002] OF HEALTHY MICROBIOTA

[0003] REFERENCE TO A SEQUENCE LISTING

[0004] This application includes a Sequence Listing with 4794 sequences submitted electronically as a text file named “231040-2 sequence listing”, created on November 4, 2024, with a size of 9930 kilobytes. The sequence listing is incorporated by reference.

[0005] FIELD OF INVENTION

[0006] The present disclosure relates to bacterial compositions having specific genotypes that are useful in antibiotic-resistant bacteria (ARB) decolonization, eradication, and colonization prevention via establishing a “colonization resistance” mechanism and (but not exclusively) simultaneously restoration of a healthy microbiome to prevent antibiotic-resistant bacteria and other (non-resistant and non- bacterial) invasive infections. More specifically, the bacterial compositions provided herein have been designed to exhibit specific properties (functions) useful in the clinical settings described above. All related methods and uses are also disclosed.

[0007] BACKGROUND OF THE INVENTION

[0008] The overuse and misuse of antibiotics, not only in healthcare but also in agriculture, animal husbandry, and other sectors, have inadvertently fostered the emergence and rapid spread of antibiotic resistance. The consequences are profound, significantly impacting medical interventions and prolonging hospitalizations due to infections, especially antibiotic-resistant ones. As the World Health Organization (WHO) has emphasized, antimicrobial resistance, encompassing antibiotic resistance, is one of the most critical threats to human health. Furthermore, the O'Neill report underscores that failure to address this issue could result in an alarming 10 million annual deaths due to antimicrobial-resistant infections by 2050, exceeding fatalities from cancer or traffic accidents.

[0009] It is scientifically proven that 70% of [systemic] bacterial, including antimicrobial resistant (“AMR”) infections originate in the gut microbiome, a bacterial ecosystem that can be thought of, and functions like an organ system. When the gut microbiome becomes unbalanced, either due to disease or unhealthy lifestyle or, mostly, antibiotic therapy, it is susceptible to becoming colonized with antibioticresistant bacteria. In favorable conditions, bacteria translocate through the gut wall to the blood or lymphatic system and cause systemic (sepsis) or localized (if the bacteria flow to the organs) infection. The gut microbiome is the largest reservoir of bacteria in the human body, and obviously is also a reservoir for ARB.

[0010] To date, no active eradication or decolonization strategies are generally accepted as valid. Current efforts to curb antibiotic resistance primarily focus on restricting antibiotic use and enhancing surveillance in various environments. Apart from diet, there is no clear strategy to maintain anti-ARB colonization resistance and its restoration if the microbiome is disrupted. In light of these challenges, it is imperative to recognize the need for innovative and effective therapeutic approaches and eradication, decolonization strategies which should offer a promising frontier in the battle against antibiotic resistance.

[0011] Eradication and decolonization efforts could involve the removal or decrease of antibiotic-resistant bacteria from the human gut using a variety of techniques. Typical techniques include selective gut decontamination, diet, probiotics, phage therapies. In addition, faecal microbiota transplantation (FMT) have shown very promising results. FMT is composed, as a faecal filtrate, of mostly unidentified matter, and could not be designed to exert specific functions - all matters from the donor’s stool composition. Live Biotherapeutic Products (LBPs), a relatively new medicinal product category, consisting of live bacteria, are strictly designed in terms of composition (in most cases, but not always, e.g. the equivalent of FMT is "standardized FMT", which, despite maintaining certain standards, is not fully characterized) being equivalent of FMT. To this day, the known LBPs are products with a maximum of a dozen or several dozen strains, which - if have a biological effect (all of them are in clinical trials and the results and effectiveness of their treatment are not known yet) - do not rebuild the full spectrum of the intestinal microbiome, and also the protective mechanisms, such as “colonization resistance", which is necessary to restore full health. These products have a chance to cure the disease, but without curing the patient and restoring the patient’s full well-being.

[0012] In view of no treatment options currently available, with FMT being the most potent one, improved methods of eradication, decolonizing and preventing ARB colonization with a healthy microbiome restoration properties are needed. There is a need to replace FMT with scalable and safe therapies.

[0013] Antibiotic-resistant bacteria (ARB) is a bacterium which evolve mechanisms that protect them from antimicrobials, which are drugs used to treat infections (mostly antibiotics). Antibiotic resistance is a subset of antimicrobial resistance. Microbiological resistance to antibiotics occurs from genes, mutated or inherited, that allow the bacteria to resist the mechanism to kill the microbe associated with certain antibiotics. This means the bacteria can pass the genetic catalyst for resistance through horizontal gene transfer: conjugation, transduction, or transformation. This allows the resistance to spread across the same species of pathogen or even similar bacterial pathogens. To eliminate ARB there is then a need to kill / eliminate the ARB bacteria or antibiotic-resistance genes (ARG). Eradication means that a bacterium possessing ARG and / or ARG itself are completely removed from the digestive tract.

[0014] Decolonization means that a bacterium possessing ARG and / or ARG itself are significantly decreased in abundance within the digestive tract.

[0015] “Colonization resistance” restoration means that a natural mechanism whereby the human microbiome protects itself against incursion by new and often harmful microorganisms (e.g. ARB) was restored.

[0016] WO2019032572A1 discloses compositions and methods for delivery of novel mixtures of bacterial strains for the decolonization and / or eradication of various pathogenic bacteria and, particularly, antibiotic-resistant bacteria (ARB).

[0017] EP3074027A1 discloses therapeutic compositions containing microbial populations for prevention, treatment and reduction of symptoms associated with a dysbiosis of a mammalian subject such as a human.

[0018] EP3388069B1 discloses novel compositions comprising specific consortia of living bacteria strains, methods of manufacturing of such compositions, particularly by co-cultivation; and use of such compositions in pharmaceutical applications, such as the treatment of diseases associated with intestinal microbiota dysbiosis, particularly intestinal infections such as CDI (Clostridium difficile infection) and IBD (Inflammatory bowel diseases)

[0019] SUMMARY OF THE INVENTION

[0020] In view of the previous findings, it is necessary to determine some minimum requirements that must be met in order to achieve the desired therapeutic effect (ARB eradication, decolonization, antiARB colonization resistance restoration with infection prevention properties) and to rebuild the full microbiome ecosystem. The present invention is aimed at addressing this need. The application developed here differs from previous ones by indicating a specific functional potential set, the delivery of which (by means of a bacterial consortium) will bring a therapeutic effect and restore microbiome ecosystem.

[0021] The method proposed by the present invention involves the production of Next Generation Biotherapeutics (NGB), which are based on creating artificial bacterial ecosystems composed of cultured bacteria, isolated (at least in vast majority of cases) from donors whose FMT products were previously tested in proof-of-concept studies and are proven to be effective - this means the “strain specificity” phenomenon preservation and are designed in silico to exert the desired biological effect, e.g. "colonization resistance" to ARB (as in this case) and rebuild the full spectrum of the functional microbiome, ensuring patient well-being and minimizing the risk of any negative microbiome-related events in the future. The NGBs are a new vision of LBP, realistically introducing a biosynthetic, donorindependent microbiome in the form of a "smart drug," i.e. a drug that restores well-being but in the same time is designed to exert a specific function against the disease or status (such as ARB colonization) it treats.

