Probiotic composition comprising live Klebsiella strain ARO112 as the only ingredient or in combination with SCFA-producing bacteria

Abstract

The present invention relates to a probiotic composition comprising live bacteria of Klebsiella strain ARO112, optionally for use as a therapeutic drug. The present invention composition is provided for use in the prevention or treatment of dysbiosis, intestinal inflammation, intestinal pathogen infection or intestinal inflammatory disease, such as Crohn's disease or ulcerative colitis.

Description

[0001] Invention Field

[0002] This invention relates to bacteria containing Klebsiella pneumoniae (Klebsiella pneumoniae) Klebsiella Probiotic compositions of strain ARO112 and probiotic compositions used as medicines, particularly for the prevention or treatment of dysbiosis, intestinal inflammation or bacterial infection. Background of the Invention

[0004] Inflammatory bowel disease (IBD) is a group of diseases caused by multiple factors, including genetic susceptibility and / or environmental factors. Limited treatment options available focus on alleviating common symptoms that severely impact patients' quality of life. The core of the disease cycle that plagues IBD patients comprises three symptoms: persistent and exacerbated intestinal inflammation, gut microbiota imbalance, and a predisposition to recurrent intestinal infections.

[0005] Patients with inflammatory bowel disease (IBD) often experience flare-ups of intestinal inflammation, typically requiring antibiotics or anti-inflammatory drugs. However, these medications can adversely affect the gut microbiota and may exacerbate the pro-inflammatory environment in the gut (Santana, 2022). Another consequence of microbiota imbalance is that it leads to a loss of the natural protection provided by the gut microbiota, thereby increasing susceptibility to infection, particularly infections by Enterobacteriaceae such as Adhesive Invasive Escherichia coli (AIEC), which can also induce flare-ups (Small, 2013). Currently, antibiotic therapy remains the gold standard for treating bacterial infections, but this can further erode the gut microbiota, thus increasing susceptibility to infection.

[0006] This complex and self-sustaining disease cycle exacerbates the chronicity of IBD, leaving patients in a prolonged state of disease-treatment-disease, urgently requiring novel and effective methods to complement existing treatments and break this vicious cycle. Probiotics have long been considered a complementary or alternative therapy to existing treatments for intestinal diseases, but few cases have demonstrated their effectiveness in complex diseases like IBD. Probiotics have been suggested for IBD treatment (Carmen, 2011); however, currently known probiotics do not target the many key symptoms of IBD. Furthermore, the efficacy of therapeutic probiotics is often unsatisfactory when tested in patient rather than laboratory settings, which can be attributed to several common limitations: 1) the probiotic strain is foreign to the target microbiota and therefore cannot colonize long enough to exert its effects; 2) probiotics persist in imbalanced microbiota, hindering the natural restoration of microbial diversity (Suez, 2018); 3) probiotic strains can lead to increased antibiotic resistance transfer (Montassier, 2021). Probiotics have been shown to delay the recovery of the gut microbiota after antibiotic treatment and to persist in the gut microbiota for a long time (Suez, 2018). In addition, even with potent therapies such as fecal microbiota transplantation, AIEC infection can hinder the recovery of the gut microbiota in IBD patients (Zhilu 2021).

[0007] Escherichia coli Nissle 1917 (EcN) has long been used as a probiotic strain and has been touted as a potential therapy for IBD (Schultz, 2008). Common probiotic strains of EcN have shown partial effects on the growth of AIEC in in vitro studies (Huebner 2011). None of the aforementioned probiotic therapies have been tested in inflammatory environments, which are highly conducive to dysbiosis, nor have they been studied in the presence of AIEC, which, as mentioned above, leads to persistent dysbiosis and hinders recovery. A recent approach found that GMO EcN strains showed promising results in inflammatory environments, but genetically modified organisms face stringent regulatory hurdles (Zhou 2022).

[0008] In previous studies, the inventors identified a gut commensal bacterium, *Klebsiella* spp. ARO112, which exhibited limited colonization resistance to *Escherichia coli* strain K-12 MG1655 in a streptomycin-induced gut microbiota dysbiosis mouse model (Oliveira, 2020). However, it had only a partial effect on the number of pathogenic bacteria, and as with other probiotics, it remains unclear whether this substitution can be achieved in an inflammatory environment similar to that of IBD patients, or whether the intervention has a positive or negative impact on microbial diversity and local inflammation. Furthermore, *Klebsiella* spp. ARO112 is a member of the genus *Klebsiella*, which contains many opportunistic pathogenic bacteria such as *Klebsiella pneumoniae* and *Klebsiella acidogenic*, thus the pathogenicity and virulence safety of *Klebsiella* spp. ARO112 remains unclear. One of the most challenging and intractable characteristics of the Enterobacteriaceae family as a whole is their ease with which they acquire antibiotic resistance from other members of the flora, particularly through horizontal gene transfer events such as plasmid conjugation. This is evident even in non-Klebsiella pneumoniae strains (Moradigaravand 2017; Luo 2022; Hubbard 2020), therefore they are not suitable for use as probiotics or for therapeutic purposes.

[0009] Based on the aforementioned prior art, the object of the present invention is to provide means and methods for preventing or treating dysbiosis and / or intestinal inflammation or related diseases such as IBD and pathogenic bacterial infections. This object is achieved through the subject matter of the independent claims of this specification, while the dependent claims, embodiments, drawings, and general description of the specification further illustrate further advantageous embodiments. Summary of the Invention

[0010] This study demonstrates how the protective Klebsiella spp. ARO112 competes with other Proteobacteria (Pseudomonadota, including Enterobacteriaceae) and suppresses intestinal inflammation. Simultaneously, this bacterium produces minimal inflammatory siderophores while maintaining the production of host-tolerant lipid transporter-2, thus potentially adapting to compete with invading bacteria in inflammatory environments without further exacerbating inflammation. Furthermore, ARO112 exhibits a strong attenuating effect on both the acquisition and maintenance of antibiotic resistance, which is typically associated with clinical Enterobacteriaceae isolates (such as Kp1012) and EcN, suggesting that this symbiotic strain is potentially safer as a probiotic or therapeutic species.

[0011] Animal model studies have demonstrated that ARO112 is a successful probiotic therapy for IBD, targeting multiple microbiome-based disease hallmarks. It can displace invading pathogenic bacteria, including clinically relevant AIECs; promote the restoration of microbiome diversity, particularly reducing Proteobacteria; and increase the proportion of butyrate-producing bacteria, thereby reducing the likelihood of pathogenic bacterial overgrowth and suppressing inflammatory responses.

[0012] These results, taken together—replacement of infectious agents (such as AIEC), non-induction of inflammation, and restoration of the microbiome—all achieved after treatment with ARO112, validate the three-pronged approach targeting these three IBD markers.

[0013] The first aspect of this invention relates to a probiotic composition comprising live Klebsiella spp. ARO112 as an active ingredient, for oral or enteral administration. The probiotic of this invention is particularly suitable for administration to subjects who have recently received a course of antibiotic treatment or who suffer from inflammatory bowel diseases such as ulcerative colitis or Crohn's disease.

[0014] A second aspect of the invention relates to a probiotic composition comprising live Klebsiella spp. ARO112 as an active ingredient, formulated for oral or enteral administration for the treatment or prevention of recurrence of inflammatory diseases, including diseases associated with intestinal flora imbalance (microbiome dysbiosis), intestinal inflammatory diseases, infections by intestinal pathogenic bacteria such as Proteobacteria (Pseudomonas, including Enterobacteriaceae), and / or flare-ups of inflammatory bowel disease.

[0015] Terms and Definitions

[0016] For ease of understanding of this specification, the following definitions apply, and where appropriate, singular terms include plural forms and vice versa. In the event of any conflict between any of the following definitions and any document incorporated herein by reference, the following definitions shall prevail.

[0017] The terms “comprising,” “having,” “containing,” and “including,” and their similar forms and grammatical equivalents as used herein, have the same meaning and are open-ended, meaning that none of these terms is intended to be an exhaustive list or limited to the listed terms. For example, an article “comprising” components A, B, and C may consist of components A, B, and C (i.e., contain only), or it may contain not only components A, B, and C but also one or more other components. Therefore, it should be understood that “comprising” and its similar forms and grammatical equivalents include embodiments that disclose “consistently consisting of” or “comprises of.”

[0018] Where a numerical range is provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value (up to one-tenth of the lower limit unit) between the upper and lower limits of the range and between any other numerical values ​​or intermediate values ​​within the range is included in this disclosure, subject to any explicitly excluded limits within the range. If the range contains one or two limits, the range that does not contain one or both limits is also included in this disclosure.

[0019] The use of "about" in this article to refer to a value or parameter includes (and describes) the various variations of that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0020] As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural references unless the context clearly specifies otherwise.

[0021] As used herein, the term “and / or” refers to a specific description of each of two particular features or components, regardless of the presence of the other feature or component. Thus, in phrases such as “A and / or B”, the term “and / or” is intended to cover “A and B”, “A or B”, “A (alone)”, and “B (alone)”. Similarly, in phrases such as “A, B, and / or C”, the term “and / or” is intended to cover the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0022] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used in molecular, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0023] In the context of this specification, the term "probiotic" refers to a pharmaceutical composition containing live or lyophilized bacteria that stimulates the growth of beneficial microorganisms in the recipient's gut.

[0024] In this instruction manual, the terms LF82 and *Escherichia coli* LF82 refer to a pathogenic *Escherichia coli* strain AIEC commonly associated with IBD patients. This strain can be identified by classification ID 591946 in the NCBI classification database, and its genome sequence can be found in multiple databases.

[0025] In this context, the term "inflammatory bowel disease" refers to inflammatory bowel diseases such as Crohn's disease (CD) or ulcerative colitis (UC). Other diseases associated with intestinal inflammation include, for example, obesity, malnutrition, and aging.

[0026] In this specification, the term "viable" refers to live bacteria. This can be confirmed by methods known in the art, such as serial dilution plating on agar plates, or by detection using a cell counter and live / dead bacteria distinguishing dyes. The term also covers lyophilized or freeze-dried bacteria that can be revived and grown upon hydration.

[0027] As used herein, the term "pharmaceutical composition" refers to a composition of the live bacteria of the present invention and at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions of the present invention are provided in a form suitable for topical, parenteral, or injectable administration.

[0028] As used herein, the term “pharmaceutically acceptable carrier” includes any solvent, dispersion medium, coating agent, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonic agent, absorption retardant, salt, preservative, drug, drug stabilizer, binder, excipient, disintegrant, lubricant, sweetener, flavoring agent, dye, and combinations thereof, as known to those skilled in the art (e.g., see Remington: the Science and Practice of Pharmacy, ISBN0857110624).

[0029] As used herein, the term "treatment" for any disease or condition (e.g., AIEC infection) in one embodiment means improving the disease or condition (e.g., slowing, stopping, or alleviating the development of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" means reducing or improving at least one physiological parameter, including parameters that may be imperceptible to the patient. In yet another embodiment, "treatment" means modulating the disease or condition, including physical modulation (e.g., stabilizing perceptible symptoms), physiological modulation (e.g., stabilizing physiological parameters), or both. Unless specifically described herein, methods for assessing the treatment and / or prevention of disease are generally known in the art.

[0030] sequence

[0031] Sequences similar to or homologous to the sequences disclosed herein (e.g., at least about 70% sequence identity) are also part of this invention. In some embodiments, amino acid-level sequence identity can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. Nucleic acid-level sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. Alternatively, the nucleic acid fragment also exhibits substantially the same identity when hybridized to the complementary strand under selective hybridization conditions (e.g., very stringent hybridization conditions). Nucleic acids can be present in intact cells, cell lysates, or in partially or substantially purified forms.

[0032] In the context of this specification, the terms "sequence identity" and "sequence identity percentage" refer to a single quantitative parameter representing the sequence alignment result determined by comparing two aligned sequences position by position. Sequence alignment methods used for comparison are well known in the art. Sequence alignment for comparison can be performed using methods such as local homology algorithms (Smith and Waterman, Adv. Appl. Math. 2:482 (1981)), global alignment algorithms (Needleman and Wunsch, J. Mol. Biol. 48:443 (1970)), similarity retrieval methods (Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988)), or computer implementations of these algorithms, including but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. The software used to perform BLAST analysis is publicly available, for example, through the National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0033] An example of amino acid sequence comparison is using the BLASTP algorithm with the default settings: expected threshold: 10; word length: 3; maximum match within the query range: 0; matrix: BLOSUM62; gap penalty: 11 for presence, 1 for extension; composition adjustment: conditional composition score matrix adjustment. A similar example of nucleic acid sequence comparison is using the BLASTN algorithm with the default settings: expected threshold: 10; word length: 28; maximum match within the query range: 0; match / non-match score: 1-2; gap penalty: linear. Unless otherwise stated, the sequence identity values ​​provided herein refer to values ​​obtained using the BLAST program suite (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) with the above-described default parameters for protein and nucleic acid comparisons, respectively.

[0034] Referring to the same sequence without specifying a percentage value indicates that the sequences are 100% identical (i.e., the same sequence).

