Decolonization of pathogenic enterobacteriaceae, enterococci and / or acinetobacter from the gut using strains of e. coli

Abstract

The present invention relates to probiotic bacteria of the species E. coli, in particular in combination with bacteria of the species Klebsiella oxytoca, that are used for a decolonization of pathogenic and / or multidrug resistant (MDR) Enterobacteria, such as pathogenic E. coli and / or Klebsiella pneumoniae (K. pneumoniae), Enterococci and / or Acinetobacter, from the gut of a subject. The decolonization can both be therapeutic, i.e. after colonization of the gut by the pathogenic and / or multi-resistant pathogen(s), or as a preventive measure before a re-colonization of the gut, as required after antibiotic treatment or treatment-induced dysbiosis.

Description

[0001] H34511WO Decolonization of pathogenic Enterobacteriaceae, Enterococci and / or Acinetobacter from the gut using strains of E. coli The present invention relates to probiotic bacteria of the species E. coli, in particular in combination with bacteria of the species Klebsiella oxytoca, that are used for a decolonization of pathogenic and / or multidrug resistant (MDR) Enterobacteria, such as pathogenic E. coli and / or Klebsiella pneumoniae (K. pneumoniae), Enterococci and / or Acinetobacter, from the gut of a subject. The decolonization can both be therapeutic, i.e. after colonization of the gut by the pathogenic and / or multi-resistant pathogen(s), or as a preventive measure before a re-colonization of the gut, as required after antibiotic treatment or treatment-induced dysbiosis. Background of the invention Multi-drug resistance in the family of Enterobacteriaceae is one of the greatest health associated problems worldwide (WHO. United Nations meeting on antimicrobial resistance. Bull World Health Organ.2016;94(9):638-639. doi:10.2471 / blt.16.020916). Especially the incidence of infections as well as the mortality rates linked to carbapenem- resistant K. pneumoniae is constantly increasing and has reached alarming rates in Europe (Cassini, Alessandro et al (2018). Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015: a population-level modelling analysis. The Lancet Infectious Diseases.19.10.1016 / S1473-3099(18)30605-4). Klebsiella pneumoniae (KLP), a Gram-negative bacterium belonging to the family of Enterobacteriaceae, is a common cause of antimicrobial-resistant opportunistic infections in hospitalized patients. KLP can colonize in the human gastrointestinal tract, especially in patients with inflammatory bowel diseases, activate caspase-11 inflammasomes, and contribute to intestinal inflammation (Zhang Q, Su X, Zhang C, Chen W, Wang Y, Yang X, Liu D, Zhang Y, Yang R. Klebsiella pneumoniae Induces Inflammatory Bowel Disease Through Caspase-11-Mediated IL18 in the Gut Epithelial Cells. Cell Mol - 2 - Gastroenterol Hepatol. 2023;15(3):613-632. doi: 10.1016 / j.jcmgh.2022.11.005. Epub 2022 Nov 25. PMID: 36436756; PMCID: PMC9871440.). Several mechanisms of colonization resistance against intestinal pathogens have been described, including competition for nutrients, short chain fatty acid (SCFA)–dependent inhibition of virulence and replication, and direct antagonism through bacteriocin production or Type VI Secretion System–mediated killing. In healthcare settings, antibiotic-mediated disruption of the microbiota can be associated with enteric expansion of members of the Enterobacteriaceae family, including E. coli and Klebsiella pneumoniae, as well as vancomycin-resistant Enterococci and Clostridium difficile (Kim S., Covington A., and Pamer E.G. 2017. The intestinal microbiota: Antibiotics, colonization resistance, and enteric pathogens. Immunol. Rev.279:90–105. 10.1111 / imr.12563). While C. difficile causes gastroenteritis, hospital-associated Enterobacteriaceae and vancomycin-resistant Enterococci strains often expand in the gut without triggering overt inflammatory responses. In these instances, the primary clinical concern is that expansion of these species in the gut increases the risk for subsequent development of a bloodstream infection (BSI) in vulnerable patient populations. For example, allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective but highly immunocompromising treatment for some forms of cancer. The high incidence and risk of bacterial infections in patients undergoing allo-HCT necessitates administration of prophylactic and empiric antibiotics, leading to destruction of the normal microbiome. In this patient population, enteric domination with Enterobacteriaceae leads to a significant increase in the risk of developing a BSI. Furthermore, an increasing proportion of clinical isolates of Enterobacteriaceae are resistant to a wide range of antibiotics, including strains that produce extended-spectrum- β-lactamases or carbapenemases. Accordingly, BSI with these highly antibiotic-resistant strains are increasingly challenging to treat. Klebsiella pneumoniae, Escherichia