[0022] This disclosure describes efforts aiming to propose elements such as bacterial taxa and certain functions coded in their genomes that hold promise for inclusion in an LBP with the objective of eradicating, decolonizing antibiotic-resistant bacteria from the human gut and rebuilding colonization resistance mechanism against them with (but not exclusively) healthy microbiota restoration.

[0023] The present invention is based on the finding that changes in specific functions provided by bacteria in the ARB-colonized or ARB colonization-prone patient's microbiome following FMT correlate with the reversal of above stated, and further with rebuilding the full microbiota ecosystem. Consequently, the clinical effect depends solely on the engrafted bacteria.

[0024] The invention is based on the inventors’ finding that recipient post-FMT microbiota consist of bacteria remaining in recipient’s gut during therapy, coming from donor samples (FMT) and other environmental sources (i.e diet).

[0025] The invention is based on the inventors’ finding that the failure to detect strains at a given time point is not evidence that those strains are not present in the sample. They remain below the detection threshold. Bacteria being below the detection threshold is equivalent to very low abundance, which limits the impact of such bacteria on the final clinical outcome in the patient.

[0026] The invention is based on the inventors’ finding that the entire set of FMT bacteria is not required to obtain therapeutic effect. In other words, only a subset of the donor strains are confirmed to contribute to the therapeutic effect. According to the literature and inventors’ findings, there has never been a recorded case where all the strains from a donor were engrafted. The engrafted strains constitute on average about 20% of all detected recipient’s strains (see Figure 1).

[0027] The invention is based on the finding that changes in specific functions provided by bacteria in the patient's microbiome colonized with ARB and / or ARG following FMT correlate with eradication or decolonization of ARB and / or ARG.

[0028] The invention is based on the finding that changes in specific functions provided by bacteria in the patient's microbiome following FMT correlate with preventing colonization of ARB and / or ARG.

[0029] The invention is based on the finding that there is a set of functions (in the context of KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology functions, hereinafter referred to simply as functions or KOs) that are missing in colonized / colonization-prone patients. References to the KOs provided in this description are compliant with the KEGG database release 110.0 of 1 April 2024, published at https: / / www.genome.jp / kegg / . There are functions present in healthy and dysbiotic microbiomes that do not have an impact on the development of the invention. More than half of the functions present in healthy individuals are functions occurring in every single healthy person. These functions have a high prevalence, which means that they occur on many bacteria, including pathogenic ones. Therefore, only functions being statistically significantly different between healthy and disrupted microbiomes were selected (see Figure 2). These functions were considered as potential components of functional consortia.

[0030] The inventors determined a set of functions, herein referred to as the first set, detected on strains engrafted in recipient microbiome from donors, and those functions are shared between eradicated and / or decolonized patients but not in not-eradicated or not-decolonized patients (see Table 1). Providing an LBP (NGB) or FMT consortium covering the first set of functions ensured eradication and / or decolonization of ARB and / or ARG.

[0031] The inventors determined a set of functions, herein referred to as the second set, detected on strains engrafted in recipient microbiome from donors, and those functions are shared between “colonization resistant” and (see Table 2). Providing an LBP (NGB) or FMT consortium covering the second set of functions in combination with the first set of functions ensured post-colonization prevention of ARB and / or ARG.

[0032] The inventors also determined a third set of functions detected on strains engrafted from donors, shared between patients with restored healthy microbiome (see Table 3). Providing an LBP (NGB) or FMT consortium covering functions provided as a third set in combination with the first set of functions ensures restoration of a healthy microbiome (based on metrics and normatives provided below).

[0033] In this patent application, the term "bacteria" is used expansively to include both typical bacterial organisms and archaea. This interpretation considers that while archaea and bacteria are distinct domains of life with unique biochemical and genetic characteristics, both share certain cellular structures and functional roles that are relevant to the intended molecular functions in this composition. Therefore, for the purposes of this application, "bacteria" is understood to encompass archaea as well, thereby including any prokaryotic organisms capable of performing the specified molecular functions within the described composition.

[0034] Selecting a minimal functional set providing clinical goals and full microbiome restoration should be considered as an optimization method, aimed at determining the minimal composition (as functional or taxonomical) with a given therapeutic effect, however, any consortium larger than the minimal one (with redundant genes coding above mentioned functions), having proposed first set of functions or the first set combined with the second set, the third set or both the second set and the third set, should still be considered as within the scope of the present invention (as even having better stability and resilience). The results were confirmed in silico, which is a generally accepted premise for drawing reliable conclusions by experts, as described by Duncker, K.E., Holmes, Z.A. & You, L. in “Engineered microbial consortia: strategies and applications” (Microb Cell Fact 20, 211 (2021). https: / / doi.org / 10.1186 / sl2934-021-01699-9).

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The figures are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labelled in every drawing. In the drawings:

[0037] Figure 1 shows a percentage distribution of engrafted and not engrafted strains for selected 10 samples with highest engrafted strains ratio. This results shows that only part of strains in post FMT recipients come from donors.

[0038] Figure 2 shows differences in functional potential (KOs) in healthy donor samples and pre-FMT patients, identified using a pangenome (all functions, even detected on unbinned contigs), binary approach. The Kruskal -Wallis test was applied to determine differences in occurrence of KOs between groups, with p-values corrected using the Benjamini -Hochberg (BH) method.

[0039] Figure 3 shows Scatter Plots showing correlation between number of species classified using MAG approach and Kraken approach in all samples. Value of R represents the correlation using Pearson. The figure shows that there is a moderate correlation between the number of species identified by Kraken approach and the number of MAGs in donor samples. The strongest correlation has been observed on the family level (RA2 = 0.77).

[0040] Figure 4 shows a comparison of unique functions counts identified in donors, patients pre-FMT and cured patients post-FMT.

[0041] Figure 5 shows a comparison of in-house and public data in terms of quality for metagenomic analyses. X-axis represents sequencing depth in bp, Y-axis represents unique functions (KO) count. The results show a significant difference in sequencing depth and quality what correlates with the number of unique features detected, in favor of in-house data.

[0042] Figure 6 shows a principal component analysis of functional potential (KOs) of selected MAGs and ATCC strains. X-axis represents the first principal component, Y-axis represents second principal component. The results show strong clustering of MAGs and ATCC strains from given taxonomy, but high variance between strains in clusters. These results make it possible to move from general analyses to very detailed analyses at the level of individual strain differences.

[0043] Figure 7 shows taxonomic diversity between in-house donor samples. These results show that most taxa at the species level differ between samples. Figure 8 shows an overlap between the first set, second set and third set. There is a minimal overlap between sets.

[0044] Figure 9 shows a comparison of engrafted functions from the first set between patients with eradication (n=l 1) and persistence (n=8) of ARB and / or ARG. These results demonstrate the discovery of a limited set of functional potential necessary to achieve full eradication / decolonization.

[0045] Figure 10 shows a comparison of engrafted functions from the second set between patients with colonization resistance (n=17) and post colonization of ESKAPE+ species with antibiotic resistance mechanisms (n=20). These results demonstrate the discovery of a limited set of functional potential necessary to achieve colonization resistance.