[0035] In the context of this invention, the term "having substantially the same biological activity" refers to one or both of the principal functions of the ARO112 composition, namely, the replacement of AIEC strains in an inflammatory Nod2- / - mouse model of IBD. In short, an ARO112 composition having substantially the same biological activity as the ARO112 strain characterized by the genome of BioProject ID PRJNA590204 in the NCBI Sequence Reading Archive (SRA), replaces pathogenic AIEC strains within 12 days using the methods described in the "Animal Experiments" section of "Materials and Methods" below. Invention Details

[0037] One aspect of the present invention relates to a probiotic composition comprising a live Klebsiella genus ARO112 strain as the sole active bacterial component, wherein the Klebsiella genus ARO112 strain is characterized in that its genome has >90% similarity to the genome of a Klebsiella genus ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

[0038] Another aspect of the invention relates to a probiotic composition comprising a live Klebsiella genus ARO112 strain as the sole active bacterial component, wherein the Klebsiella genus ARO112 strain comprises the genome of a Klebsiella genus ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

[0039] The inventors have demonstrated that probiotic compositions containing Klebsiella spp. ARO112 strain as the sole active bacterial component promote the recovery of short-chain fatty acid (SCFA) producing bacteria. Therefore, the inventors anticipate that probiotic compositions containing live Klebsiella spp. bacteria and SCFA-producing bacteria will further promote and accelerate the SCFA increase described herein.

[0040] Therefore, another aspect of the present invention provides a probiotic composition comprising: (i) live Klebsiella bacteria; and (ii) bacteria that produce short-chain fatty acids (SCFAs).

[0041] In some embodiments of the probiotic composition, the short-chain fatty acid (SCFA) is butyrate. In some embodiments, the bacteria that produce butyrate are selected from the bacterial families comprising the group consisting of: Lachnospiraceae, Ruminococciaceae, Oscillospiraceae, Butyricicoccaceae, and Bacteroidaceae.

[0042] In some embodiments of the probiotic composition, the Klebsiella bacteria include a Klebsiella ARO112 strain, wherein the Klebsiella ARO112 strain is characterized in that its genome has >90% similarity to the genome of a Klebsiella ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

[0043] In some embodiments of the probiotic composition, the Klebsiella spp. ARO112 strain contains the genome of the Klebsiella spp. ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

[0044] In one embodiment, the probiotic composition contains 10 4 Up to 10 20 10 live cells 5 Up to 10 14 10 live cells 6 Up to 10 13 One live cell or 10 7 Up to 10 12 10 live cells. In some embodiments, the probiotic composition contains 10 8 Up to 10 10 One living cell.

[0045] In some embodiments, the probiotic composition comprises Klebsiella bacteria and SCFA-producing bacteria in a ratio of Klebsiella bacteria to SCFA-producing bacteria of 1:1 to 100,000:1, 10:1 to 10,000:1, or 100:1 to 1,000:1.

[0046] In some embodiments, the probiotic composition comprises SCFA-producing bacteria and Klebsiella bacteria in a ratio of SCFA-producing bacteria to Klebsiella bacteria of 1:1 to 100,000:1, 10:1 to 10,000:1, or 100:1 to 1,000:1.

[0047] In some embodiments, the probiotic composition comprises Klebsiella bacteria and SCFA-producing bacteria in a ratio of approximately 1:1, 10:1, 100:1, 1000:1, or 10000:1.

[0048] In some embodiments, the probiotic composition comprises SCFA-producing bacteria and Klebsiella bacteria in a ratio of approximately 1:1, 10:1, 100:1, 1000:1, or 10000:1.

[0049] In some embodiments, the probiotic composition may be administered to the subject more than once. In some embodiments, the probiotic composition may be administered to the subject 2 to 10 times. The more than one administration of the probiotic composition may be continuous or discontinuous.

[0050] Another aspect of the invention relates to a probiotic composition comprising live Klebsiella bacteria and short-chain fatty acid (SCFA) producing bacteria, for use as a medicine.

[0051] In some embodiments where a probiotic composition comprising live Klebsiella bacteria and short-chain fatty acid (SCFA)-producing bacteria is used as a drug, the composition is intended to promote bacterial diversity in mammalian or human subjects.

[0052] In some embodiments where a probiotic composition comprising live Klebsiella bacteria and short-chain fatty acid (SCFA) producing bacteria is used as a medicine, the composition is used to prevent or treat intestinal flora imbalance in human subjects.

[0053] In some embodiments where a probiotic composition comprising live Klebsiella bacteria and short-chain fatty acid (SCFA)-producing bacteria is used as a drug, the composition is used to prevent or treat intestinal inflammation in human subjects.

[0054] Another aspect of the invention relates to a probiotic composition comprising live Klebsiella bacteria as the sole active bacterial component for the prevention or treatment of intestinal inflammation in mammalian subjects (particularly human subjects).

[0055] Another aspect of the invention relates to a probiotic composition comprising live Klebsiella bacteria as the sole active bacterial component, for increasing the relative abundance of butyrate-producing bacteria in the gut of a subject.

[0056] Another aspect of the invention relates to a probiotic composition comprising live Klebsiella bacteria as the sole active bacterial component, for increasing butyrate levels in the gut of a subject.

[0057] In some implementations, butyrate-producing bacteria have been depleted by antibiotics.

[0058] In some embodiments of the probiotic composition, the Klebsiella bacteria include a Klebsiella ARO112 strain, wherein the Klebsiella ARO112 strain is characterized in that its genome has >90% similarity to the genome of a Klebsiella ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

[0059] In some embodiments of the probiotic composition, the Klebsiella spp. ARO112 strain contains the genome of the Klebsiella spp. ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

[0060] One aspect of the present invention relates to a probiotic composition comprising a live Klebsiella spp. ARO112 strain as the sole active bacterial component, wherein the Klebsiella spp. ARO112 strain is characterized in that its genome has >90% similarity to the genome of a Klebsiella spp. ARO112 strain submitted to the NCBI Sequence Reading Archive (SRA) with BioProject ID PRJNA590204; and / or a probiotic composition comprising live Klebsiella spp. bacteria and short-chain fatty acid (SCFA) producing bacteria for the prevention or treatment of intestinal infections caused by pathogenic bacterial species in human subjects.

[0061] In some implementations, the pathogenic bacterial species are members of the phylum Proteobacteria (Pseudomonas).

[0062] In some implementations, the pathogenic bacterial species are antibiotic-resistant bacterial species, especially multidrug-resistant bacterial species.

[0063] In some implementations, the pathogenic bacterial species are selected from Escherichia coli (E. coli). Escherichia ), Citrobacter ( Citrobacter ),salmonella( Salmonella ) or Vibrio ( Vibrio Members of the genus *Clostridium difficile*; Clostridioides difficile Vancomycin-resistant Enterococci ( Enterococcus ) bacterial strains; and / or selected from Enterococcus faecalis ( Enterococcus faecium Staphylococcus aureus ( Staphylococcus aureus ), Klebsiella pneumoniae ( Klebsiella pneumoniae Acinetobacter baumannii ( Acinetobacter baumannii ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Enterobacteriaceae ( Enterobacter ) bacterial strains and Escherichia coli ( E. coli Multidrug-resistant strains of pathogenic bacteria.

[0064] In some implementations, the pathogenic bacterial species is Vibrio cholerae (Cholera vibrio). Vibrio cholerae Adherent-invasive Escherichia coli E. coli AIEC), enterohemorrhagic Escherichia coli (AIEC) Escherichia coli EHEC), enteroaggregative Escherichia coli E. coli EAEC), enteropathogenic Escherichia coli E. coli EPEC) or uropathogenic Escherichia coli E. coli UPEC), especially the pathogenic bacteria, which is AIEC Escherichia coli LF82 strain.

[0065] In some embodiments, the present invention relates to a probiotic composition comprising a live Klebsiella spp. ARO112 strain as the sole active bacterial component, wherein the Klebsiella spp. ARO112 strain is characterized in that its genome has >90% similarity to the genome of a Klebsiella spp. ARO112 strain submitted to the NCBI Sequence Reading Archive (SRA) with BioProject ID PRJNA590204; and / or a probiotic composition comprising live Klebsiella spp. bacteria and short-chain fatty acid (SCFA) producing bacteria, for use in subjects who have received a course of antibiotic treatment in the past month (especially within the past 48 hours).

[0066] In some implementations, the course of antibiotic treatment includes vancomycin, gentamicin, metronidazole, ciprofloxacin, and / or rifaximin.

[0067] In some embodiments, the present invention relates to a probiotic composition comprising a live Klebsiella spp. ARO112 strain as the sole active bacterial component, wherein the Klebsiella spp. ARO112 strain is characterized in that its genome has >90% similarity to the genome of a Klebsiella spp. ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI, and / or a probiotic composition comprising live Klebsiella spp. bacteria and short-chain fatty acid (SCFA) producing bacteria, for use in patients diagnosed with inflammatory bowel disease.

[0068] In some implementations, inflammatory bowel disease is inflammatory bowel disease, particularly Crohn's disease or ulcerative colitis.

[0069] In some implementations, the Klebsiella spp. ARO112 strain is characterized by a genome similarity of >95%, >97%, >98%, >99%, >99.5%, or particularly >99.9% to the genome of the Klebsiella spp. ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI. 。

[0070] In some implementations, the probiotic composition is administered orally.

[0071] The inventors have demonstrated that the Klebsiella spp. ARO112 strain possesses an unexpected and unique ability to enhance intestinal microbial diversity and suppress intestinal inflammation. Klebsiella spp. ARO112 is a non-spore-forming facultative anaerobic bacterium. The inventors have demonstrated that this bacterium exhibits surprisingly high safety because it typically does not acquire antibiotic resistance genes, promotes the growth of butyrate-producing anti-inflammatory bacteria, and is gradually cleared from the intestines after oral administration.

[0072] Probiotic ARO112 Composition

[0073] One aspect of the present invention relates to a probiotic composition comprising (in other words, comprising as the sole active bacterial component) a live Klebsiella genus ARO112 strain. The Klebsiella genus ARO112 strain is characterized by having a genome similarity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher to the genome of a Klebsiella genus ARO112 strain with BioProject ID PRJNA590204 submitted to the NCBI Sequence Reading Archive (SRA). Typically, the Klebsiella genus ARO112 strain is characterized by having a genome similarity >90% to the genome of a Klebsiella genus ARO112 strain with BioProject ID PRJNA590204 submitted to the NCBI Sequence Reading Archive (SRA).

[0074] Another aspect of the invention relates to a probiotic composition comprising live Klebsiella bacteria as the sole active bacterial component for the prevention or treatment of intestinal inflammation in mammalian subjects (particularly human subjects).

[0075] Probiotics are bacterial cultures that are consumed orally to enhance gut health. In some embodiments, the probiotic compositions of the present invention are formulations of live Klebsiella spp. ARO112 strains and buffers or excipients. In some embodiments, the probiotic compositions of the present invention are in the form of conveniently packaged probiotic foods such as dairy products, or dietary supplements such as vitamins or calcium, or fiber, supplemented with live colony-forming units of Klebsiella spp. ARO112 strains. In some embodiments, the probiotics of the present invention are in the form of packaged foods manufactured to provide a dose of active live Klebsiella spp. ARO112 strains per serving, for example, containing a total of 100 million live cells per serving.

[0076] Another aspect of the invention relates to a probiotic composition comprising a live Klebsiella spp. ARO112 strain as the sole active bacterial component, for use as a medicine.

[0077] In some embodiments of the probiotic composition, it comprises a Klebsiella strain characterized in that its genome has greater than 97% identity with the genome of a Klebsiella strain ARO112 with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

[0078] In some embodiments, the probiotic composition comprises Klebsiella spp. ARO112 strain, whose genome shares >97%, >98%, >99%, >99.5%, or especially >99.9% similarity with the aforementioned strain, and has the same or substantially the same biological activity, namely, the ability to prevent long-term colonization by pathogenic Proteobacteria (Pseudomonas), including Enterobacteriaceae. 。

[0079] In some embodiments where the probiotic ARO112 composition is used as a medicine, the composition comprises a Klebsiella genus ARO112 strain derived from a strain characterized by the genome of a BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

[0080] In a particular embodiment of the probiotic ARO112 composition of the present invention, it is provided in a form suitable for oral administration, such as capsules containing the bacterial preparation, or lyophilized bacteria. It should be understood that, in addition to live Klebsiella spp. ARO112 strains, the probiotic composition may also contain a proportion of dead bacteria.

[0081] In some embodiments where the probiotic composition ARO112 is used as a drug, the composition is used to promote bacterial diversity in a human subject. Examples Figure 8 and Figure 9This indicates that the compositions of the present invention can enhance overall microbial diversity, particularly beneficial butyrate-producing bacterial members.

[0082] In some embodiments, the bacterial composition for treating or preventing cancer recurrence comprises isolated live bacteria. Here, "isolated" refers not to bacteria as part of a fecal transplant, but to bacteria obtained by isolating or extracting (and optionally culturing) bacteria from a natural source. In certain embodiments, the probiotic composition does not contain fecal matter. In other words, the composition contains isolated strains produced in an industrial environment, rather than isolated or cultured from human fecal samples.

[0083] The probiotic ARO112 composition is used to prevent or treat intestinal flora imbalance and / or inflammation.

[0084] In some embodiments of the probiotic ARO112 composition of the present invention used as a medicine, the ARO112 composition is used to prevent or treat intestinal flora imbalance in human subjects.