coli, and other members of the Enterobacteriaceae family are common human pathogens that have acquired broad antibiotic resistance, rendering infection by some strains virtually untreatable. Enterobacteriaceae are intestinal residents, but generally represent <1% of the adult colonic microbiota. - 3 - Sorbara et al. (in: Sorbara MT, et al. Inhibiting antibiotic-resistant Enterobacteriaceae by microbiota-mediated intracellular acidification. J Exp Med.2019 Jan 7;216(1):84-98. doi: 10.1084 / jem.20181639. Epub 2018 Dec 18) demonstrate that an antibiotic-naive microbiota suppresses growth of antibiotic-resistant clinical isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis by acidifying the proximal colon and triggering short chain fatty acid (SCFA)-mediated intracellular acidification. High concentrations of SCFAs and the acidic environment counter the competitive edge that O2and NO3respiration confer upon Enterobacteriaceae during expansion. Reestablishment of a microbiota that produces SCFAs enhances clearance of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis from the intestinal lumen and represents a potential therapeutic approach to enhance clearance of antibiotic-resistant pathogens. Fecal microbiota transplant (FMT), also known as a stool transplant, is the process of transferring fecal bacteria and other microbes from a healthy individual into another individual. FMT involves restoration of the colonic microflora by introducing healthy bacterial flora through infusion of stool via colonoscopy, enema, orogastric tube, or by mouth in the form of a capsule containing feces from a healthy donor, which in some cases is freeze-dried. FMT, for example, is an effective treatment for Clostridioides difficile infection (CDI) and may be more effective than vancomycin treatment. Side effects may include a risk of infections; therefore the donor should be screened. With CDI becoming more common, FMT is gaining increasing prominence, with some experts calling for it to become the first-line therapy for CDI. FMT has been used experimentally to treat other gastrointestinal diseases, including colitis, constipation, irritable bowel syndrome, and neurological conditions, such as multiple sclerosis and Parkinson's. In the United States, human feces has been regulated as an experimental drug since 2013. Caballero et al. (in: Silvia Caballero, et al., Cooperating Commensals Restore Colonization Resistance to Vancomycin-Resistant Enterococcus faecium, Cell Host & Microbe, Vol 21, 5, 2017, pp. 592-602.e4, https: / / doi.org / 10.1016 / j.chom.2017.04.002.2017) demonstrate that a precisely defined - 4 - consortium of commensal bacteria containing the Clostridium cluster XIVa species Blautia producta and Clostridium bolteae restores colonization resistance against VRE and clears VRE from the intestines of mice. O. Ljungquist et al. (in: Probiotics for intestinal decolonization of ESBL-producing Enterobacteriaceae: a randomized, placebo-controlled clinical trial. Clinical Microbiology and Infection 26 (2020) 456e462 https: / / doi.org / 10.1016 / j.cmi.2019.08.019) disclose a randomized, placebo-controlled, single-blinded clinical superiority trial, where the probiotic Vivomixx®, a mixture of 8 different living bacterial strains or placebo was given to adult outpatients intestinally colonized for at least 3 months with extended spectrum b-lactamase (ESBL)-producing Enterobacteriaceae (EPE). Successful EPE eradication was observed only in very few individuals. Lagrafeuille et al. (in: Lagrafeuille R, et al. Opposing effect of Lactobacillus on in vitro Klebsiella pneumoniae in biofilm and in an in vivo intestinal colonization model. Benef Microbes. 2018 Jan 29;9(1):87-100. doi: 10.3920 / BM2017.0002. Epub 2017 Oct 12. PMID: 29022382) analyze the anti-biofilm activity of 140 neutralized Lactobacillus supernatants was assessed against K. pneumoniae. Among the 13 strains whose supernatant significantly impaired biofilm formation, Lactobacillus plantarum CIRM653 was selected because it was also able to impair K. pneumoniae preformed biofilm, independently of a bactericidal effect. EP2040724B1 discloses a biotherapeutic composition comprising a pharmaceutically effective amount of a probiotic Escherichia coli strain, a pharmaceutically effective amount of an anaerobic bacteria antibiotic to which said Escherichia coli strain is resistant, and a pharmaceutically acceptable carrier, wherein said antibiotic is metronidazole, and wherein said Escherichia coli strain and said antibiotic act in synergy. WO 2021 / 030198A1 discloses a composition comprising (1) one or more purified bacterial strains belonging to the phylum Firmicutes or Bacteroidetes and (2) one or more purified bacterial strains selected from the group consisting of an Escherichia species and - 5 - a Fusobacterium species. Also disclosed are methods of suppressing colonization of the intestine of a subject with oral microbiome bacteria. US7018629 B2 