[0046] Figure 11 shows a comparison of engrafted functions from the third set between restored (n=17) and non-restored (n=20) healthy microbiome triads. These results demonstrate a set of functional potential necessary to achieve restoration of a healthy microbiome.

[0047] Figure 12 shows a Principal Component Analysis (PCA) of engrafted bacterial compositions (derived from FMT), computed NGB consortia and other artificial consortia.

[0048] DETAILED DESCRIPTION

[0049] In the following passages, different aspects of the invention are defined in more detail. Each aspect described herein may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0050] Analyses were performed to determine the presence of antibiotic-resistance mechanisms among patients treated with FMT in a proof-of-concept studies. Many bacteria carrying resistance mechanisms (ARB - Antibiotic Resistant Bacteria) were identified, as well as resistance mechanisms that could not be assigned to specific bacterial strains (ARG - Antibiotic Resistant Genes).

[0051] There are many genes classified as antibiotic-resistance mechanisms, but only some of them have phenotypic properties. This is currently regulated by the World Health Organization (WHO). The list of resistance mechanisms analyzed herein is given in Table 3.

[0052] Resistance to antibiotics can be carried by various bacteria, including non-pathogenic bacteria. However, the dissemination of resistance mechanisms should always be avoided. WHO also indicates a list of bacteria of special concern, which is the main therapeutic target for the Inventors - mainly composed by the ESKAPE bacteria list (Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas and Enterobacter; ESKAPE is a commonly used abbreviation of the most dangerous bacteria in the clinical environment, especially antibiotic-resistant ones). For the purposes of internal research, the inventors have created an extended list of bacteria that pose a clinical threat, especially those that often carry resistance genes to clinically relevant antibiotics, including: ESBL-resistant bacteria (Extended-Spectrum P-Lactamases), CRE (carbapenem-resistant Enterob acteriaceae, sometimes (if particular resistance gene is identified) referred to as carbapenemase- producing Enterobacteriaceae - CPE), VRE (vancomycin-resistant enterococci), MDR (multidrug resistance), XDR (extensively drug resistant), PDR (pandrug-resistant) or other clinically relevant mechanisms like cephalosporin or fluoroquinolones resistance mechanisms, which for the purpose of this patent application are referred to as ESKAPE+. Additionally, a list of ARB containing all species for which ARG were detected in in-house data was prepared. Species assigned to the above-mentioned lists are presented in Table 5.

[0053] Patients were analyzed in “triads” (the term explained in detail below) to determine the effect of FMT on the eradication and / or decolonization of ARB and / or ARG. Eradication, as stated above, was defined as identification of ARB and / or ARG in the pre-FMT patient’s sample which was no identified in the 30-days-post-FMT sample, and decolonization was defined as identification of ARB and / or ARG in the pre-FMT patient’s sample which relative abundance significantly dropped in the 30-days-post- FMT sample or other post-FMT sample if 30-days-post-FMT sample was not available.

[0054] Even use of techniques as accurate as very deep ONT (Oxford Nanopore Technologies) sequencing does not always allow to bin the operons responsible for resistance mechanism with full bacterial genomes. Therefore, two levels of eradication and decolonization of resistance mechanisms were distinguished, at the strain level (ARB) and at the gene level (ARG). At the strain level, Metagenome-Assembled Genomes (MAGs, described in detail below) with identified resistance mechanisms were defined. To assess eradication and / or decolonization MAGs were compared between samples in a given triad using fastANI software to determine their similarity. For ARG, contigs were compared using BLAST software.

[0055] It has been observed that in some cases FMT administration causes eradication and / or decolonization of ARB and ARG (see Table 7). Additionally in some cases, FMT confers postcolonization resistance to ARB and ARG, which are defined herein as the status of the microbiota that contains the appropriate functions generating “colonization resistance” if particular ARB was introduced to this microbiota (gastrointestinal tract) and persistent colonization does not occur. For the purposes of this study, the post-colonization resistance is treated as equivalent to the most proven case of “colonization resistance”, as confirmed by a real example of the introduction of a resistant bacterium (e.g. during FMT or accidentally around FMT with food) and it fail to engraft (see Table 8).

[0056] The invention relates to the therapeutic bacterial compositions, each comprising one or more bacteria, e.g. a consortium of defined bacterial isolates. The compositions are useful in the ARB and / or ARG eradication and / or decolonization and / or prevention of colonization with ARB and / or ARG (post- colonization resistance as “colonization resistance”) and (but not exclusively) in restoration of a healthy microbiome.

[0057] The bacterial composition is selected based on the ability of the live bacterial product to induce or stimulate a desired response when administered to a subject (e.g., an ARB colonized patient).

[0058] The invention relates to compositions comprising a plurality of bacteria, e.g. a consortium of defined bacterial isolates (LBP; NGB) or a consortium of bacteria in the form of gut microbiota obtained from a donor (e.g. as a stool sample) for faecal microbiota transplantation (FMT) procedure. Plurality of bacteria present in the composition comprises plurality of genes encoding plurality of molecular functions. The compositions are useful in the ARB and / or ARG eradication and / or, decolonization and / or prevention of colonization with ARB and / or ARG and (but not exclusively) in restoration of a healthy microbiome..

[0059] Isolated bacteria can be (and to achieve “strain specificity phenomenon” preservation must be) obtained from gut microbiota obtained from a donor whose faecal microbiota transplantation (FMT) preparations were used in proof-of-concept studies showing its therapeutic efficacy. Bacterial strains with beneficial therapeutic properties that are capable of engrafting into the recipient microbiota can be identified based on metagenomic analyses, which allow (by high quality, deep sequencing) for the indication of MAGs belonging to the same strains that occurred in the donor material and later engrafted into the recipient microbiota. These strains can then be identified among the isolated strains from the donor material based on genomic analyses (e.g. average nucleotide identity (ANI) analysis or DNA- DNA digital hybridization (dDDH)). Then, the strains identified in this way with beneficial properties can become a component of a bacterial composition consisting of isolated bacteria. In such a composition, isolated bacteria from one donor can be combined, but it is also possible to combine isolated bacteria from multiple donors if a particular strain in different donors exhibits better functions or more abundant functions than in other donors. Combining isolated bacterial strains from different donors allows for an increase in the diversity of the composition and can help increase the effectiveness of therapy using such a bacterial composition.

[0060] In a preferred, but non-limiting embodiment, the bacterial compositions of the invention include isolated bacteria and are not therefore faecal microbiota transplants (FMT). They do not contain faecal material, but contain defined mixtures of bacterial isolates free of faecal material. Preparations that contain a defined bacterial mixture are generally accepted to be a safer treatment than FMT. An advantage of the present composition in such an embodiment is that it comprises only fully defined and characterized bacteria proven to exert particular biological role in a previously performed proof-of- concept FMT study (the same strains provided in FMT from the in-house donor biobank compose the final drug composition) and no undefined or unwanted components, which may be present in donor stools, thereby allowing the therapeutic composition to be standardized and increase safety of the composition, and additionally preserving the “strain specificity” phenomenon. Thus, in some aspects relating to bacterial compositions, the invention may exclude the administration of faecal transplants.