[0085] Intestinal dysbiosis refers to a range of symptoms, including but not limited to abdominal pain, diarrhea, postprandial discomfort, and malnutrition, all associated with gut microbiota imbalance (also known as dysbiosis syndrome). Dysbiosis is caused by disruption of the gut microbiome, leading to an imbalance, alteration of its functional composition and metabolic activity, or changes in its local distribution. Prevention of dysbiosis is particularly important for patients who have recently received antibiotic treatment. Diagnosis and subsequent treatment of dysbiosis may include treating the aforementioned symptoms or providing a diagnosis based on fecal microbiome analysis.

[0086] In some embodiments of the probiotic ARO112 composition of the present invention used as a medicine, the ARO112 composition is used to prevent or treat intestinal inflammation in human subjects. Intestinal inflammation encompasses a variety of conditions, the most common being inflammatory bowel disease, such as Crohn's disease or ulcerative colitis. Other conditions associated with intestinal inflammation include, for example, obesity, malnutrition, and aging, which are also associated with a range of symptoms, including but not limited to abdominal pain, diarrhea, postprandial discomfort, malnutrition, bloating, and intestinal tissue damage and bleeding.

[0087] The probiotic ARO112 composition is used for the prevention or treatment of intestinal bacterial infections.

[0088] In some embodiments of the use of the probiotic ARO112 composition of the present invention, it is used to prevent intestinal infections caused by pathogenic bacteria. This is particularly relevant to certain high-risk patients, such as those with inflammatory bowel disease (e.g., IBD), or those with a recent history of intestinal bacterial infection (e.g., within the past year, especially within the past two months), or those with post-antibiotic dysbiosis and related discomfort symptoms.

[0089] In some embodiments, the probiotic ARO112 composition may be administered to patients prior to hospital admission to prevent hospital-acquired infections by increasing gut microbiota diversity. In some embodiments, the probiotic ARO112 composition is used after a course of antibiotic treatment, particularly oral antibiotic treatment, to prevent or more quickly clear pathogenic infections that may occur during the recovery of the gut microbiota. In some embodiments where the probiotic composition of the present invention is used as a medicine, it is provided to patients diagnosed with a pathogenic bacterial infection. In certain embodiments, the infectious bacteria are members of the Enterobacteriaceae family.

[0090] In certain embodiments, pathogenic bacteria are infectious agents typically associated with inflammatory diseases characterized by persistent intestinal bacterial infections. In some embodiments, the infection is caused by microorganisms characterized by resistance to antibiotic treatment, such as antibiotic-resistant adhesive invasive Escherichia coli or AIEC (a multidrug-resistant Escherichia coli), and Klebsiella pneumoniae.

[0091] In some implementations, the pathogen is a bacterial species that frequently infects patients after antibiotic treatment, such as Vibrio cholerae or Clostridium difficile. 。

[0092] In some embodiments of the probiotic composition of the present invention used as a medicine, it is used for the prevention or treatment of intestinal infections caused by antibiotic-resistant bacterial species. In a specific embodiment, the ARO112 composition is used for the prevention or treatment of infections caused by multidrug-resistant bacterial species.

[0093] In specific embodiments of the ARO112 compositions used as medicines in the above aspects of the invention, the ARO112 compositions are used to treat or prevent infections caused by common nosocomial pathogenic bacteria. Prophylactic treatment may be particularly useful for preventing recurrent infections and may be provided to the subject several weeks or months prior to scheduled hospital admission.

[0094] In some embodiments, the probiotic compositions of the present invention are used to prevent or treat infections caused by pathogenic bacterial species of the genus *Escherichia coli*, *Citrobacter*, or *Salmonella*.

[0095] In an optional implementation, the probiotic ARO112 composition is used to prevent or treat subjects with an infection caused by Clostridium difficile.

[0096] In some embodiments, the probiotic ARO112 composition is used to prevent or treat intestinal infections caused by vancomycin-resistant Enterococcus species.

[0097] In some embodiments of the probiotic ARO112 composition, it is used to prevent or treat intestinal infections caused by clinically relevant ESKAPEE multidrug-resistant strains selected from Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli.

[0098] In specific embodiments, the probiotic composition ARO112 is used to prevent or treat intestinal infections caused by strains of Adhesive Invasive Escherichia coli (AIEC). These strains are capable of adhering to the mucosal layer of the intestine (ileum and colon), particularly in patients with IBD (CD and UC), causing mucosal damage and inflammation, leading to typical inflammatory flare-ups. These infections are common in IBD patients, especially in CD (Rolhion 2007), as demonstrated in Nod2- / - mice where ARO112 provides beneficial anti-inflammatory effects, as shown in the data provided herein. In some embodiments, the probiotic composition is used to treat infections caused by the AIEC strain LF82, the prototype strain of the pathogenic type of AIEC, to which a single dose of the composition of the present invention can completely replace (…). Figure 9 , Figure 10 This strain has been clinically isolated globally and is the most common pathogenic strain associated with IBD patients (Palmela 2018).

[0099] In some embodiments, the ARO112 composition is used to prevent or treat intestinal infections caused by pathogenic bacteria classified as: enterohemorrhagic Escherichia coli (EHEC), enteroaggregative Escherichia coli (EAEC), enteropathogenic Escherichia coli (EPEC), or urinary tract pathogenic Escherichia coli (UPEC).

[0100] A probiotic ARO112 composition is used to treat antibiotic-associated diseases.

[0101] In some embodiments, the probiotic ARO112 composition is administered to human subjects after a course of antibiotic treatment. In a particular embodiment, the subject has completed a course of antibiotic treatment within the previous month. In a more specific embodiment, the subject has been given the last dose of antibiotic within 48 hours prior to administration of the ARO112 composition. The inventors have successfully tested the administration of a single dose of probiotic 48 hours after the last dose of antibiotic to prevent subsequent Enterobacteriaceae or Vibrio cholerae infections. In an alternative embodiment, the ARO112 composition is used to therapeutically treat patients with persistent recurrent dysbiosis symptoms following antibiotic treatment. In a particular embodiment, the patient has been diagnosed with inflammatory bowel disease such as IBD and has been receiving antibiotics to treat acute exacerbations of symptoms.

[0102] In some embodiments where the ARO112 composition is used as a probiotic or medicine, it is administered to a patient after a course of antibiotic treatment comprising vancomycin, gentamicin, metronidazole, ciprofloxacin, and / or rifaximin. In some embodiments, the composition is used to combat dysbiosis that may occur in patients after receiving combination therapy with vancomycin and gentamicin or metronidazole and ciprofloxacin, which are typically given to patients with IBD. Vancomycin and gentamicin have been successfully tested in a cohort of infants with early-onset IBD, and are therefore a viable and validated treatment for IBD (Lev-Tzion 2017). Metronidazole and ciprofloxacin (alone or in combination) and rifaximin have been used for IBD, and other clinically effective combinations are known in the art (Xiong 2015). The ARO112 composition is sensitive to antibiotics, and it should be understood that administration before or after an antibiotic dose is most appropriate.

[0103] A probiotic ARO112 composition is used to treat inflammatory bowel disease.

[0104] In some embodiments where the probiotic composition containing strain ARO112 is used as a medicine, it is administered to a subject or patient diagnosed with an inflammatory bowel disease. This covers a wide range of diseases, most notably inflammatory bowel diseases such as Crohn's disease or ulcerative colitis. Other diseases associated with bowel inflammation include, for example, obesity, malnutrition, and aging.

[0105] In some embodiments, the probiotic ARO112 composition is used in patients diagnosed with IBD / inflammatory disease and other clinical conditions described herein (e.g., pathogenic bacterial infection or post-antibiotic dysbiosis), particularly in patients with IBD. The efficacy of this strain in reducing inflammation in IBD patients was demonstrated in examples using a Nod2- / - mouse model, a surprising result given the common belief that probiotics exacerbate disease (especially in IBD patients).

[0106] In certain embodiments of the probiotic composition, it is used to treat patients with inflammatory bowel disease, i.e., to alleviate their symptoms, reduce markers of dysbiosis or intestinal pathology, or reduce the frequency of associated intestinal infections. In a particular embodiment, the disease is Crohn's disease. In other particular embodiments, the disease is ulcerative colitis.

[0107] Medical treatment

[0108] Similarly, the present invention also relates to a method for treating dysbiosis, intestinal inflammation, or inflammatory bowel disease in patients in need, the method comprising administering to the patient a probiotic composition comprising a live Klebsiella spp. ARO112 strain as described above.

[0109] Pharmaceutical composition, administration / dosage form and salt

[0110] According to one aspect of the probiotic composition of the present invention, it is provided as a pharmaceutical composition, a pharmaceutical administration form, or a pharmaceutical dosage form. The pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form comprises a live Klebsiella spp. ARO112 strain of the present invention and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0111] In some embodiments of the invention, the live Klebsiella pneumoniae ARO112 strain of the invention is typically formulated into a pharmaceutical dosage form to provide an easily controlled drug dosage and to provide patients with a compact and easy-to-use product.

[0112] The present invention also includes a pharmaceutical composition comprising the live Klebsiella pneumoniae ARO112 strain of the present invention and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

[0113] Some embodiments of the present invention relate to dosage forms for enteral administration, such as nasal, buccal, rectal, transdermal, or oral administration, or suppositories. Furthermore, the pharmaceutical compositions of the present invention can be formulated in solid forms (including, but not limited to, capsules, tablets, pills, granules, powders, or suppositories) or liquid forms (including, but not limited to, solutions, suspensions, or emulsions).

[0114] The dosage regimens of the compounds of this invention will vary depending on known factors, such as the pharmacodynamic properties of the particular agent and its method and route of administration; the recipient's species, age, sex, health status, medical condition, and weight; the nature and severity of symptoms; the type of concomitant treatment; the frequency of treatment; the route of administration; the patient's renal and hepatic function; and the desired effect. In some embodiments, the compounds of this invention may be administered as a single daily dose, or the daily dose may be divided into two, three, or four doses.

[0115] Examples demonstrate that delivering a single dose of a total of 100 million live cells in an active cell culture of the probiotic strain ARO112, 48 hours after the last antibiotic treatment, can shorten the duration of multiple pathogenic infections and increase microbial diversity. Technicians can determine the effective therapeutic dose level by using animal disease models or by referring to live bacterial doses delivered via similar routes of administration in human clinical trials.

[0116] In one set of embodiments, the probiotic composition is formulated for oral delivery to the small or large intestine of a subject, where most of the gut microbiota resides. One such embodiment involves an enteric coating that protects the bacterial composition from the high pH of the stomach and dissolves upon arrival at the intestine. Examples of such coatings include, but are not limited to, polymers and copolymers, such as eudragit (Evonik).

[0117] In similar embodiments, the probiotic composition can be delivered to a specific region of the intestine in the form of a buffered sachet, or via a coating that dissolves within a pH range specific to that particular region of the intestine. For example, formulations that dissolve in a pH range of 6.8 to 7.5 are advantageous for delivery to the colon (for a full description of targeted delivery to the gastrointestinal region, see Villena et al 2015). Int J. Pharm. 487 (1-2):314-9.).

[0118] In another similar embodiment, the probiotic composition may be specifically administered to the intestine via a delayed-release method that takes into account the time required for the probiotic composition to pass through the stomach, small intestine, and colon. Delayed-release formulations include hydrogel formulations and biodegradable, water-soluble, hydrolyzable, or enzymatically degradable polymers. Examples of coating materials suitable for delayed-release formulations include, but are not limited to, cellulose-based polymers, acrylic polymers, and vinyl polymers.

[0119] In another embodiment where the probiotic composition is formulated for delivery to the colon, the formulation comprises a coating that can be removed by enzymes present in the human gut, such as carbohydrate reductases. Examples of enzyme-sensitive coatings include amylose, xanthan gum, and azo polymers.

[0120] In embodiments of the invention relating to rectal administration, the probiotic composition may be formulated as a suppository, enema, or delivered as part of an endoscopic or colonoscopy procedure.

[0121] The therapeutically effective dose of probiotic or pharmaceutical compositions depends on the species, weight, age, and individual condition of the subject, as well as the disease or condition being treated and its severity. A physician, clinician, or veterinarian with general skills can easily determine the effective dose of each active ingredient required to prevent, treat, or inhibit the progression of a disease or condition.

[0122] The probiotic and pharmaceutical compositions of the present invention may contain conventional inert diluents, lubricants or buffers, and adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers. They can be produced by standard processes, such as conventional mixing, granulation, dissolving, or lyophilizing processes. Many such processes and methods for preparing pharmaceutical compositions are known in the art, see, for example, L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

[0123] The manufacturing method and treatment method of the present invention

[0124] Another aspect of the invention also includes the use of the live Klebsiella genus ARO112 strain identified herein in the manufacture of a medicament for the treatment or prevention of diseases such as dysbiosis, intestinal inflammation or inflammatory bowel disease, or in the prevention of post-antibiotic pathology.

[0125] Similarly, the present invention includes a method for treating a patient diagnosed with a disease associated with intestinal inflammation. The method includes administering to the patient an effective amount of a probiotic composition comprising a live Klebsiella spp. ARO112 strain identified herein.

[0126] For any individual feature listed herein as an “implementation” (e.g., the genome coding sequence of the live Klebsiella spp. ARO112 strain used in the composition, a medical indication, or a medical symptom), regardless of any alternatives, it should be understood that these alternatives can be freely combined to form independent embodiments of the invention disclosed herein. Therefore, any alternative implementation for symptoms such as intestinal inflammation can be combined with any alternative implementation for a patient diagnosed with IBD, and these combinations can be combined with any medical indication or formulation of the live Klebsiella spp. ARO112 strain composition mentioned herein.