relates to pharmaceutical compositions of probiotic E. coli strains and uses thereof for treating inflammatory bowel disease. The search for alternative intervention strategies new therapeutics is therefore urgently required. Other objects and advantages will become apparent to the person of skill when studying the present description of the present invention. In a first aspect of the present invention, the above object is solved by a bacterium selected from the group of consisting of at least one of E. coli strains MR010, MK031, MR043, MR045, MR102, MR134, MR158, MK241, MK249, MK267, MK270, MK289, MR107, MR050, MK254, MR174, MR084, MK251, MK062, MR097, MK270, MR138, MK262, and MK179 for use in the prevention and / or treatment of pathogenic and / or antibiotic resistant Enterobacteriaceae, such as E. coli and / or Klebsiella pneumoniae, in particular multi-resistant (MDR) Enterobacteriaceae, Enterococci and / or Acinetobacter. Preferred is the bacterium for use according to the present invention, wherein said use is in combination with at least one bacterium selected from the group of Klebsiella michiganensis and / or Klebsiella oxytoca, in particular from the strain MK01, MR08, MR050 and species related to these strains, and wherein preferably the combination shows a synergistic effect in the prevention and / or treatment. “Combination” shall mean both a mixture of the strains or separate preparations that are given jointly or sequentially to the patient, in order to act together, in particular synergistically. Preferred is the bacterium for use according to the present invention, wherein said prevention and / or treatment is through decolonization of said Enterobacteriaceae, Enterococci and / or Acinetobacter, in particular Acinetobacter baumannii. The present invention provides an alternative treatment strategy that allows for an effective decolonization of multi-resistant bacteria and / or can prevent a colonization with such bacteria. - 6 - There is currently no medically recommended treatment strategy for intestinal decolonization with pathogenic and / or multi-resistant K. pneumoniae, Enterococci and / or Acinetobacter, and E. coli strains. Reserve antibiotics are currently used for this purpose, but they further worsen the problem of resistance development and also have strong effects on the natural protective effect of the microbiome. Alternative approaches such as fecal microbiota transplantation (FMT), used to treat Clostridioides difficile infections, have not been shown to be effective in treating MDR enterobacteria. In addition, it has already been shown that probiotics such as bifidobacteria, lactobacilli and E. coli Nissle can have a positive effect on the host. However, there are currently no probiotics available on the market that could displace MDR E. coli from the intestine. FMT is an experimental therapy approach that is only used as a last resort because there are serious safety concerns with this form of therapy. Since a complex undefined bacterial community is transmitted, MDR bacteria can also be transmitted. The administration of probiotics has not yet been proven to have any effect on colonization with MDR enterobacteria. Furthermore, recent studies have shown that the probiotic strain E. coli Nissle produces the toxin colibactin. In a second aspect of the present invention, the above object is solved by the bacterium for use as above in reestablishing colonization resistance after antibiotic treatment or treatment-induced dysbiosis in the microbiome of an animal, in particular a mammalian subject. A third aspect of the present invention then relates to a method for preventing and / or treating pathogenic and / or antibiotic resistant Enterobacteriaceae, such as E. coli and / or Klebsiella pneumoniae, in particular multi-resistant (MDR) Enterobacteriaceae, Enterococci and / or Acinetobacter, in the microbiome of an animal, in particular an avian or mammalian subject, such as a human, comprising administering to said animal, in particular said avian or mammalian subject an effective amount of a bacterium selected from the group consisting of at least one of E. coli strains MR010, MK031, MR043, MR045, MR102, MR134, MR158, MK241, MK249, MK267, MK270, MK289, MR107, - 7 - MR050, MK254, MR174, MR084, MK251, MK062, MR097, MK270, MR138, MK262, and MK179. It was surprisingly found that isolates of the bacterium E. coli, and in particular combinations thereof with Klebsiella michiganensis and / or Klebsiella oxytoca and thus species related to these strains, constitute an effective probiotic for the prevention and / or treatment of pathogenic and / or antibiotic-resistant Enterobacteriaceae, such as Klebsiella pneumoniae, Enterococci and / or Acinetobacter in the microbiome of an animal, in particular an avian or mammalian subject. Acinetobacter baumannii, particularly carbapenem-resistant strains (CRAB), is a major cause of healthcare-associated infections, thriving in clinical settings