[0061] As used herein, the term “species” refers to a taxonomic entity as conventionally defined by genomic sequence and phenotypic characteristics. A “strain” is a particular instance of a species from a given sample, for which genome assembly (MAG) was prepared and taxonomy was assigned. Additionally unbinned contigs were also included in analysis as an undefined bacteria containing a specific set of functional properties.

[0062] There is a problem with defining the parameters of a healthy microbiome. Scientists obtain different results, e.g. the number of bacteria detected in the human gut. This has been discussed in a number of publications:

[0063] Yang J, Pu J, Lu S, Bai X, Wu Y, Jin D, Cheng Y, Zhang G, Zhu W, Luo X, Rossell6-M6ra R, Xu J. “Species-Level Analysis of Human Gut Microbiota With Metataxonomics” (Front Microbiol. 2020 Aug 26; 11 :2029. doi: 10.3389 / fmicb.2020.02029. PMID: 32983030; PMCID: PMC6479098), Gupta VK, Kim M, Bakshi U, Cunningham KY, Davis JM 3rd, Lazaridis KN, Nelson H, Chia N, Sung J. “A predictive index for health status using species-level gut microbiome profiling” (Nat Commun. 2020 Sep 15;11(1):4635. doi: 10.1038 / s41467-020-18476-8. PMID: 32934239; PMCID: PMC6492273),

[0064] Sheng Y, Wang J, Gao Y, Peng Y, Li X, Huang W, Zhou H, Liu R, Zhang W. “Combined analysis of cross-population healthy adult human microbiome reveals consistent differences in gut microbial characteristics between Western and non-Western countries” (Comput Struct Biotechnol J. 2023 Nov 28;23: 87-95. doi: 10.1016 / j .csbj .2023.11.047. PMID: 38116074; PMCID: PMC9730331), Quigley EM “Gut bacteria in health and disease” (Gastroenterol Hepatol (N Y). 2013 Sep;9(9):560- 9. PMID: 24729765; PMCID: PMC3983973).

[0065] This is due to differences resulting from sequencing depth, sequencing technology, used software and adopted thresholds. Therefore, several definitions and normatives of the healthy microbiome will be defined for the purposes of the present description, as elaborated in detail below. This allows for the diagnosis of each sample and identification of whether the patient has a healthy or abnormal microbiome (see. Table 9). It is particularly difficult to determine the exact number of bacteria in a healthy sample. Metagenome-assembled genomes (MAGs) and Kraken-detected (species detected using Kraken software) strains / species counts will not perfectly reflect the real number of bacteria in a healthy microbiome. MAGs underrepresent and Kraken-detected species overrepresent a real number of species in a sample. However, there is a correlation (see Figure 3) between these counts. Both metrics will be therefore included to describe a healthy microbiome. The advantage of the approach presented herein lies in focusing on functions rather than on composition of bacteria in the context of taxonomic names, in contrast to descriptions commonly presented in other patent applications. Functions, rather than just the composition of the microbiome, are a key element in the functioning of the human gut ecosystem. It is widely known that the same functions can be encoded by completely unrelated bacteria and still possess the same benefits to gut microbiome. Bacterial compositions presented herein, that encode a set of functions present in a healthy microbiome, i.e., glycosylation of mucins, short-chain fatty acids production, biotransformation of bile acids, optimal carbon source metabolism, ascorbate metabolism, methane metabolism, degradation of benzoate and aromatic compounds, porphyrin metabolism and biosynthesis of amino acids encoded by protein, disclose a set of KOs that are crucial for ARB and / or ARG eradication and / or, decolonization and / or prevention of colonization with ARB and / or ARG and (but not exclusively) in restoration of a healthy microbiome.

[0066] Donors with different taxonomic compositions produce the same positive clinical outcome. Therefore, taxonomic analyses do not allow defining patterns and principles responsible for patient cure. Functional analysis of the microbiome has enabled the determination of common features that are the basis for creating effective consortia for ARB and / or ARG eradication and / or, decolonization and / or prevention of colonization with ARB and / or ARG and (but not exclusively) restoration of a healthy microbiome, which is the primary inventors’ finding revealed here. It is worth mentioning that the diversity or large number of functions itself may be related to the pathological status, not health (see Figure 4). In the FMT treatment procedure and the method proposed herein, it is important to deliver / engraft specific functions that cause a therapeutic effect.

[0067] As explained herein, the bacterial compositions of the invention have a therapeutic effect when administered to a subject and can be used in the ARB and / or ARG eradication and / or, decolonization and / or prevention of colonization with ARB and / or ARG and (but not exclusively) in restoration of a healthy microbiome. Thus, the compositions as described herein are therapeutic / prophylactic compositions. Thus, the invention also extends to pharmaceutical compositions comprising a composition of bacteria as described herein and methods of use thereof.

[0068] Identification of therapeutic components in total stool microbiota allows the identification of donors and stool samples with increased therapeutic / prophylactic efficacy.

[0069] All donor samples used in proof-of-concept studies were, if included in colonization resistance mechanisms unraveling, free of any clinically relevant (as were identified in ESKAPE+ group) ARB. It was confirmed with both methods: genomic (PCR (Polymerase Chain Reaction) and deep ONT NGS (Oxford Nanopore Technologies Next Generation Sequencing)) and culture. Therefore it is plausible that their microbiome composition on both functional and taxonomic levels grants colonization resistance against ARB and / or ARG and hence was used to determine functional and (afterwards) taxonomic sets of compositions granting colonization resistance against ARB and / or ARG.

[0070] Samples used for analysis were sequenced using ONT and very deep (>20 Gbp per sample). Methods generally accepted by experts skilled in the art and in-house tools, were used to process these data. ONT allows obtaining results with high accuracy, thanks to the very long reads guaranteed by that technology (see Figure 5). Nanopore technology, which enables long-read sequencing, is particularly advantageous for reconstructing complete bacterial genomes from metagenomic samples. Unlike shortread sequencing technologies, which often struggle with resolving repetitive regions and complex genome structures, long reads generated by nanopore sequencing can span these difficult areas, allowing for more accurate assembly of full genomes. This is especially important in metagenomics, where DNA from multiple organisms is mixed, and achieving high-resolution assemblies is critical for identifying and characterizing microbial species and their functions. As a result, nanopore sequencing enhances the ability to reconstruct complete bacterial genomes with greater continuity and fewer gaps. The inventors found that the metagenome-assembled genomes (MAGs) reconstructed from the samples used, in most cases, contain fully recovered genomes when compared to reference genomes from the same bacterial species. By aligning and comparing the assembled MAGs with these reference genomes, a high degree of completeness and structural accuracy was observed, confirming that the long-read sequencing approach successfully captures the full genomic content of the bacteria present in the samples used. This validation highlights the robustness of the reconstruction process in accurately representing bacterial genomes (see Figure 6). This also allows for drawing reliable conclusions.