[0127] The following embodiments and accompanying drawings further illustrate the present invention, from which further embodiments and advantages can be derived. These embodiments are intended to illustrate the invention and not to limit its scope. Attached Figure Description

[0128] Figure 1 This section presents a comparison of Enterobacteriaceae strains from the genera *Klebsiella* and *Escherichia coli*, including clinical isolates of common pathogenic species (*Klebsiella pneumoniae* and *Escherichia coli*), as well as symbiotic species. a) Phylogenetic tree based on whole-genome analysis of the 15 strains shown. b) PCA performed using quantification of genes encoding predicted virulence factors in their genomes, based on a set of public databases.

[0129] Figure 2 This section displays predicted virulence factors encoded by genomes obtained using multiple public databases. Genomic analysis was performed in the PATRIC web browser, from which hit counts for different virulence factor databases were obtained. a) Virulence factor databases (Victors, PATRIC, VFDB); b) Drug target and transporter gene databases (TCDB, DrugBank, TTD); c) Antibiotic resistance databases (PATRIC, CARD, NDARO). d) Comparison of hit counts for different bacterial groups in each database. Two-way ANOVA was used for multiple comparisons with Tukey correction. p<0.0001); different letters indicate significant differences between strains (p<0.05).

[0130] Figure 3 This study demonstrates the phenotypic assessment of nosocomial virulence factors important for host invasion and colonization. A cohort of nine strains (four *Escherichia coli*, three *Klebsiella pneumoniae*, and two non-*Klebsiella pneumoniae*) were tested in enriched and minimal media for the following: a) biofilm formation; b) urease activity; c) siderophore production; and d) resistance to human serum killing or inhibition. Different letters indicate significant differences (p<0.05) between different strains in the same medium, and # indicates significant differences (p<0.05) between different media for the same strain. Kruskal-Wallis test was used for multiple comparisons with Dunn correction. Biofilm was tested in 15 replicates from each group across three independent experiments.

[0131] Figure 4 The results show a) antibiotic resistance profiles in enriched and minimal media, with multidrug-resistant Enterobacteriaceae strains shown in bold (resistant to more than 3 classes of antibiotics). The ability of selected strains to acquire or retain RP4 conjugating plasmids in enriched and minimal media (b) and to retain non-conjugating plasmids in enriched and minimal media (d) were assessed. Multiple comparisons were performed using the Kruskal-Wallis test with Dunn correction. (b) Multiple comparisons were performed using two-way ANOVA with Sidak correction. p<0.05; p<0.01; p<0.0001; c, d). e) Comparison of HGT capacity of selected strains. The ability of selected strains to receive non-conjugated plasmid (pMP7605) was tested by direct or reverse mobilization using the conjugation plasmid (RP4). The ability of ARO112 and EcN to retain the natural multidrug resistance plasmid obtained by conjugation from clinical isolate Ec1898 was tested. Kruskal-Wallis test was used for multiple comparisons with Dunn correction. p<0.05. Two-way ANOVA was used for multiple comparisons with Sidak correction. p<0.01; p<0.0001).

[0132] Figure 5 This study demonstrates the phenotypic analysis of bacteria isolated from fecal samples of single-strain colonized mice. Germ-free mice were colonized with one of the selected strains for 5 days, and bacteria were then isolated from fecal samples. a) urease activity and b) resistance to human serum were assessed. c) Induction of lipid transporter-2 / NGAL in fecal supernatant was evaluated. d) Plasmid retention after bacterial colonization in the intestine was also assessed. Multiple comparisons were performed using the Kruskal-Wallis test and Dunn correction. p<0.05; p<0.01; p<0.001; p < 0.0001; be). Results represent at least three independent experiments. The growth capacity of the indicated strain in the presence of elevated concentrations of lipid transporter-2 / NGAL was assessed in six replicates of two experiments, in enriched or minimal medium.

[0133] Figure 6The following data are displayed: a) Vibrio cholerae load in gnotobiotic mice before (pre-treatment) and one week after treatment (+ARO112). b) Multidrug-resistant Escherichia coli (Ec1898) load in gnotobiotic mice before (pre-treatment) and one week after treatment (+ARO112). c) AIEC CFU per gram of feces in gnotobiotic mice before (pre-treatment) and one week after treatment (+ARO112). d) AIEC CFU per gram of feces in SPF wild-type mice treated with streptomycin (5 g / L in drinking water) for two weeks before (pre-treatment) and one week after treatment (+ARO112). e) AIEC strain load in SPF wild-type mice previously treated with gentamicin (3 mg / kg / d, gavage) and vancomycin (40 mg / kg / d, gavage) once daily for three consecutive days, before treatment with the probiotic strain (pre-treatment) and after 10 and 20 days of treatment (+ARO112) or without this treatment (+PBS). Multiple comparisons were performed using the Kruskal-Wallis test and Dunn correction (e) or the Mann-Whitney test (ac). p<0.05; p<0.01; (p<0.001). Fecal load of Vibrio cholerae in the gnotobiotic model was detected in a total of 8 (pre-treatment) and 6 (+ARO112) samples from two independent experiments (a). Fecal load of Ec1898 in the gnotobiotic model was detected in a total of 8 (pre-treatment) and 5 (+ARO112) samples from two independent experiments (b). Fecal load of AIEC in the gnotobiotic model was detected in a total of 9 (pre-treatment) and 9 (+ARO112) samples from two independent experiments (c). Fecal load of AIEC in the streptomycin-treated model was detected in a total of 14 (pre-treatment) and 7 (+ARO112) samples from two independent experiments (d). Fecal load of AIEC in the gentamicin + vancomycin-treated model was detected in a total of 6 (pre-treatment) and 3 (+ARO112) samples from one experiment (e).

[0134] Figure 7This study demonstrates the protective efficacy of ARO112 and EcN against AIEC colonization in IBD mouse models previously treated with antibiotics. a) Experimental setup: IBD mice were treated with antibiotics, subsequently infected with AIEC, and treated with probiotic strains (ARO112, EcN) or untreated (control). b) AIEC load during the experiment. c) Percentage of mice colonized with AIEC throughout the experiment. d) ARO112 load in the IBD mouse model experiment, and (below) colonization of AIEC (as shown in c) and ARO112 in IBD mice infected with AIEC and treated with ARO112. Two-way ANOVA with Tukey correction was used for multiple comparisons to test for overall differences between treatment groups, and Sidak correction was used to test for differences between different treatment groups at different time points. p<0.05, p<0.001, p<0.0001; 15 (control), 12 (+ARO112) or 9 (+EcN) animals from at least 2 independent experiments (ad).

[0135] Figure 8 This shows the a) richness and b) diversity of the gut microbiota in mice treated with or without probiotics after antibiotic treatment and AIEC infection. c) The relative abundance changes of the most dominant taxa after antibiotic treatment (post-AB), in the treatment group (+ARO112), or in the untreated group (control) compared to pre-antibiotic treatment (pre-AB) levels. The relative abundance changes of d) Pseudomonas members and e) butyrate-producing bacteria depleted by antibiotic treatment are also shown. Multiple comparisons were performed using the Kruskal-Wallis test with Dunn correction (a, b); two-way ANOVA with Tukey correction was also used for multiple comparisons. p<0.05; p<0.01; p<0.001; p<0.0001; ce). f) PCA of microbial community composition. g) Relative abundance changes of taxa associated with butyrate production, h) Pseudomonas and i) other taxa shown. A total of 14 animals from 2 independent experiments (Nod2) - / -Fecal microbiota composition analysis was performed on 14 (before AB), 15 (after AB), 7 (+ARO112), or 7 (control) animals from two independent experiments. Two-way numerical signs (#) indicate significant differences between treatment groups (control vs. ARO112; p < 0.1). Plus signs (+) indicate taxa typically associated with butyrate production.

[0136] Figure 9 This study illustrates intestinal inflammation in mice treated with different probiotics (ARO112, EcN) or untreated (control). a) Intestinal inflammation was measured throughout the experiment using lipid transporter-2 in fecal supernatant. Two-way ANOVA and Tukey's test were used for multiple comparisons. p<0.001; p<0.0001; a total of 11 (control), 8 (+ARO112), or 9 (+EcN) samples from at least 2 experiments.

[0137] Figure 10 The following are experimental setups: a) Wild-type mice treated with streptomycin and colonized with AIEC, then treated with the probiotics ARO112 or EcN. b) AIEC colonization in wild-type mice treated with streptomycin. c) Inflammation in wild-type mice treated with streptomycin and colonized with AIEC before (D4) and after (D12) treatment with the probiotics ARO112 or EcN. d) Wild-type germ-free mice colonized with Klebsiella pneumoniae (Kp1012) and treated with the probiotics ARO112 or EcN. e) Klebsiella pneumoniae colonization in wild-type germ-free mice. f) Inflammation in wild-type germ-free mice colonized with Klebsiella pneumoniae before (D4) and after (D12) treatment with the probiotics ARO112 or EcN. g) Experimental setup of wild-type germ-free mice colonized with *E. coli* (AIEC or Ec1898) and treated with the probiotic ARO112. h) Colonization of *E. coli* (AIEC or Ec1898) in wild-type germ-free mice. i) Inflammation status of wild-type germ-free mice colonized with *E. coli* (AIEC or Ec1898) before (D4) and after (D12) treatment with ARO112. j) IL10 KO mice were treated with vancomycin and gentamicin, colonized with AIEC, and in experimental settings either treated or untreated with the probiotic ARO112. (k) Treatment with vancomycin and gentamicin IL10AIEC colonization in KO mice. l) Mice treated with vancomycin and gentamicin and colonized with AIEC. IL10 Inflammation status in KO mice before (D0) and after (D20) probiotic treatment with ARO112, or in untreated (control) mice.

[0138] Figure 11 This study shows the effects of different probiotic treatments (ARO112, EcN) or no treatment (control) on SCFA levels and intestinal inflammation in mice. a) Metabolomics of butyrate, isobutyrate, acetate, propionate, and succinate in fecal samples from Nod2- / - IBD mouse models that were untreated (control) or treated with probiotics ARO112 (+ARO112) or EcN (+EcN) at the end of the experiment. b) Changes in the relative abundance of potential butyrate-producing bacteria on day 5 post-AIEC infection, which were depleted by antibiotics but recovered during the experiment. c) Metabolomics of butyrate in fecal samples from Nod2- / - IBD mouse models that were untreated (control) or treated with probiotics ARO112 (+ARO112) or EcN (+EcN) on day 5 post-infection (day 4 of treatment). In a and c, the bar charts represent the median. Data analysis employed the Kruskal-Wallis test and used Dunn correction for multiple comparisons. p<0.05; p<0.01; p<0.001; p < 0.0001. Metabolomics analyses were performed in 12 (control), 8 (+ARO112), and 10 (+EcN) samples. In b, different colors represent the log2 fold change for each mouse relative to the pre-AB level—severe depletion (< -5), depletion (< -1), no change or recovery (-1 to 1), enrichment (>1), and severe enrichment (>5). Data analysis was performed using two-way ANOVA with Dunnett correction for multiple comparisons. p<0.1; p<0.01; p<0.001; p<0.0001). Fecal microbiota composition was analyzed in a total of 14 (control), 10 (+ARO112), or 4-10 (+EcN) samples from 2-4 independent experiments.

[0139] Figure 12Mice treated with DSS showed that ARO112 probiotics had a protective effect against colitis. a) Experimental protocol. b) Changes in mouse body weight during the experiment. c) Changes in mouse body weight on day 4 and day 7 (at the end of DSS treatment). d) Disease activity score, derived from the sum of body weight change score and stool consistency score (see supplementary table below; mixed-effects analysis with Sidak correction for multiple comparisons). p<0.01). e) Survival curves show mice euthanized upon reaching the experimental endpoint (weight loss >20% and severe diarrhea with bloody stools); Log-rank (Mantel-Cox) test; p<0.05). f) Colon length of mice that reached the experimental endpoint and were sacrificed at the end of the experiment (Mann-Whitney test); p<0.05).

[0140] Example

[0141] Example 1: The ARO112 strain showed that its genome-encoded predictive virulence factors were fewer than those of the probiotic EcN.