such as intensive care units (ICUs) due to its persistence and adaptability (7-10). The pathogen's ability to colonize the intestine and evade the host immune system by forming a bacterial reservoir has been recognized as a significant risk factor for subsequent infections, especially in immuno-compromised patients or those undergoing medical procedures. In the context of the present invention, probiotic Escherichia coli strains were found to be a non- antibiotic approach to mitigating A. baumannii colonization in the intestinal environment. Apparently, E. coli, particularly in combination with Klebsiella michiganensis and / or Klebsiella oxytoca and species related to these strains outcompete said Enterobacteriaceae, Enterococci and / or Acinetobacter and leads to effective decolonization of Enterobacteriaceae, Enterococci and / or Acinetobacter which is particularly useful in case of multidrug resistant (MDR) Enterobacteria, such as Klebsiella pneumoniae, and thus causes a prevention and / or treatment of said pathogen(s). The decolonization can both be therapeutic, i.e. after colonization of the gut by the (multi-resistant) pathogen(s), or as a preventive measure before a re-colonization of the gut, as required after antibiotic treatment (see also Examples below). The prevention and / or treatment of pathogenic and / or antibiotic resistant Enterobacteriaceae, such as Klebsiella pneumoniae, Enterococci and / or Acinetobacter in the microbiome of an animal, in particular an avian or mammalian subject can be preferably embodied as a long-lasting effect in combination with at least one bacterial species selected from the group consisting of Bacteroidetes, Actinobacteria, and Firmicutes spec., and preferably - 8 - from the families Lachnospiraceae (in particular the genus Schaedlerella, Chodladocola), Acutalibacteraceae (in particular the genus Eubacterium_R), Lactobacillaceae (in particular the genus Ligilactobacillus), Eggerthellaceae (in particular the genus Adlercreutzia), and Rikenelleaceae (in particular genus the Alistipes). Preferred is therefore the bacterium for use according to the present invention, wherein said prevention and / or treatment is through decolonization of said Enterobacteriaceae and / or Acinetobacter. In the context of the present invention, the term “pathogenic” in particular in the context with the Enterobacteriaceae, Enterococci and / or Acinetobacter as herein, shall generally relate to bacteria that cause undesired infections or inflammations in the gut of a subject. Preferred examples are hypervirulent strains of Acinetobacter or Klebsiella pneumoniae strains that can cause IBD. In the context of the present invention, the term “Escherichia coli” or “E. coli”, in particular in the context with the phrase “bacterium for use” as herein, shall generally relate to the E. coli strains MR010, MK031, MR043, MR045, MR102, MR134, MR158, MK241, MK249, MK267, MK270, MK289, MR107, MR050, MK254, MR174, MR084, MK251, MK062, MR097, MK270, MR138, MK262, and MK179, and strains that are related (e.g., because of genetic modifications), but still show the effect of outcompeting the antibiotic resistant Enterobacteriaceae, as described herein. “Related” in the context of the present invention further relates to strains that have a genome that is at least 95% identical, preferably at least 99% identical, to the genomes of the strains as indicated in table 1 (see below). The term includes individual strains or isolates or mixtures thereof. In the context of the present invention, the term “Klebsiella oxytoca”, in particular in the context with the phrase “bacterium for use” as herein, shall generally relate to the species of the Gram-negative bacterium including species related to Klebsiella oxytoca, and includes individual strains or isolates or mixtures thereof. “Related” in the context of the present invention further relates to strains that have a genome that is at least 95% identical, preferably at least 99% identical, to the genomes of the strains as indicated in table 1 (see below). The term shall further include Klebsiella oxytoca and species related - 9 - to Klebsiella oxytoca that outcompete the Enterobacteriaceae for the metabolic use of beta-glucosidic sugars such as sucrose and / or cellobiose. The term also includes commensal Klebsiella strains, K. michiganensis, K. grimontii, K. aerogenes, and Klebsiella that have a carbohydrate utilization pattern substantially identical to K. oxytoca strain MK01, and in particular human commensals. Klebsiella oxytoca is naturally resistant to ampicillin, amoxicillin, ticarcillin and to antibiotics to which other Enterobacteriaceae are also intrinsically resistant (herein referred to as “naturally occurring ampicillin / amoxicillin-resistance phenotype”). Examples in the context of the present invention are the isolates K. oxytoca strain MK01 isolated from donor MK1903 (K. oxytoca MK01), MR08, K. oxytoca strain MK02 isolated from donor