[0071] All analyzed samples were divided into “triads” (cases), which means sets of at least 3 samples from a single treatment. A triad consists of at least one sample from a patient before FMT (regardless of how many timepoints were set pre-FMT; usually 1 or 2), at least one sample from the donor stool used as FMT and at least one sample from the recipient after FMT (regardless of how many timepoints were set post-FMT; usually 3 to 8 within a year post FMT). Only a complete triad allows for drawing conclusions about causality and allows for correlation of treatment outcomes with changes occurring as a result of this treatment.

[0072] The new finding presented herein is a functional mechanism responsible for the therapeutic effect, which includes ARB and / or ARG eradication and / or, decolonization and / or prevention of colonization with ARB and / or ARG and (but not exclusively) restoration of a healthy microbiome. It has been proven that the FMT procedure significantly accelerates ARB and / or ARG eradication and / or, decolonization and / or prevention of colonization with ARB and / or ARG and (but not exclusively) restoration of a healthy microbiome. Each therapy is significantly different because two completely different metagenomes are combined. Defining the engrafted strains in triads and identifying common features between them has allowed for defining the previously unknown functional potential of the microbiome needed for a therapeutic effect for ARB and / or ARG eradication and / or, decolonization and / or prevention of colonization with ARB and / or ARG and (but not exclusively) restoration of a healthy microbiome.

[0073] It was also tested whether there were some common features of FMT between patients with ARB and / or ARG eradication and / or decolonization. As previously discussed, FMT donor samples differed taxonomically (see Figure 7). The question remained whether functional similarity could be determined. To determine this, strain sets identified as independently engrafted for each triad were functionally coded. Subsequently determining the common part of all engrafted strain (functional) sets was possible. Additionally, a narrowing was applied only to features that were statistically significant in differentiating donor and pre-FMT samples (see Figure 3). The first set containing Kos associated with eradication and decolonization of ARB and / or ARG was selected and is presented in Table 1.

[0074] It is to be stated that the aforementioned first set of functions introduced through a number of strains, where each strain encodes many more functions than those composing the first set, provides overall many more functions within the therapeutic NGB consortium than necessary for eradication and decolonization of ARB and / or ARG, thereby providing a “natural” therapeutic effect.

[0075] It was tested whether there were some common features of FMTs performed in proof-of-concept studies, between patients who had ARB and ARG post-colonization resistance (a “colonization resistance” proof). Patients with a post-colonization resistance were selected as previously described. To determine whether functional similarities existed, strain sets identified as independently engrafted for each triad were functionally coded. Subsequently, it was possible to determine the common part of all engrafted strain (functional) sets. The second set containing Kos significant in the ARB and ARG postcolonization resistance was selected and is presented in Table 2.

[0076] A healthy microbiome was defined as one whose metrics (normatives), measured using molecular diagnostics (especially with the NGS method), fall within acceptable norms established through population-based analyses (conducted especially for the invention described herein). The populationbased analyses were conducted on a sample of 328 donors from different geographical regions. These metrics that have been developed relate to species richness, the level of taxonomic diversity, the presence of dominant bacteria and the ratio of Bacillota to Bacteroidota. Using these metrics, it is possible to determine the health status of the microbiome of any sample that has metagenomic sequencing results. All used metrics are presented in Table 9.

[0077] It was tested whether there were some common features of FMTs performed in proof-of-concept studies, between patients who restored a healthy microbiome. Patients with a healthy microbiome restoration were selected based on the health metrics previously described. To determine whether functional similarities existed, strain sets identified as independently engrafted for each triad were functionally coded. Subsequently, it was possible to determine the common part of all engrafted strain (functional) sets. The third set containing KOs significant in the restoration of the healthy microbiome was selected and is presented in Table 3.

[0078] It was shown that the ARB and / or ARG eradication and / or, decolonization and / or prevention of colonization with ARB and / or ARG and (but not exclusively) restoration of a healthy microbiome could occur independently (see Table 6). Additionally, it was shown that there is only a slight overlap between the first set, second set and the third set (see Figure 8). However there are engrafted consortia, and therefore NGB consortia, that have the ability to simultaneously eradicate and / or decolonize ARB and / or ARG, prevent ARB and / or ARG post-colonization and restore a healthy microbiome (see Table 6).

[0079] It was shown that the consortium encoding aforementioned first set needs to be supplemented by additional bacterial species encoding functions from the second set to provide ARB and / or ARG postcolonization resistance.

[0080] It was shown that the consortium encoding aforementioned first set needs to be supplemented by additional bacterial species encoding functions from the third set to provide full restoration of a healthy microbiome.

[0081] It was shown that the consortium encoding aforementioned first set needs to be supplemented by additional bacterial species encoding functions from the second set and the third set to achieve ARB and / or ARG eradication and / or, decolonization and prevention of colonization with ARB and / or ARG and in restoration of a healthy microbiome.

[0082] EXAMPLES

[0083] Example 1. Identification of bacterial isolates, engrafted strains consortia and antibiotic resistant mechanisms genes

[0084] Metagenomic ONT sequencing and bioinformatic analysis was performed on patient and donor samples collectively composing 33 triads (204 samples in total), across multiple timepoints, from a proof-of-concept study using faecal microbiota transplantation (FMT) in those 33 patients (in-house data).

[0085] Using the microbiome profile of both FMT donor material and patient samples, several tools were used to determine which bacterial species from the donor material engrafted in the recipient and are associated with clinical remission and recurrence prophylaxis. To assess strain similarity between donors and post-FMT samples, fastANI software was used. For strains engraftment thresholds of ani value > 99 and mapped fragments > 0.90 were set. Based on engrafted MAGs, engrafted consortia were selected for each triad. Donor MAGs shared with MAGs from at least one of post-FMT samples were selected as engrafted consortia.

[0086] Antibiotic resistant genes were detected using AMR Finder software (>90% Coverage of reference sequence and >90% Identity to reference sequence thresholds were set). The detected genes were limited to those indicated by WHO as particularly dangerous to health. The list of genes found in in-house samples is given in Table 7 and Table 8.

[0087] Example 2, In-silico identification of CDS (coding sequences - proteins) and KOs

[0088] Protein functions were determined using the KEGG database. This means that each protein could have an assigned specific KO identifier. Proteins without an assigned KO identifier have as yet unknown functions. Proteins with the same KO identifier encode similar functional properties. Table 10 contains the KO identifiers with their assigned sequence IDs.

[0089] The KO identifiers used herein are intended to serve as the functional names for the protein sequence clusters. However, it is recognized that the designated protein clusters may be known by other functional names that are not listed here. Therefore, any functional name that describes any subset of the protein sequences in a cluster shown in Table 10 should be considered the same as the cluster names used.

[0090] Proteins encoding each function identified with a KO identifier shown in Table 10 have been clustered within each function. Subclusters aligned by > 80% of total length and with E-value < 1.000E- 03 have been saved, and the most representative sequence is shown for each subcluster. Such methodology is typically used for exhaustive reporting of protein clusters in the field (and it also results from the limitations of the patent application handling system).

[0091] Example 3, In-silico identification of engrafted functions

[0092] Engrafted functions are a unique set of KOs that consist of all KOs assigned to proteins encoded on the MAGs from the engrafted consortium. Table 11 shows engrafted functions for each triad.