[0142] Genome-wide comparisons were performed on 15 strains: 5 *E. coli* strains, including two of the most common laboratory strains (strain B and strain K-12 MG1655), one commonly used probiotic strain (Nissle 1917), one pathogenic *E. coli* AIEC strain commonly associated with IBD patients (strain LF82), and one *E. coli* strain isolated from a human patient (Ec1898); and 5 non-*Klebsiella pneumoniae* strains, including one acid-producing *Klebsiella pneumoniae* isolated from mouse fecal samples. K. oxytoca (MBC025), a Klebsiella acidogenic strain (DSM5175), and a Klebsiella micranthae strain (McC025). K. michiganensis The type strain (DSM25444) and a strain of Grimalcian lebotomyces ( K. grimontii The model strain (06D021) and a Klebsiella pneumoniae strain ARO112 isolated from mouse fecal samples; and five Klebsiella pneumoniae strains, including a model strain (NCTC9633), two strains isolated from human samples and commonly used in laboratory studies (ATCC43816 and MH258), and two strains isolated from human patients (Kp1012 and Kp834). Phylogenetic tree ( Figure 1a) showed that *E. coli* Nissle 1917 and AIEC strains were closely related, and more distantly related to laboratory strains and human isolate Ec1898. In the *Klebsiella pneumoniae* clade, three human isolates (MH258, Kp1012, and Kp834) clustered together, with two other strains forming another cluster. Among non-*Klebsiella pneumoniae* strains, MBC025 clustered with the *Klebsiella acidogenic* model strain, while *Klebsiella pneumoniae* ARO112 clustered with the *Klebsiella grimonosa* model strain, the latter clustering more closely with the *Klebsiella micrantha* model strain. Nine databases reported in the PATRIC Genome Browser software were used as proprietary genes in each of the above genome overview tabs ( Figure 2 After evaluating the predictive virulence factors encoded by the genome, principal component analysis (PCA) was performed. Figure 1 b). PCA results based on genome-predicted virulence factors showed that *E. coli* strains were clearly separated from *Klebsiella* strains, and slight separation was also observed between *Klebsiella pneumoniae* and non-*Klebsiella pneumoniae* strains, while strain ARO112 was even more distant from *Klebsiella pneumoniae* strains. Interestingly, when analyzing each strain cluster according to different virulence factor databases (virulence factor databases (…),… Figure 2 The results showed that significant differences were only observed in non-Klebsiella pneumoniae strains, with acid-producing Klebsiella strains being significantly different from the Klebsiella grimonis type strain. ARO112 was the only strain that showed no significant differences from the other five strains in the cluster. Klebsiella pneumoniae strains showed some significant differences in assessing predicted drug targets and transporters. Figure 2 b). However, antibiotic resistance databases show significant differences between strains in the Klebsiella pneumoniae and Escherichia coli clusters ( Figure 2 c), of which 3 strains of Klebsiella pneumoniae and 1 strain of Escherichia coli isolated from human patients showed the predicted total number of antibiotic resistances.

[0143] Example 2: Nosocomial infection and colonization-related virulence factors exhibited environment-dependent variability.

[0144] Using this genomic analysis and results from databases as a scaffold, a subset of nine strains were tested in the laboratory to detect virulence factors commonly associated with Enterobacteriaceae, with a focus on features that may be associated with increased colonization and / or nosocomial infections. Figure 3 a).

[0145] The strains included four *Escherichia coli* strains (MG1655, AIEC, EcN, Ec1898), three *Klebsiella pneumoniae* strains (MH254, Kp1012, Kp834), and two non-*Klebsiella pneumoniae* strains (MBC025, ARO112), and were tested for selected characteristics commonly associated with nosocomial infection and colonization. Bacterial biofilms are generally considered a virulence factor, a view supported by extensive studies comparing pathogenic strains to non-pathogenic or symbiotic strains. The ability of these strains to form biofilms in enriched and minimal media was tested. Unsurprisingly, the *E. coli* strains exhibited weak biofilm-forming ability, producing low biomass communities in all test media. We measured the phenotype at 37°C, simulating the human body temperature, while reports indicate that *E. coli* strains show increased biofilm biomass even in enriched media at lower temperatures (approximately 25°C) (Mathlouthi, 2018). In fact, in enriched media, most strains produced low (EcN, Kp834, MBC025, ARO112) or very low (Ec1898, KpC) biofilm biomass. Figure 3 a), only MG1655, AIEC and Kp1012 have moderate biofilm formation capabilities ( Figure 3 a). In minimal culture media, E. coli strains exhibit low biofilm formation ability, similar to Kp834 ( Figure 3 a), while MH254, MBC025, and ARO112 formed medium-sized biofilms, and Kp1012 exhibited very strong biofilm biomass (a). Figure 3 a).

[0146] Urease production is another phenotype closely associated with virulence, particularly in Enterobacteriaceae strains. This enzyme catalyzes the hydrolysis of urea into ammonia and carbon dioxide, leading to phosphate formation and alkalization of urine. These salts deposit within the catheter, making bacteria more susceptible to adhesion and biofilm formation, thus hindering treatment. Interestingly, after 24 hours of incubation in enriched media, urease activity was undetectable in all strains except MBC025 and ARO112 (which showed milder urease activity). Figure 3 b). However, when urease activity was measured in cultures cultured in minimal medium, all Klebsiella strains (but not Escherichia coli strains) showed significantly elevated values, with Kp1012 exhibiting the highest activity, followed by MBC025 (a known urease-producing strain), while Kp834, MH254, and ARO112 showed low activities. Figure 3(b) It is expected that *E. coli* strains will lack urease activity, as this species is known not to produce urease, although some clinical isolates of pathogenic *E. coli* (e.g., *EHEC*) have shown activity. As previously mentioned, these two virulence factors can be interrelated in urinary tract infections, and strains possessing both may pose a problem. However, individually, these properties can be used to protect intestinal functions, such as the stress-resistant bacterial flora resulting from biofilm formation, and also to protect the host epithelium from bacterial invasion; or to degrade intestinal urea into carbon dioxide and ammonia, which bacteria can use as a nitrogen source, potentially giving them a competitive advantage over invading bacteria.

[0147] Another interaction between symbiotic bacteria, pathogenic bacteria, and the host is driven by iron availability. Iron is essential for a variety of ubiquitous physiological processes, and the host regulates iron availability in several ways, such as by quenching siderophores and by secreting lipid transporter 2 (Lcn2). Lcn2 is also associated with increased inflammation and is the most commonly used biomarker for assessing intestinal inflammation in fecal samples. Siderophores unaffected by Lcn2 quenching capacity are another potential virulence factor, and their production was detected in enriched and minimal media. Enriched media (iron-free) reduced siderophore production in all strains. Figure 3 c). In minimal culture, siderophore production was generally increased. Except for MG1566, MBC025, and ARO112, most strains showed a significant increase in siderophore production ( Figure 3 d), while siderophore production of MG1566, MBC025 and ARO112 remained at low levels during growth in enriched media ( Figure 3 c), which suggests that limiting iron availability does not appear to affect their growth.

[0148] Some bacteria can resist the killing effect of innate immune complement, for example, through the O antigen in bacterial lipopolysaccharide. Serum resistance tests performed in enriched and minimal media showed that only *Klebsiella pneumoniae* and strain MBC025 exhibited resistance and were able to proliferate in the presence of mixed human serum. Figure 3 (d) This indicates their potential ability to survive and multiply in patient blood. Conversely, most E. coli strains (MG1655, AIEC, EcN) are highly sensitive to human serum, while strains Ec1898 and ARO112, although resistant to human serum, cannot multiply in it. Figure 3 d). Interestingly, tests on the same strain cultured in minimal medium revealed that MH258 exhibited significantly enhanced resistance and proliferation, increasing from 40-fold to 140-fold. Figure 3d); however, what is even more surprising is the change in EcN: EcN showed strong sensitivity to human serum when cultured in enriched medium (100-fold decrease), but showed strong resistance and proliferation (70-fold increase) when cultured in minimal medium; Figure 3 d). Typically, human serum susceptibility testing is performed by culturing bacteria in enriched media. These results suggest that for some bacteria, such as EcN and MH258, resistance is significantly enhanced when strains are cultured in minimal media, potentially biasing previous analyses of this virulence-related phenotype. Finally, ARO112 did not show signs of being killed by human serum, but its proliferation levels in both media did not reach those of most other tested strains. Figure 3 d). Since the importance of this virulence factor is not located in the gut, there is no reason to link it to the potential protective function of symbiotic bacteria. In fact, even for symbiotic or protective bacteria, the ability to proliferate in response to human serum is not an ideal property, as there is no foreseeable benefit to the host, and the disadvantages of the presence of proliferating bacteria in the blood are obvious: they provide pathways for bacterial infection of different organs or lead to sepsis.

[0149] Example 3: Antibiotic resistance in clinically isolated bacteria differs from that in non-clinically isolated microbiome members.

[0150] The main clinical problem for Enterobacteriaceae is antibiotic resistance and their ability to efficiently acquire antibiotic resistance genes from other bacteria through mechanisms such as horizontal gene transfer. Antibiotic resistance was analyzed in enriched and minimal media for these strains. MG1655, EcN, MBC025, and ARO112 were not multidrug resistant because they were resistant to fewer than three classes of antibiotics. Figure 4 This differs from clinically isolated bacteria. Interestingly, EcN and ARO112 showed increased resistance to both classes of antibiotics (aminoglycosides and macrolides) when tested in minimal media, but exhibited sensitivity when tested in enriched media. Figure 4 a). The other five tested strains exhibited resistance to three (AIEC) or more than three antibiotics (Ec1898, Kp1012, Kp834, MH254), which was expected given that these strains were all clinical isolates, some of which were known to be multidrug resistant to antibiotic treatment. Compared to culture in enriched medium, Ec1898, Kp1012, and KpC showed an increase in the number of antibiotic classes they were resistant to when cultured in minimal medium, while only Kp834 lost its resistance phenotype when cultured in minimal medium. Figure 4 a). These results suggest that the stress from antibiotics, coupled with the additional stress from minimal culture media, can enhance the resistance phenotype.

[0151] However, the absence of resistance in symbiotic nonclinical strains does not preclude the possibility of antibiotic resistance acquisition, a common occurrence among Enterobacteriaceae in complex microbiota, such as the gut microbiota. To assess the ability of ARO112, EcN, and Kp1012 to acquire resistance from other bacteria via horizontal gene transfer, we examined the conjugation of a commonly used plasmid (RP4) from the donor strain (E. coli K-12 MG1655). Interestingly, the Kp1012 strain, already possessing multidrug resistance, showed the highest acquisition rate, with almost all CFUs indicating plasmid acquisition from the donor strain. Figure 4 b). The transfer rate of EcN was significantly low, with a median of 25% of the bacterial population acquiring plasmids and thus acquiring antibiotic resistance. Figure 4 b), while ARO112 showed strong acquisition resistance, with a median plasmid acquisition rate of less than 4% (b). Figure 4 b). After daily subculturing for 5 days in enriched or minimal medium, the retention of the obtained plasmids was examined. The results showed that the multidrug-resistant strain Kp1012 almost completely lost its plasmids, while the antibiotic resistance strain obtained from EcN showed higher plasmid loss, with 39% and 66% of the bacterial population retaining the plasmids in enriched and minimal mediums, respectively. Figure 4 c). ARO112 again exhibited the highest antibiotic resistance transfer, with only 13% and 35% of the bacterial population retaining the acquired resistance in enriched and minimal media, respectively. Figure 4 c). Although half of the known plasmids appear to be non-transferable, they can still be shared between bacteria through conjugation events, or transferred via conjugation plasmids from donor strains (direct mobilization). Figure 4 e), or through the conjugation pathway provided by the recipient strain's own conjugation plasmid (reverse mobilization). Figure 4 e). Therefore, these plasmids can enter other bacterial cells, providing them with an increased spectrum of antibiotic resistance. The ability of ARO112, EcN, and Kp1012 to retain the non-conjugating plasmid (pMP7605) carrying antibiotic resistance indicates that the conjugating plasmid Kp1012 was retained in almost all cells, whether in enriched or minimal media. Figure 4 d). Similar to the retention of conjugating plasmids, only 58% of EcN colonies retained non-conjugating plasmids in enriched media. Figure 4 c), but unlike the results with conjugating plasmids, all EcN colonies retained the non-conjugating plasmid when cultured in minimal medium. Figure 4 c). Strain ARO112 again exhibited the highest plasmid clearance rate, with 17% and 49% of the colony retaining non-conjugating plasmids in enriched and minimal media, respectively. Figure 4 c).

[0152] Direct and reverse mobilization of plasmids showed that the transfer rates to all three strains via these mechanisms were extremely low, with the direct mobilization rate of ARO112 being significantly lower than that of Kp1012. Figure 4 e). We also determined the retention capacity of a natural plasmid transferred from a multidrug-resistant clinical isolate, Ec1898, in ARO112 and EcN (Kp1012 was not tested due to its multidrug resistance). Natural plasmids are known to offer a certain adaptive advantage, allowing them to persist, despite the transfer costs (Alonso-del Valle, 2021). EcN retained the plasmid in all tested CFUs, while ARO112 lost the plasmid in more than 15% (enriched medium) and 30% (minimum medium) of the colony. Figure 4 e).

[0153] Intestinal colonization in mice can alter phenotypic traits.

[0154] To determine whether enriched or minimal laboratory media more closely approximate the expected phenotypic characteristics of bacteria in the mammalian gut, bacteria isolated directly from monocolonized mice were used for testing, without the need for culture. Three representative strains (ARO112, EcN, and Kp101) were colonized in germ-free mice for 5 days, after which strains were extracted from fecal samples without screening or culture. Urease activity ( Figure 5 a) and resistance to human serum ( Figure 5 b) showed that the urease activity of Kp1012 was as elevated as that of cultures cultured in minimal medium, while EcN reproduced the low urease activity of laboratory cultures. ARO112, isolated from fecal samples, exhibited a variable phenotype, with half of the samples showing low urease activity, similar to the results obtained from laboratory cultures, while the other half showed elevated urease activity. Figure 5 a). Although this result is surprising due to the low urease activity level of ARO112 cultured in the laboratory, it also suggests that ARO112 has potential competitive properties that could be beneficial to both the bacteria and the host, especially given its low biofilm-forming ability, unlike Kp1012, which produces large amounts of urease and also forms strong biofilms, thus increasing the likelihood of urinary tract infections.

[0155] The same samples were used to test for resistance to human serum after intestinal colonization, and the results were similar to those obtained from laboratory-cultured cultures. Figure 5 b). The resistance and proliferation characteristics of ARO112 and Kp1012 were similar to those of laboratory-cultured cultures; EcN isolated from the intestine exhibited resistance and proliferated after 3 hours of treatment with human serum ( Figure 5(b) This is similar to cultures cultured in minimal medium, but different from sensitive cultures cultured in enriched medium. In fact, the resistance profile of ARO112 extracted from the mouse intestine colonized by a single bacterium was significantly lower than that of EcN ( Figure 5 b).

[0156] As mentioned above, host production of Lcn2 is a strategy to control bacterial growth, and elevated levels of this protein in feces are an indicator of increased intestinal inflammation. We then examined the ability of three selected bacterial strains (ARO112, EcN, and Kp1012) to induce Lcn2 production and intestinal inflammation in mice colonized with a single strain. Fecal supernatant five days after colonization showed that, unlike colonization with EcN and Kp1012 (which induced even greater inflammation), colonization with ARO112 did not induce inflammation. Figure 5 d). Interestingly, siderophore production was detected in the same fecal supernatant, and all three strains showed low values ​​(data not shown). For symbiotic or protective bacteria, the lack of significant siderophore production and the absence of host-induced Lcn2 and inflammation are host-friendly characteristics, as bacterial-induced inflammation is a major health concern, particularly in certain inflammatory bowel diseases such as IBD and obesity. However, low siderophore production and low Lcn2 induction levels suggest that strain ARO112 may be more sensitive to elevated Lcn2 levels in an inflamed gut compared to strains that produce higher levels of siderophores and induce Lcn2 (such as EcN and Kp1012). ARO112, EcN, and Kp1012 were cultured in enriched and minimal media, respectively, in the absence of Lcn2 or without increased Lcn2 concentrations. Interestingly, ARO112 exhibited higher overall resistance to elevated Lcn2 levels. Figure 5 (e) This indicates that its inability to produce higher levels of siderophores or induce inflammation does not make it more sensitive to the gut environment. For potentially protective bacteria, the ability to compete with invading bacteria for iron without having to compete with other symbiotic microorganisms and the host is an advantage.

[0157] By colonizing germ-free mice with strains carrying the non-conjugating plasmid, and quantifying the proportion of bacteria retaining the plasmid 5 days after colonization, the retention of the non-conjugating plasmid in the mouse intestine was detected. Kp1012 and EcN retained the plasmid in almost all cells, while ARO112 retained it in less than 1% of its cells. Figure 5 (d) This indicates that the resistance of ARO112 to the acquired antibiotic resistance increases exponentially in vivo.

[0158] In summary, these results indicate that commonly used probiotic strain EcN exhibits higher resistance and proliferation potential in the presence of human serum, while also demonstrating significant ability to acquire and retain antibiotic resistance.

[0159] Example 4: ARO112 promotes faster recovery after infection

[0160] Previous research by the inventors showed that ARO112 could partially replace the single bacterial strain *Escherichia coli* MG1655 in the intestines of germ-free mice (Oliveira, 2020). Surprisingly, in further testing, we found that two other *E. coli* strains, namely the multidrug-resistant clinical isolate *Ec1898* and the IBD-associated pathogenic commensal *AIEC*, as well as the non-Enterobacterial human pathogen *Vibrio cholerae*, were also replaced even more rapidly, with *Vibrio cholerae* being replaced by ARO112 more than 37-fold. Figure 6 a). The tested E. coli strains showed different effects, with Ec1898 being replaced by ARO112 by more than 4 times, and by EcN by more than 5 times. Figure 6 b). Under these conditions, neither ARO112 nor EcN produced clinically significant replacement of AIEC ( Figure 6 c).

[0161] This prompted the inventors to test another colonization model in mice, namely SPF wild-type mice treated with streptomycin, in which ARO112 was also able to replace MG1655 (Oliveira, 2020). Similar to the results obtained in the sterile model, neither ARO112 nor EcN could replace AIEC (…). Figure 6 d). The inventors tested a similar rat intestinal colonization model by treating SPF wild-type mice with a combination of non-absorbable antibiotics, vancomycin and gentamicin, which closely resembled the clinical presentation of AIEC colonization and had been shown to affect intestinal colonization resistance. Under this antibiotic regimen, AIEC was displaced from the rat intestine regardless of the presence of ARO112. Figure 6 This is consistent with published data (Drouet, 2012). This difference between antibiotic regimens in the same mouse model suggests that permanent colonization of AIEC depends on the microbiome composition resulting from antibiotic treatment and its subsequent recovery.

[0162] Previously published experiments also showed that, under this antibiotic regimen, the gene-based IBD mouse model Nod2 - / - Mutant mice were more susceptible to AIEC colonization, and this pathogenic commensal bacterium exhibited a slower replacement process (Drouet, 2012). To examine whether ARO112 or EcN could aid in the slow recovery of colonization-resistant microbiota function in this mouse model (…), Figure 7a) We simulated a common IBD-related episode: Nod2 was treated with the antibiotics vancomycin and gentamicin via gavage once daily for three consecutive days. - / - Mice (Drouet, 2012) were then administered AIEC strain LF82 via gavage. The following day, mice were administered PBS (control), ARO112 strain, or probiotic EcN strain via gavage, respectively. Figure 7 a). Subsequently, we tracked the colonization of AIEC using a selective inoculation method and found that the decolonization rate of AIEC in mice treated with ARO112 was significantly faster. After 8 days of infection (7 days of treatment), the median number of AIEC had dropped to undetectable levels; while most mice that did not receive probiotic treatment required a median of 14 days to clear the infection. Figure 7 b). Interestingly, the treatment with the probiotic EcN yielded worse results, with all test mice remaining infected throughout the 20-day experimental period. Figure 7 b). Assessment of the number of mice clearing infection under each condition revealed that EcN treatment promoted persistent AIEC infection, while only half of the mice without probiotic treatment cleared the infection. This was still worse than the results with ARO112 treatment, where 10 out of 12 mice (>83%) treated with ARO112 had no AIEC by the end of the experiment. Figure 7 c). Interestingly, ARO112 itself was cleared from the gut microbiota throughout the experiment. It took a median of 10 days after AIEC infection for the ARO112 load in most mice to drop below detectable levels. By the end of the experiment, ARO112 had disappeared in 9 out of 12 mice (75%). Figure 7 d).

[0163] Example 5: ARO112 treatment promotes the restoration of the microbiome from antibiotic-induced imbalance and promotes butyrate production. Recovery of bacteria

[0164] Antibiotic treatment leads to gut microbiota dysbiosis, typically resulting in reduced microbial community diversity and richness, allowing invasive bacteria (such as AIEC strains) to colonize the gut. Mice treated with ARO112 showed restored microbiota parameters in both richness and diversity indices, while these parameters continued to decline in mice not treated with probiotics, the latter being similar to the low levels induced by antibiotic treatment. Figure 8 a, 8b). ARO112 treatment promotes microbiome restoration ( Figure 8 c) could explain the faster replacement of AIEC in this group and the replacement of ARO112 itself due to the colonization resistance provided by the restored microbial community.

[0165] Of the 18 taxa with the highest abundance in our experimental environment, 10 (56%) were depleted due to antibiotics, 6 (33%) were enriched, and 2 (11%) remained unchanged. Figure 8 c). In the control group mice that did not receive any probiotic treatment, 11 (61%) and 4 (22%) taxa were depleted or enriched at the end of the experiment, respectively, while 3 (17%) taxa showed levels similar to those before antibiotic treatment. Figure 8 c). In mice treated with strain ARO112, 6 (33%) and 3 (17%) taxa showed depletion or enrichment, respectively, and 9 (50%) taxa recovered to pre-antibiotic levels. Figure 8 c). Furthermore, among the taxa depleted by antibiotic treatment, five taxa recovered ( Figure 8 c). PCA showed that antibiotic treatment (after AB) resulted in a significant deviation of the microbiota composition from the original microbiota before antibiotic treatment (before AB). Mice that did not receive probiotic treatment (control) showed only partial recovery and were very random, with no clustering, while mice treated with ARO112 (+ARO112) resulted in microbiota clustering that was closer to the pre-treatment samples. Figure 8 f).

[0166] IBD patients often exhibit a high proliferation of Pseudomonas species, particularly after antibiotic treatment, which is concerning because several members of this phylum have pathogenic potential. Our experiments show that antibiotics can lead to the enrichment of three taxa within the Pseudomonas phylum, including Proteus (…). Proteus ) and Escherichia coli / Shigella ( Escherichia / Shigella At the end of the experiment, the levels were significantly reduced, regardless of whether probiotic treatment was received. Figure 8 c, 8d). However, only ARO112 treatment resulted in low levels similar to those before antibiotic treatment. Antibiotics did not increase other Pseudomonas taxa, while Klebsiella pneumoniae were enriched after probiotic treatment, which was expected since ARO112 belongs to that taxa ( Figure 8 g).

[0167] Further analysis of the bacterial genome affected by antibiotic treatment revealed that, among the seven known butyrate-producing taxa, five were depleted due to antibiotics. Figure 8 e), while the other two are enriched ( Figure 8 g). ARO112 treatment resulted in a significant recovery of three butyrate-producing bacteria that had been depleted by antibiotics, namely the Trichophyceae family ( Lachnospiraceae Rumenococci ( Ruminococcaceae ) and Vibrio spp. ( Oscillibacter () Figure 8e). Interestingly, mice that did not receive ARO112 treatment did not recover any depleted butyrate-producing bacteria ( Figure 8 e). The effect of ARO112 treatment in restoring and increasing butyrate-producing bacteria depleted by antibiotics is very encouraging, as studies have shown that butyrate is an important metabolite of IBD, and even that oral administration of butyrate can clinically improve IBD in patients. ARO112 has the advantage of promoting producers in the microbiome, and therefore has the potential to lead to a sustained increase in butyrate in the microbiome without the need for butyrate administration.

[0168] like Figure 11 As shown, the SCFA levels and intestinal inflammation in mice after receiving different probiotic treatments (ARO112, EcN) or no treatment (control) are illustrated. Figure 11 a shows the metabolomics of butyrate, isobutyrate, acetate, propionate, and succinate in fecal samples of Nod2- / - IBD mouse models that were untreated (control) or treated with probiotics ARO112 (+ARO112) or EcN (+EcN) at the end of the experiment. Figure 11 b shows the relative abundance changes of potential butyrate-producing bacteria on day 5 after AIEC infection, which were depleted after antibiotic treatment but recovered during the experiment. Figure 11 Figure c shows the metabolomics of butyrate in fecal samples from Nod2- / - IBD mouse models on day 5 post-infection (day 4 of treatment): untreated (control), treated with the probiotic ARO112 (+ARO112), or treated with EcN (+EcN). The bars in Figures a and c represent medians. Data analysis was performed using the Kruskal-Wallis test with Dunn correction for multiple comparisons. p<0.05; p<0.01; p<0.001; p < 0.0001. Metabolomics analysis was performed in 12 (control), 8 (+ARO112), and 10 (+EcN) samples. In Figure b, different colors represent the log2 fold change for each mouse relative to the pre-AB level—severe depletion (< -5), depletion (< -1), no change or recovery (-1 to 1), enrichment (>1), and severe enrichment (>5). Data analysis was performed using two-way ANOVA with Dunnett correction for multiple comparisons. p<0.1; p<0.01; p<0.001; (p<0.0001). Fecal microbiota composition was analyzed from a total of 14 (control), 10 (+ARO112), or 4-10 (+EcN) samples from 2-4 independent experiments. Indeed, as the data above show, the inventors found that an increase in butyrate-producing bacteria led to an increase in the production of butyrate and other short-chain fatty acids, which were promoted by ARO112 treatment.

[0169] Example 6: Unlike EcN, ARO112 does not induce intestinal inflammation.

[0170] Although treatment with the potential probiotic ARO112 has shown increased microbiota restoration and pathogen clearance, for this treatment to be beneficial to IBD patients, it should not exacerbate IBD symptoms, such as intestinal inflammation. ARO112 reduces the production of Lcn2 (an indicator of intestinal inflammation) in microbiota-deficient mice; however, IBD mice have resident microbiota and therefore exhibit different inflammatory characteristics. Therefore, the inventors evaluated the intestinal inflammatory response in IBD mice infected with AIEC and untreated, treated with ARO112, or treated with EcN. Interestingly, EcN treatment not only led to persistent AIEC infection but also to a persistent exacerbation of intestinal inflammation. Figure 9 This differs from the untreated control group, whose intestinal inflammation remained at a low level, with a significant and transient increase only occurring on day 5 of infection. Figure 9 a). More importantly, mice treated with ARO112 showed persistently low levels of intestinal inflammation, which remained largely unchanged compared to pre-antibiotic levels. Figure 9 a). The inventors tested intestinal inflammation in multiple models and found that treatment with the probiotic ARO112 did not increase inflammation in any of the tested models, regardless of whether the pathogen was cleared. In contrast, no treatment or treatment with the probiotic EcN appeared to lead to increased inflammation. Figure 10 g-10i).

[0171] Materials and Methods

[0172] Genomic-based virulence factor prediction

[0173] The genomes of all strains were uploaded to the PATRIC BV-BRC browser software (v3.29.20) for analysis. In the overview tab of each genome, the number of predicted virulence factor genes hit in the plotted database was quantified using proprietary gene features.