[0093] Example 4, Validation of eradication, persistence and post colonization of ARB and ARG after FMT,

[0094] Validation of ARB and / or ARG eradication / decolonization / persistence were performed using computational and bioinformatics methods.

[0095] To assess ARB eradication / decolonization or persistence MAGs were compared between pre-FMT and post-FMT samples using fastANI software. For MAGs persistence thresholds ani value > 99 and mapped fragments > 0.95 were set. Lack of persistence of ARB MAGs means eradication of ARB. To assess ARG eradication / decolonization or persistence contigs similarity were calculated using BLAST software to assess if contigs containing same resistance genes have high nucleotide similarity to distinguish between persistence and post colonization. For contigs with resistance mechanism persistence thresholds similarity > 99% and query coverage > 50% and subject coverage > 50% were set.

[0096] A sample of patients after antibiotic therapy but before FMT administration, whose microbiome metagenome indicated colonization with ARB and / or ARG with ESBL mechanism and at least simultaneous colonization with bacteria with a vancomycin-resistant mechanism of resistance was selected for further analyzes to check whether multiple ARB and ARG could be decolonize at the same time, during single therapy. On this basis, 19 triads met selected conditions (11 triads with eradication status and 8 with persistence status). Triads were classified as eradicated if all ARB and ARG from the pre-FMT sample were eradicated by day 30 post-FMT. Remaining at least one ARB or ARG by day 30 post-FMT resulted in a persistence classification.

[0097] Post colonization status (“colonization resistance”) was assigned to samples based on MAGs and contigs, if ARB and / or ARG was detected on post-FMT samples and persistence status was excluded (only samples with “new” colonization were assessed). On this basis, 20 triads with post colonization positive status (with “new” ARB and / or ARG colonization) and 17 with resistance to post colonization (“anti-ARB / and / or ARG colonization resistance”) were selected. Functions needed to achieve “colonization resistance” in such cases were computed.

[0098] Example 5, The engrafted consortia encoding the functional first set provide eradication and / or decolonization of ARB and / or ARG

[0099] Engrafted functions were selected for each triad, i.e. the functions that were present on the engrafted strains. The overlap of functions engrafted between triads with ARB and / or ARG eradication / decolonization was computed and the first set of functions (KO) associated with ARB and / or ARG eradication / decolonization was obtained. To exclude a false positive result the presence of these functions was checked on triads with ARB and / or ARG persistence (see Figure 9). The analysis showed a lack of these functions in engrafted consortia of patients with ARB and / or ARG persistence.

[0100] ARB and ARG for which eradication / decolonization was confirmed using the first set are listed in Table 12. ARB and ARG for which eradication / decolonization was confirmed using the partial first set are listed in Table 13. Example 6, The engrafted consortia encoding the functional second set provide ARB and ARG postcolonization resistance (antiARB “colonization resistance”)

[0101] Engrafted functions were selected for each triad, i.e. the functions that were present on the engrafted strains. The overlap of functions engrafted between triads with ARB and / or ARG postcolonization resistance (“colonization resistance”) was computed and the second set of functions (KO) associated with ARB and / or ARG post-colonization resistance (“colonization resistance”) was obtained. To exclude a false positive result the presence of these functions was checked on triads with ARB and / or ARG post-colonization (see Figure 10). The analysis showed a lack of these functions in engrafted consortia of patients with ARB and / or ARG presence in case when it was not persistent from pre-FMT.

[0102] Example 7, The engrafted consortia encoding the functional third set restore a healthy microbiome

[0103] Engrafted functions were selected for each triad, i.e. the functions that were present on the engrafted strains. All above mentioned functions were then analyzed for correlation with the metrics and normatives of a healthy microbiome described and calculated above.

[0104] The overlap of functions engrafted within triads with a healthy microbiome restoration was computed, and the third set of functions (KO) associated with restoration of a healthy microbiome was obtained. To exclude a false positive result the presence of these functions was checked on all triads (see Figure 11). The analysis showed a lack of these functions in engrafted consortia in patients without healthy microbiome restoration.

[0105] Example 8, In silico consortia prediction

[0106] Computed functions were permuted into different bacterial consortia and tested in silico for association with clinical response. The consortium of strains which maximized this effect was selected. As it is not known how many strains compose the healthy microbiome and whether providing functional completeness to exert the aimed biological role could be delivered within a smaller number of strains, this therapeutic consortia consisting of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 bacteria (as inhouse internal normative showed 300 taxons as a median of the healthy population, and as few as 10 strains could encode all the functional potential necessary to exert the aimed role) were computed and included in Table 14.

[0107] Comparison of an in silico computed, real-life engrafted, random and other generated consortia were shown using PCA on Figure 12. Only specific consortia cluster together and separately from random, possibly non-therapeutic consortia. TABLES

[0108] Table 1. List of Kegg Orthology functions engrafted within donor strains into recipients associated with ARB and ARG eradication and or decolonization (first set).

[0109] Table 2. List of Kegg Orthology functions engrafted within donor strains into recipients associated with resistance o post colonization of ARB and ARG (second set).

[0110] Table 3. List of Kegg Orthology functions engrafted within donor strains into recipients associated with restoration of healthy microbiome (third set). Table 4. Antibiotic-resistance genes (ARG) from the WHO list marked as dangerous for human health.

[0111] Table 5. ESKAPE+ and ARB containing ARGs included in WHO alarming bacteria species list. ARB list contains all species for which ARG were detected on in house samples.

[0112] Table 6. Characteristics of eradication / decolonization and post-colonization resistance (“colonization resistance”) status across recipients. For eradication / decolonization analyzes only samples colonized with at least two (VRE and ESBL) mechanisms were included for discovery of features associated with eradication / decolonization of multiple mechanisms in parallel. (Abbreviations: ERA: Eradication, DEC: Decolonization, PC-RES: PostColonization Resistance, HMR: Healthy Microbiota Restoration)

[0113] Table 7. Detailed characterics of eradication and persistence across patients. Table contains ARB and ARG relative abundance across multiple timepoints. (ERA: Eradication, PER: Persistence, NA: sample not aval able)

[0114] Table 8. Detailed characteristics of post colonization cases across patients. Table contains ARB relative abundance

[0115]

[0116] Table 9. Detailed information of metrics used to compose normatives, associated with healthy microbiome. Analyses were performed on 328 donors from different geographical regions.

[0117] Table 10. KO with representative assigned seq IDs encoding them. Odd IDs correspond to nucleotide sequence encoding aminoacid sequence with the following even ID.

[0118] Table 11. A list of Sequence IDs (KO's equivalents) encoded in engrafted strains.

[0119] Table 12. List of ARB and corresponding ARG eradicated / decolonized using full first set.

[0120]

[0121] Table 13. List of ARB and corresponding ARG eradicated / decolonized using partial first set. Table 14. Example NGB consortia composed of different number of strains. Names assigned to consortia according

Claims

CLAIMS1. A composition comprising plurality of bacteria comprising genes encoding a plurality of molecularKEGG Orthology functions from a first set comprising: K03828, K21470, K06015, K21557, K13821,K07343, K20922, K02116, K03623, K01046, K08961, K07004, K05873, K19419, K08981, K19166,K00684, KI 1261, K19165, K17675, K18430, K14091, K14188, K14087, K05884, K01596, K01308,K06927, K03298, K07800, K07318, K05521, K01429, K01054, K03190, K18704, K02053, K02052,K11751, K01919, K02042, K03189, K14534, K01205, K21449, K03188, K01419, K22210, K07713,K14089, K01518, K15024, K03817, K19159, K16650, K04088, K23144, K09692, K09769, K14088,K24131, K06188, K00841, KOI 138, K06952, K02413, K24258, K18675, K07321, K02652, K20742,K19350, K00728, KI 1936, K05566, K21030, K19225, K25155, KOI 167, K12243, K02401, K25950,KOI 183, KOI 118, K01488, K06351, K16044, K01580, K06198, K02044, K05820, K16923, K09939,K25154, KOI 192, K19997, K04087, K02566, K21467 and K02473 functions.

2. The composition according to claim 1, wherein the plurality of molecular functions further compriseKEGG Orthology functions from a second set comprising: K00657, K01046, K01464, K12527, K06351, K12941, K09769, K07469, K23124, K06416, K08296, K07160, K02116, K09798, K18446, K22210,K13051, K02566, K02588, K02118, K19405, K02443, K19411, K09825, K01751, K02122, K02654,K02119, K03298, K15024, K06905, K02117, K02120, K01494, K03733, K01295, K02055, K07450,K01646, K01271, K00428, K01644, K19119, K03724, K06218, K02221, K07045, K17472, K03272,K18122, K03325, K05521, K13542, K14392, K07451, K00128, K01643, K07590, K19575, K19117,K21755, K13789, K06895, K02049, K19118, K14982, K02825, K03708, K07321, K14415, K01958,K02226, K01523, K08978, K07571, K01487, K18346, K02052, K01304, KI 1529, K06317, K16052,KI 1071, K09807, K03640, K22900, K06206, K01007, K02019, K07474, K01507, K21903, K07507,K07099, K23753, K07402, K04034, K07339, K09797 and K00652 functions.

3. The composition according to claim 1 or 2, wherein the plurality of molecular functions further comprise KEGG Orthology functions from a third set comprising: K19956, K06606, K02626, K22958,K06903, K02800, K08260, K07313, K06907, K15726, K17723, K03769, K21453, K07492, K07679,K01975, K03187, K09684, K09780, K11051, K23356, K11904, K02810, K10710, K00171, K11050,K01266, K18828, K07071, K21011, K15527, K00005, K09974, K19309, K13990, K00819, K03337,K22757, K12941, K06330, K00564, K21744, K23253, K16153, K03436, K10119, KOI 174, K18011,K01843, K00556, K21012, K15555, K19310, K10192, K01235, K00156, K19137, K01035, K03335,K07494, K05813, K25114, K16150, K05986, K10212, K18012, K01844, K02757, K00641, K07397,K09384, K15531, K03297, K00294, K25067, K03390, K17948, K13012, K22960, K01838, K00853, K10974, K13018, K13017, K09992, K06416, K02456, K18014, K10118, K16927, K06726, K00074, KOI 160, K02007, K13049, K24948, K06284, K09775, K15519 and K22699 functions.

4. The composition according to any of claims 1-3, wherein the plurality of bacteria comprises a consortium selected from the group consisting of: consortium Cl, C2, C3, C4, C5, C6, C7, C8, C9, CIO, Cl l, C12, C13, C14 and C15 of Table 14.

5. The composition according to any of claims 1-3, wherein the plurality of bacteria comprises a consortium selected from the group consisting of: consortium HBI09, HBI14, HBI116, HBI95, HBI07, HBI08, HBI11, HBI35, HBI178, HBI69, HBI27, HBI30, HBI70, HBI38, HBI81, HBI147A, HBI16, HBI52, HBI181, HBI46, HBI74, HBI02AA, HBI77, HBI193, HBI57A, HBI68, HBI177, HBI238A, HBI226A and HBI226 of Table 11.

6. A pharmaceutical composition comprising the composition of any one of the preceding claims, further comprising a pharmaceutically acceptable excipient.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is formulated for delivery to the intestine.

8. The composition according to any one of claims 1-7 for use as a medicament.

9. The composition according to claim 8, wherein the use comprises administering the composition in a form of faecal microbiota transplantation.

10. The composition according to claim 8, wherein the use comprises administering the composition in a form of an isolated bacteria consortium.

11. The composition according to any one of claims 1-7 for use in eradication and / or decolonization of antibiotic-resistant bacteria (ARB) and / or antibiotic-resistance genes (ARG).

12. The composition according to any one of claims 2-7 for use in post-colonization prevention of antibiotic-resistant bacteria (ARB) and / or antibiotic-resistance genes (ARG).

13. The composition according to any one of claims 3-7 for use in eradication and / or decolonization and / or prevention of post-colonization of antibiotic-resistant bacteria (ARB) and / or antibiotic-resistance genes (ARG), as well as restoration of a healthy microbiome.

14. The composition according to claim 11, 12 or 13, wherein the ARB and / or ARG are selected from the group consisting of the following ARB and corresponding ARG: Klebsiella pneumoniae - blaSHV- 28, blaSHV-28, blaCTX-M-3, qnrSl, blaSHV-1; Enterococcus B faecium - vanA, vanB, erm(B); Escherichia coli - blaTEM-1, blaCTX-M-15; Parasutterella gallistercoris - blaTEM-1, blaTEM; Clostridium AQ innocuum - vanB, erm(B); Faecalimonas umbilicata - mef(A), msr(D); Streptococcus gordonii - msr(D), mef(A); Streptococcus salivarius D - msr(D), mef(A); Dialister invisus - vanB; Prevotella melaninogenica - cfxA3; Agathobacter rectalis - cfxA3; Klebsiella oxytoca - blaOXY-2-4; Morganella morganii - blaDHA-1; Dielma fastidiosa - erm(B); Blautia A wexlerae - erm(B); Dorea formicigenerans - mef(A); Acutalibacter omithocaccae - erm(B); Protoclostridium gallicola - erm(B); Enterococcus D gallinarum - vanC; Fimisoma sp000435715 - erm(B); Lachnotalea soehngenii - vanG; Limivivens intestinipullorum - erm(B); Enterococcus D casseliflavus - vanC; Merdimonas faecis - vanB; Rikenella microfusus - erm(B); Roseburia intestinalis - erm(B); Anaerotignum lactatifermentans- mef(A); Flavonifractor avistercoris - erm(B); Ligilactobacillus salivarius - erm(B); Bacteroides uniformis - mef(A); Hominimerdicola aceti - erm(B); Unclassified - blaTEM-1, blaTEM-135, blaSHV- 1, blaSHV-28, blaSHV-11, blaCTX-M-15, blaKLUC-1, blaOXY-2-1, blaOXY-1-1, blaOXY-1-2, blaOXY-2-7, blaOXY-2-2, vanC, vanA, erm(B), qnrSl, mef(A) as well as Pseudomonas aeruginosa - blaPDC-3; Klebsiella grimontii - blaOXY-4-1; Bilophila wadsworthia - erm(B); Enterococcus faecalis- vanA, erm(B); Proteus mirabilis - blaTEM-1; Klebsiella neumoniae - blaSHV-11, blaNDM-1, blaSHV- 207; Facklamia hominis - erm(B); Bacillus J hisashii - msr(D), mef(A); Escherichia coli - blaDHA-1, qnrB19; Klebsiella michiganensis - blaOXY-1-9; Veillonella nakazawae - msr(D), mef(A); Streptococcus parasanguinis L - msr(D), mef(A); Unclassified - cfxA3, msr(D), blaOXY-1-11, blaDHA-1, blaOXY-1-9, VanG, vanB as well as any ARG of Table 4 and any ARB of Table 5.