[0174] Biofilm formation

[0175] In summary, the biofilm formation process is as follows: Bacterial cultures were cultured at 37°C with shaking for 24 hours in either enriched medium (lysogen broth, LB) or minimal medium (M9 salt). The following day, each culture was washed once with sterile PBS, and the final OD was determined. 600 Adjust the pH to 0.05 in an appropriate culture medium, then aliquot 200 μL of culture into each well of a 48-well plate. After bacterial adhesion, carefully remove and discard the supernatant, and use 200 μL of water... Wash each well once with 500 μL of sterile PBS, then carefully add 500 μL of sterile, fresh, appropriate culture medium to each well. Incubate the plate at 37°C for 24 hours, then remove and discard the supernatant, and carefully wash the bottom of each well with 500 μL of sterile PBS. Carefully stain each well with 0.1% crystal violet solution and incubate the plate at room temperature in the dark for 20 minutes. After washing and drying, destain each well with 33% glacial acetic acid and incubate at room temperature for 15 minutes, then read the OD. 580 (Peak of crystal violet staining) to quantify biofilm biomass.

[0176] Urease activity

[0177] Cultures were incubated at 37°C with shaking for 24 hours in either enriched or minimal medium, washed once with sterile PBS, and OD was collected. 600 The value was adjusted to 1, and then each culture was diluted 20-fold in urease medium and incubated at 37°C with shaking for 24 hours. The OD values ​​of the cultures were then measured. 560 Urease activity was quantified. For the determination of urease activity in bacteria extracted from single-cell colonized mouse fecal samples, a concentration of 2.5 × 10^7 cells / ml was used. For the determination of urease activity in bacteria extracted from single-cell colonized mouse fecal samples, a cell suspension was prepared directly from the extracted bacteria, as described above, instead of using cultured cultures.

[0178] Resistance to pooled human serum

[0179] Cultures were incubated at 37°C with shaking in either enriched or minimal medium for 24 hours. The cells were washed once with sterile PBS and adjusted to a concentration of 2 × 10^7 cells / ml. Then, 25 μl of the cell suspension in PBS was added to a final concentration of 75 μL. The cultures were mixed with human serum solution. After incubation at 0 hours and 37°C for 3 hours, the cultures were plated to assess bacterial killing, resistance, or proliferation in the presence of human serum.

[0180] Ferrocarrier generation

[0181] Cultures were incubated at 37°C with shaking for 24 hours in either enriched or minimal medium. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter. The cell-free supernatant was mixed with Cas-PIPES solution at a 1:1 (v:v) ratio and incubated at room temperature for 20 minutes, followed by measurement of OD. 630 The value is used to calculate the percentage of ferrocarrier production.

[0182]

[0183] Lipid transporter protein-2 / NGAL enzyme-linked immunosorbent assay (ELISA)

[0184] Fecal samples were resuspended in sterile PBS and mechanically homogenized. Samples were centrifuged at 18,000 xg for 15 minutes at 4°C, and then homogenized using 0.22... Filtration using a membrane filter. The ELISA for Lipocarrier Protein-2 / NGAL (Human Lipocarrier Protein-2 / NGAL Duoset ELISA) was performed according to the manufacturer's instructions.

[0185] In vitro growth of lipid transport protein-2

[0186] The cultures were placed in enriched or minimal medium and incubated at 37°C with shaking for 24 hours. They were then washed once with sterile PBS and adjusted to the final OD value in appropriate mediums with different concentrations of lipid transporter-2 protein. 600 The value was 0.05. The culture was incubated in 96-well plates for 24 hours, and the OD was measured periodically using a Biotek Synergy H1 microplate reader. 600 The final OD600 value was compared with the growth of the control without lipotransporter-2. If the growth was higher than 80% of the control, it was considered resistant; higher than 40% of the control, it was considered moderate; and lower than 40% of the control, it was considered sensitive.

[0187] Antibiotic spectrum

[0188] The cultures were incubated at 37°C with shaking for 24 hours in either enriched or minimal medium, washed once with sterile PBS, and then adjusted to the final OD value in appropriate media containing different concentrations of antibiotics. 600 The value was 0.05. After incubating the culture in a 96-well plate with shaking for 24 hours, the OD was measured using a Multiskan microplate reader. 600 Value. If a strain grows at least 80% of the growth of the control (without antibiotics) in the presence of antibiotics, it is considered resistant. If a strain grows more than 40% of the growth of the control in the presence of antibiotics, it is considered moderate; if it grows less than 40% of the growth of the control, it is considered sensitive.

[0189] Plasmid conjugation experiment

[0190] The culture was cultured in enriched medium at 37°C with shaking for 24 hours, then washed once with sterile PBS. Donor and recipient strains were thoroughly mixed at a 1:1 ratio (total cell count 10^8 cells), centrifuged at 14,000 rpm for 30 seconds, and resuspended in 20 μL PBS. Two 10 μL drops were inoculated onto an agar plate, air-dried, and incubated at 37°C for 1 hour. After incubation, the droplets were thoroughly removed with an inoculation loop and resuspended in 1 mL sterile PBS. Serial dilutions were performed and plated to count CFUs in recipient strains carrying (selective plasmid plasmid-borne antibiotic resistance) and those not carrying (non-selective or selective plasmid plasmid plasmid-borne antibiotic resistance) the test plasmid. The conjugation plasmid (RP4) exhibits resistance to tetracycline (10^6)... g / ml), kanamycin (50 g / ml) g / ml) and ampicillin (100 g / ml) Drug resistance (g / ml).

[0191] plasmid retention experiment

[0192] The bacterial strain carrying the test plasmid was cultured at 37°C with shaking in an antibiotic-enriched medium or minimal medium (for antibiotic resistance of the plasmid) for 24 hours, then washed with sterile PBS and adjusted to the final OD value in an appropriate antibiotic-free medium. 600 The value was 0.05. Serial dilutions were inoculated onto selective and non-selective LB agar plates to assess CFUs with (plasmid retention) and without (plasmid loss) antibiotic resistance. Cultures were incubated at 37°C with shaking for 24 hours. Every 24 hours, the culture was diluted 1:100 in fresh, appropriate medium and re-incubated, and then serial dilutions of the 24-hour culture were plated as described above. The experiment ended after 5 days. The non-conjugating plasmid (pMP7506) carried resistance to gentamicin (30 μg / ml).

[0193] Fecal microbiota extraction

[0194] Fecal particles were collected from mice colonized with single bacteria for the extraction of live intestinal bacteria. The particles were weighed and 500 μl of sterile PBS was added. The particles were then mechanically disrupted using an electric grinder, followed by the addition of another 500 μl of sterile PBS. The samples were subjected to four replicates of vortexing for 15 seconds, centrifugation at 1000 rpm for 30 seconds at room temperature, and 750 μl of fragment-free cell-containing supernatant was collected into a new tube (this volume was replaced with sterile PBS before the next replicate). After recovering 3 ml of each sample, the separated cells were centrifuged at 4000 g for 5 minutes at room temperature, the supernatant was discarded, and the cell fractions were resuspended in 1 ml of sterile PBS supplemented with glycerol and cysteine ​​(final concentrations of 20% and 0.1%, respectively).

[0195] Animal experiments

[0196] At the Gulbenkian Institute of Science All mouse experiments conducted by the IGC (Institute of Infectious Diseases, IGC) were approved by the institution's ethics committee and the Portuguese National Entity (Dire). o Geral de Alimenta oe Veterin The approval of (ria; 015190) complies with European Council Directive 86 / 609 / EEC. It uses 6-8 week old male C57BL / 6J mice (wild-type or Nod2). - / - They were then randomly assigned to experimental and control groups. All experiments used C57BL / 6J mice.

[0197] C57BL / 6J Nod2 were raised under SPF conditions - / - Mice. Gentamicin (3 mg / kg / day) and vancomycin (40 mg / kg / day) were administered orally once daily for three consecutive days (Drouet, 2012). Two days after the last oral administration of antibiotics, mice were administered approximately 10 mg / kg / day orally. 8 The AIEC strain of CFU (LF82-strepR) was administered to mice orally via gavage the following day, either as a control or approximately 10 mg / L PBS. 8 CFU of ARO112 or EcN strains. Fecal samples were collected at the time points shown in the corresponding charts and inoculated into selective media to assess the colonization level of AIEC. In addition, fecal samples were used to detect intestinal inflammation levels (lipocaloric transporter-2 / NGAL ELISA) and to determine the composition of the gut microbiota.

[0198] C57BL / 6J germ-free mice were housed and bred in a sterile isolator (La Calhene / ORM) and then transferred to a sterile ISOcage (Tecniplast). Approximately 10 mg of [unspecified substance] was administered orally to the animals. 8 CFU containing the AIEC strain (LF82-strepR) was administered again the following day at approximately 10... 8 CFU of ARO112 or EcN strains. Fecal samples were collected at the time points shown in the corresponding charts and inoculated into selective media to assess the colonization level of AIEC.

[0199] C57BL / 6J mice were housed under SPF conditions in ventilated cages equipped with HEPA particulate filters. Streptomycin (5 g / L) was provided freely in drinking water for 15 days, with the water changed every 3 days. After streptomycin treatment, mice drank untreated water for 4 days, followed by oral administration of AIEC strain (LF82-strepR) (approximately 10 mg / L). 8 CFU / mouse). Three days later, either the ARO112 strain or the EcN strain (approximately 10 CFU / mouse) was administered orally by gavage. 8 (CFU / mouse). Fecal samples were collected at the time points shown in the corresponding charts and inoculated into selective media to assess the colonization level of AIEC.

[0200] Fecal microbiota analysis

[0201] Sequences were processed using Mothur v.1.32.1. Sequences were converted to FASTA format. Sequences shorter than 220 bp, containing homopolymers longer than 8 bp, or with undetermined bases not perfectly matched to either the forward or reverse primers, as well as barcodes that are not complementary to each other or not aligned with the appropriate 16S rRNA variable region, were excluded from the analysis. Quality scores higher than 30 (ranging from 0 to 40, where 0 represents an ambiguous base) were used to process sequences, and a sliding window technique was used for trimming within 50 bp windows. Sequences were trimmed from the 3′ end until the quality score criteria were met, and then merged. 20,000 to 50,000 sequences were obtained per sample. 16S rRNA gene sequences were aligned using the SILVA template reference method. ChimeraSlayer was used to remove potentially chimeric sequences. Using an average neighborhood algorithm, sequences with a distance-based similarity greater than 97% were merged into the same OTU. All samples were diluted to the same number of sequences (10,000) for diversity analysis. Samples with fewer than 10,000 reads were excluded from the analysis (including samples). A Bayesian classifier algorithm was used for each sequence, with a 60% bootstrap threshold set; sequences were assigned to genus-level classifications whenever possible, otherwise to the closest genus-level classification. Using the top 100 OTUs, the taxonomic groups were plotted by merging OTUs with the same classification, resulting in 18 distinct taxonomic groups. Less representative OTUs / taxonomic groups were merged and labeled "Others". The Clearcut algorithm was used to infer phylogenetic trees based on 16S rRNA sequence alignment, and the unweighted and weighted UniFrac distances between each pair of samples were calculated using the phylogenetic tree.

[0202] Example 7: The protective potential of ARO112 against inflammation / colitis in the absence of infection.

[0203] To test the potential of ARO112 probiotic treatment against inflammatory colitis in the absence of infection, we treated Nod2- / - mutant mice with drinking water containing 2% sodium dextran sulfate (DSS) for 7 days to induce experimental colitis. Figure 12 As shown in the experimental protocol of a, mice were treated with PBS (control) or ARO112 on days 4, 8 and 9, respectively.

[0204] like Figure 12 As shown in b, mice treated with ARO112 probiotics did not experience weight loss, and as... Figure 12As shown in c, no severe diarrhea (with bloody stools) was observed. All mice treated with ARO112 (6 out of 6) survived the experiment because they did not develop severe diarrhea with bloody stools. Mice treated with PBS (control) experienced greater weight loss and developed severe diarrhea with bloody stools; 4 out of 6 mice reached the endpoint on day 7-8 and required euthanasia (weight loss exceeding 20%, accompanied by severe diarrhea and bloody stools, such as...). Figure 12 (as shown in d and Table 1). At the end of the experiment (day 15), surviving mice were dissected, colon length was measured, and compared with the euthanized mice to serve as an indicator of colitis (colitis causes colon shortening). The results showed that the colon length of mice treated with PBS (control) was significantly shorter compared to mice treated with ARO112. Figure 12 As shown in e.

[0205] Table 1. Weight loss, fecal characteristics, and disease activity score. The disease activity score was obtained by adding the scores of each sub-score (weight loss and fecal characteristics). A disease activity score of 8 (weight loss >20% and severe diarrhea with bloody stools) was the endpoint, at which point the mice were euthanized.

[0206]

[0207] As discussed, the findings above support the inventors' envisioned strategy of using the probiotic ARO112 to target intestinal inflammation / colitis (including prevention and treatment) and subsequent symptoms / complications such as diarrhea, weight loss, and bacterial infection. Many diseases share intestinal inflammation / colitis as a core symptom associated with microbiome imbalance (dysbiosis), such as inflammatory bowel disease (Crohn's disease and ulcerative colitis), obesity, colorectal cancer, diabetes, and malnutrition. Indeed, the inventors have found in experimental models of colitis that the probiotic ARO112 provides protection against inflammation / colitis and diarrhea, even in the absence of infection.

[0208] Example 8 – Probiotic composition comprising live Klebsiella spp. bacteria and short-chain fatty acids (SCFAs). Live bacteria.

[0209] The probiotic composition of this invention can be used in combination with helper bacteria (i.e., short-chain fatty acid (SCFA) producing bacteria) to achieve a superior enhancing effect compared to using Klebsiella bacteria alone. See also Figure 11 As in Example 5, the inventors have demonstrated that probiotic compositions containing a live Klebsiella spp. ARO112 strain (as the sole active bacterial component) can promote the recovery of short-chain fatty acid (SCFA) producing bacteria. In fact, the inventors found that ARO112 treatment promoted an increase in butyrate-producing bacteria, which in turn led to an increase in the production of butyrate and other short-chain fatty acids.

[0210] Therefore, the inventors anticipate that the probiotic composition containing live Klebsiella spp. ARO112 strain bacteria and SCFA-producing bacteria will further promote and accelerate the increase of exemplary SCFA, thereby more quickly replacing pathogenic bacterial species and associated infections, and enhancing anti-inflammatory effects.

[0211] The inventors envision combining Klebsiella bacteria (e.g., ARO112 strain) and SCFA-producing bacteria into a probiotic composition. The inventors envision selecting SCFA-producing bacteria from the Trichophyceae family (…). Lachnospiraceae ), Rumenococci ( Ruminococcaceae ), Vibriospirillumaceae ( Oscillospiraceae ), Butyric acid cocci ( Butyricicoccaceae ) and Bacteroidetes ( Bacteroidaceae Any of the bacterial families listed. The inventors further envision that a single application of this probiotic composition would contain approximately 10 8 Up to 10 10 The inventors also envision that the ratio of Klebsiella bacteria to SCFA-producing bacteria in the probiotic composition is approximately 1:1, 1:10, 1:100, 10:1, 100:1, 1000:1, or 10000:1. Furthermore, the inventors envision administering the probiotic composition more than once, for example, 2 to 10 consecutive times. The inventors anticipate that this multiple-application strategy will increase the successful colonization rate of the bacterial components (i.e., Klebsiella bacteria and SCFA-producing bacteria), thereby producing a more rapid therapeutic effect. Additionally, the inventors envision that the SCFA-producing bacteria produce any SCFA, including butyrate.

[0212] The inventors were able to evaluate the effect of the above-mentioned probiotic composition (containing live Klebsiella spp. bacteria and SCFA-producing bacteria) on increasing SCFA-producing bacteria and SCFA content. Therefore, by comparing the obtained data with... Figure 11 By comparing the data shown and discussed in Example 5, the inventors will be able to understand the enhancing effect produced by using both Klebsiella bacteria and SCFA-producing bacteria, which is superior to using a probiotic composition containing only live Klebsiella bacteria.

[0213] Referenced existing technical documents: All scientific publications and patent documents cited in this specification are incorporated herein by reference.

[0214] Santana 2022 – Santana, PT et al., 2022, Int. J. Mol.Sci.,23,3464.

[0215] Small, 2013 – Small, CL., et al., 2013, Nat Commun 4, 1957

[0216] (Schultz, 2008) – Schultz, M., 2008, Inflamm Bowel Dis. Jul;14(7):1012-8.

[0217] Carmen, 2011 –Carmen, S., 2011, Ulcers, vol. 2011, Article ID 841651,13 pages

[0218] (Suez, 2018); 3) – Suez, J. et al., 2018, Cell 174, 1406–1423

[0219] (Montassier, 2021) – Montassier, M. et al., 2021, Nat Microbiol, VOL6, 1043–1054

[0220] Oliveira, 2020 – Oliveira, RA, et al., 2020, Nat Microbiol, Apr;5(4):630-641.

[0221] Moradigaravand 2017 – Moradigaravand D., et al., 2017, mBio 8:e01976-16

[0222] Luo 2022 – Luo, X., et al., 2022, Infect Drug Resist, Apr 13;15:1831-1843

[0223] Hubbard 2020 – Hubbard, ATM, et al., 2020, Microbiologyopen, Jun;9(6):1128-1134.

[0224] Zhou 2022 – Zhou, J., et al., 2022, Nat Commun, Jun 14;13(1):3432

[0225] Zhilu 2021 – Zhilu, X.,et al., 2021, Gut Microbes, Jan-Dec;13(1)

[0226] Huebner 2011 – Huebner, C., et al., 2011, Appl Environ Microbiol.,Apr; 77(7): 2541–2544

[0227] Drouet, 2012 – Drouet, M., et al., 2012, Inflamm Bowel Dis., Oct;18(10):1923-31

[0228] Rolhion 2007 PMID17476674 – Rolhion, N., et al., 2007, InflammatoryBowel Diseases, Volume 13, Issue 10, 1, Pages 1277–1283

[0229] Palmela 2019 PMID 29141957 – Palmela C, et al. Gut 2017;0:1–14.

[0230] (Lev-Tzion 2017). PMID 26056382 – Lev-Tzion, R., et al., 2017,Digestion, 95:310–313

[0231] (Xiong 2015). PMID 28564649 – Xiong H, et al., 2015, Infect Immun 83:3418 –3427

[0232] item

[0233] 1. A probiotic composition comprising live Klebsiella spp. ARO112 strain bacteria as the sole active bacterial component.

[0234] 2. A probiotic composition comprising live Klebsiella spp. ARO112 strain bacteria as the sole active bacterial component, for use as a pharmaceutical.

[0235] 3. The probiotic composition used according to item 2 for promoting bacterial diversity in mammals.

[0236] 4. The probiotic composition used according to item 2, for promoting bacterial diversity in a human subject.

[0237] 5. The probiotic composition used according to item 2 or 4 for the prevention or treatment of intestinal flora imbalance in human subjects.

[0238] 6. The probiotic composition used according to any one of items 2 to 5 for the prevention or treatment of intestinal inflammation in a human subject.

[0239] 7. The probiotic composition used according to any one of items 2 to 6, for the prevention or treatment of intestinal infections caused by pathogenic bacterial strains in a human subject.

[0240] 8. The probiotic composition used according to any one of items 2 to 7, wherein the pathogenic bacteria are antibiotic-resistant bacterial strains, particularly multidrug-resistant bacterial strains.

[0241] 9. The probiotic composition used according to item 7 or 8, wherein the pathogenic bacterial species is: - Selected from Escherichia coli ( Escherichia ), Citrobacter ( Citrobacter ) or Salmonella ( Salmonella Members of the genus ( ), - Clostridium difficile ( Clostridioides difficile ), - Vancomycin-resistant enterococci; and / or - Selected from Enterococcus faecalis ( Enterococcus faecium Staphylococcus aureus ( Staphylococcus aureus ), Klebsiella pneumoniae ( Klebsiella pneumoniae Acinetobacter baumannii ( Acinetobacter baumannii ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Enterobacter strains and Escherichia coli ( E. coli ) pathogenic bacteria with multidrug resistance.

[0242] 10. The probiotic composition used according to any one of items 7 to 9, wherein the pathogenic bacterial species is Adhesive Invasive Escherichia coli (AIEC), Enterohemorrhagic Escherichia coli (EHEC), Enterocytic Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), or Uropathogenic Escherichia coli (UPEC), particularly wherein the pathogenic bacteria is AIEC Escherichia coli LF82 strain.

[0243] 11. The probiotic composition used according to any one of items 2 to 10, for use in a human subject who has received a course of antibiotic treatment in the previous month (especially the first 48 hours).

[0244] 12. The probiotic composition used according to item 11, wherein the course of antibiotic treatment includes vancomycin, gentamicin, metronidazole, ciprofloxacin and / or rifaximin.

[0245] 13. The probiotic composition used according to any one of items 2 to 12, for use in a patient diagnosed with an inflammatory bowel disease.

[0246] 14. The probiotic composition used according to item 13, wherein the inflammatory bowel disease is inflammatory bowel disease, particularly Crohn's disease or ulcerative colitis.

[0247] 15. The probiotic composition according to any one of items 1 to 14, or the probiotic composition used therein, wherein the Klebsiella genus ARO112 strain is characterized by having a genome with >97%, >98%, >99%, >99.5%, or particularly >99.9% similarity to the genome of the Klebsiella genus ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

[0248] 16. The probiotic composition used according to any one of items 2 to 15, wherein the probiotic composition is administered orally.

Claims

1. A probiotic composition comprising live Klebsiella spp. ( Klebsiella sp. The ARO112 strain of bacteria is the only active bacterial component, wherein the Klebsiella genus ARO112 strain is characterized by having a genome with >90% similarity to the genome of the Klebsiella genus ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

2. The probiotic composition according to claim 1, wherein the Klebsiella genus ARO112 strain comprises the genome of a Klebsiella genus ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

3. A probiotic composition comprising: (i) live Klebsiella bacteria; and (ii) Live bacteria that produce short-chain fatty acids (SCFA).

4. The probiotic composition according to claim 3, wherein the short-chain fatty acid (SCFA) is butyrate, optionally wherein the bacteria producing butyrate are selected from the following bacterial families: Trichophyceae, Ruminococciaceae, Oscillatoriaceae, Butycocciaceae, and Bacteroidetes.

5. The probiotic composition according to claim 3 or 4, wherein the Klebsiella bacteria comprises Klebsiella ARO112 strain, and wherein the Klebsiella ARO112 strain is characterized in that its genome has >90% similarity to the genome of Klebsiella ARO112 strain submitted to the Sequence Reading Archive (SRA) of the NCBI database with BioProject ID PRJNA590204.

6. The probiotic composition according to any one of claims 3 to 5, wherein the Klebsiella genus ARO112 strain comprises the genome of a Klebsiella genus ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

7. The probiotic composition according to any one of claims 1 to 6, used as a medicine.

8. The probiotic composition according to any one of claims 1 to 6, for use in: (i) promoting bacterial diversity in a mammalian or human subject; and / or (ii) preventing or treating intestinal flora imbalance in a human subject.

9. The probiotic composition according to any one of claims 1 to 6, for the prevention or treatment of intestinal inflammation in a human subject.

10. A probiotic composition comprising live Klebsiella bacteria as the sole active bacterial component for the prevention or treatment of intestinal inflammation in mammalian subjects, particularly human subjects.

11. A probiotic composition comprising live Klebsiella bacteria as the sole active bacterial component, for use in: (i) increasing the relative abundance of butyrate-producing bacteria in the gut of a subject, and / or (ii) increasing the amount of butyrate in the gut of a subject.

12. The probiotic composition according to claim 11, wherein the butyrate-producing bacteria have been depleted by antibiotics.

13. The probiotic composition according to any one of claims 10 to 12, wherein the Klebsiella bacteria comprises Klebsiella ARO112 strain, and wherein the Klebsiella ARO112 strain is characterized in that its genome has >90% similarity to the genome of Klebsiella ARO112 strain submitted to the Sequence Reading Archive (SRA) of the NCBI database with BioProject ID PRJNA590204.

14. The probiotic composition according to any one of claims 10 to 13, wherein the Klebsiella genus ARO112 strain comprises the genome of a Klebsiella genus ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

15. The probiotic composition according to any one of claims 1 to 6, for the prevention or treatment of intestinal infections caused by pathogenic bacterial strains in human subjects.

16. The probiotic composition according to claim 15, wherein the pathogenic bacterial species is a member of the phylum Proteobacteria (Pseudomonas).

17. The probiotic composition according to claim 15 or 16, wherein the pathogenic bacterial strain is an antibiotic-resistant bacterial strain, particularly a multidrug-resistant bacterial strain.

18. The probiotic composition according to any one of claims 15 to 17, wherein the pathogenic bacterial species is: - Selected from members of the genera *Escherichia coli*, *Citrobacter*, *Salmonella*, or *Vibrio*. - Clostridium difficile, - Vancomycin-resistant enterococci; and / or - Selected from pathogenic bacteria and multidrug-resistant strains of Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae, and Escherichia coli.

19. The probiotic composition according to any one of claims 15 to 18, wherein the pathogenic bacterial species is Vibrio cholerae, Adhesive Invasive Escherichia coli (AIEC), Enterohemorrhagic Escherichia coli (EHEC), Enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), or Uropathogenic Escherichia coli (UPEC), particularly wherein the pathogenic bacteria is AIEC Escherichia coli LF82 strain.

20. The probiotic composition according to any one of claims 7 to 19, for use in a human subject who has received a course of antibiotic treatment in the previous month (especially the first 48 hours), optionally wherein said course of antibiotic treatment includes vancomycin, gentamicin, metronidazole, ciprofloxacin and / or rifaximin.

21. The probiotic composition according to any one of claims 7 to 20, for use in a patient diagnosed with an inflammatory bowel disease, optionally, wherein the inflammatory bowel disease is inflammatory bowel disease, particularly Crohn's disease or ulcerative colitis.

22. The probiotic composition according to any one of claims 1 to 6 or the probiotic composition used according to any one of claims 7 to 21, wherein the Klebsiella genus ARO112 strain is characterized in that its genome has >95%, >97%, >98%, >99%, >99.5%, or particularly >99.9% similarity to the genome of the Klebsiella genus ARO112 strain with BioProject ID PRJNA590204 submitted to the Sequence Reading Archive (SRA) database of NCBI.

23. The probiotic composition used according to any one of claims 7 to 22, wherein the probiotic composition is administered orally.

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