15. A method for eradication and / or decolonization of antibiotic-resistant bacteria (ARB) and / or antibiotic-resistance genes (ARG), in a subject in need for thereof, comprising administering a composition according to any one of claims 1-7 to the subject in an amount effective to eradicate and / or decolonize ARB and / or ARG.

16. A method for post-colonization prevention of antibiotic-resistant bacteria (ARB) and / or antibioticresistance genes (ARG) in a subject in need for thereof, comprising administering a compositionaccording to any one of claims 2-7 to the subject in an amount effective to prevent post-colonization of ARB and / or ARG.

17. A method for eradication and / or decolonization and / or prevention of post-colonization of antibioticresistant bacteria (ARB) and / or antibiotic-resistance genes (ARG), as well as restoration of a healthy microbiome in a subject in need for thereof, comprising administering a composition according to any one of claims 3-7 to the subject in an amount effective to eradicate and / or decolonize and / or prevent post-colonization ARB and / or ARG, as well as restore a healthy microbiome.

18. The method according to claim 15, 16 or 17, wherein the ARB and / or ARG are selected from the group consisting of the following ARB and corresponding ARG: Klebsiella pneumoniae - blaSHV-28, blaSHV-28, blaCTX-M-3, qnrSl, blaSHV-1; Enterococcus B faecium - vanA, vanB, erm(B); Escherichia coli - blaTEM-1, blaCTX-M-15; Parasutterella gallistercoris - blaTEM-1, blaTEM; Clostridium AQ innocuum - vanB, erm(B); Faecalimonas umbilicata - mef(A), msr(D); Streptococcus gordonii - msr(D), mef(A); Streptococcus salivarius D - msr(D), mef(A); Dialister invisus - vanB; Prevotella melaninogenica - cfxA3; Agathobacter rectalis - cfxA3; Klebsiella oxytoca - blaOXY-2-4; Morganella morganii - blaDHA-1; Dielma fastidiosa - erm(B); Blautia A wexlerae - erm(B); Dorea formicigenerans - mef(A); Acutalibacter omithocaccae - erm(B); Protoclostridium gallicola - erm(B); Enterococcus D gallinarum - vanC; Fimisoma sp000435715 - erm(B); Lachnotalea soehngenii - vanG; Limivivens intestinipullorum - erm(B); Enterococcus D casseliflavus - vanC; Merdimonas faecis - vanB; Rikenella microfusus - erm(B); Roseburia intestinalis - erm(B); Anaerotignum lactatifermentans- mef(A); Flavonifractor avistercoris - erm(B); Ligilactobacillus salivarius - erm(B); Bacteroides uniformis - mef(A); Hominimerdicola aceti - erm(B); Unclassified - blaTEM-1, blaTEM-135, blaSHV- 1, blaSHV-28, blaSHV-11, blaCTX-M-15, blaKLUC-1, blaOXY-2-1, blaOXY-1-1, blaOXY-1-2, blaOXY-2-7, blaOXY-2-2, vanC, vanA, erm(B), qnrSl, mef(A) as well as Pseudomonas aeruginosa - blaPDC-3; Klebsiella grimontii - blaOXY-4-1; Bilophila wadsworthia - erm(B); Enterococcus faecalis- vanA, erm(B); Proteus mirabilis - blaTEM-1; Klebsiella neumoniae - blaSHV-11, blaNDM-1, blaSHV- 207; Facklamia hominis - erm(B); Bacillus J hisashii - msr(D), mef(A); Escherichia coli - blaDHA-1, qnrB19; Klebsiella michiganensis - blaOXY-1-9; Veillonella nakazawae - msr(D), mef(A); Streptococcus parasanguinis L - msr(D), mef(A); Unclassified - cfxA3, msr(D), blaOXY-1-11, blaDHA-1, blaOXY-1-9, VanG, vanB as well as any ARG of Table 4 and any ARB of Table 5.

19. A method for assessing a suitability of a composition comprising plurality of bacteria comprising genes encoding plurality of molecular functions for use in eradication and / or decolonization and / orprevention of post-colonization of antibiotic-resistant bacteria (ARB) and / or antibiotic-resistance genes (ARG), as well as restoration of a healthy microbiome, comprising: isolating DNA from the composition to obtain a DNA isolate; sequencing the DNA isolate using nucleic acid sequencing methods to obtain a metagenome sequence; identifying in the metagenome sequence sequences of genes; assigning functions to the gene sequences based on their sequences; comparing whether the functions assigned to the gene sequences comprises KEGG Orthology functions from the first set of claim 1, the second set of claim 2 or the third set of claim 3; and if the gene sequences comprises all the functions of the first set, the composition is considered suitable for use in eradication and / or decolonization and / or prevention of post-colonization of antibioticresistant bacteria (ARB) and / or antibiotic-resistance genes (ARG), as well as restoration of a healthy microbiome; otherwise, the composition is not considered suitable for use in eradication and / or decolonization and / or prevention of post-colonization of antibiotic-resistant bacteria (ARB) and / or antibiotic-resistance genes (ARG), as well as restoration of a healthy microbiome.

20. A method for assessing a suitability of a stool sample comprising plurality of bacteria comprising genes encoding plurality of molecular functions for use in eradication and / or decolonization and / or prevention of post-colonization of antibiotic-resistant bacteria (ARB) and / or antibiotic-resistance genes (ARG), as well as restoration of a healthy microbiome, comprising: isolating DNA from the stool sample to obtain a DNA isolate; depleting human DNA in the DNA isolate; sequencing the DNA isolate using nucleic acid sequencing methods to obtain a metagenome sequence; removing from the metagenome sequence sequences belonging to human genome; identifying in the metagenome sequence gene sequences; assigning functions to the gene sequences based on their sequences; comparing whether the functions assigned to the gene sequences comprises KEGG Orthology functions from the first set of claim 1, the second set of claim 2 or the third set of claim 3; and if the gene sequences comprises all the functions from the first set, the stool sample is considered suitable for use in eradication and / or decolonization and / or prevention of post-colonization of antibioticresistant bacteria (ARB) and / or antibiotic-resistance genes (ARG), as well as restoration of a healthy microbiome; otherwise, the stool sample is not considered suitable for use in eradication and / ordecolonization and / or prevention of post-colonization of antibiotic-resistant bacteria (ARB) and / or antibiotic-resistance genes (ARG), as well as restoration of a healthy microbiome.

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