MK1901 (K. oxytoca MK02), and K. michiganensis MR050. Further preferred is the bacterium for use according to the present invention, wherein the combination is selected from the group of E. coli strains MR102, and MK289 with Klebsiella michiganensis and / or Klebsiella oxytoca MK01, preferably MK289 with Klebsiella oxytoca MK01 or MR102 with Klebsiella oxytoca MK01 and / or MR08. A combination of E. coli MR102 with other probiotic strains, such as K. oxytoca MK01, achieves an enhanced pathogen suppression, compared to a single strain probiotic application. This provides a multi-strain therapeutic formulation according to the present invention that is capable of targeting a broader range of multidrug-resistant pathogens without weakening single strain probiotic efficacy. Further preferred is the bacterium for use according to the present invention, wherein said Enterobacteriaceae is a bacterium that comprises genes (chromosomal and / or extrachromosomal) that encode for at least one multi-drug resistance mechanism, for example OXA-48, NMD-1, NDM-5, OXA-244 or IMP-14 and is selected from at least one of E. coli strains ST617 (strain MHH), ST361 (56922), ST940 (56236), ST648 (56589), ST156 (56231), ST167 (55652), ST38 (strain 55838), ST405 (strain 54519), ST1284 (strain 55476), ST1702 (strain 56035), ST131 (strain 21236), ST38 (strain 26260), ST46 (strain 53221), ST617 (strain 54875), ST2197 (strain 44583), ST410 (strain 51853), ST131 (strain PBIO729), ST648 (strain PBIO730), and EPEC (strain O127:H6 - 10 - strain E2348 / 69), preferably MHH, 56922, 56231, 21236, 53221, PBIO729, and EPEC (see also https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6113867 / for designations). Further preferred is the bacterium for use according to the present invention, wherein said pathogenic and / or antibiotic resistant Acinetobacter is selected from at least one of Acinetobacter baumannii, preferably strains A. baumannii FR4326 and FR3462, and carbapenem-resistant (CRAB) strains, preferably CRAB-strains Further preferred is the bacterium for use according to the present invention, wherein said use is in combination with supplemented lactose and / or maltose, for example with about 10g / L of each sugar, preferably when outcompeting / treating Acinetobacter. The use of specific carbohydrates, such as maltose or lactose, to enhance the probiotic activity of candidates like E. coli MR102 or K. oxytoca MK01 or MR08. Such an approach enables customized probiotic therapy, allowing for targeted manipulation of the gut environment to maximize suppression of A. baumannii. Further preferred is the bacterium for use according to the present invention, wherein said species related to Klebsiella oxytoca is selected from the group consisting of K. michiganensis, K. grimontii, K. aerogenes, and Klebsiella that are related and / or have a carbohydrate utilization pattern substantially similar to K. oxytoca strain MK01. Further preferred is an E. coli strain or a Klebsiella oxytoca for use according to the present invention, wherein said strain and / or Klebsiella oxytoca for use is a strain or an isolate isolated from a human. Even further preferred is an E. coli strain or a Klebsiella oxytoca for use according to the present invention that are genetically modified. The genetic modification can include any suitable modification that does not negatively interfere with the purpose of the strains in the context of the present invention. The genetically modified E. coli strain or a Klebsiella oxytoca for use according to the present invention may include mutations that improve the safety of the strains, e.g. a mutant lacking functional gene(s) involved in the production of toxins, or lacking genes involved in the pathogenicity of the strain(s) (Darby, Alison et al. “Cytotoxic and pathogenic properties of Klebsiella oxytoca isolated - 11 - from laboratory animals.” PloS one vol. 9,7 e100542. 24 Jul. 2014, doi:10.1371 / journal.pone.0100542). Resistant Enterobacteriaceae, such as Klebsiella pneumoniae, in the context of the present invention, shall mean any species or strain (or mixtures thereof) of Enterobacteriaceae that has acquired or has acquired an additional resistance phenotype, compared to a sensitive strain (disregarding the naturally occurring resistance phenotypes). Such antibiotic resistance is more available than ever before to organisms such as Escherichia coli and Klebsiella pneumoniae, Enterococci and / or Acinetobacter that are important causes of major sepsis (see, for example, Iredell Jon, Brown Jeremy, Tagg Kaitlin. Antibiotic resistance in Enterobacteriaceae: mechanisms and clinical implications BMJ 2016; 352:h6420). Examples are resistant strains belonging to Citrobacter, Enterobacter, Hafnia, Klebsiella, Proteus, Providencia, Salmonella, and Yersinia. Additional examples of antibiotic resistant Enterobacteriaceae are multidrug resistant Enterobacteriaceae (MDR), a concerning problem in clinical environments in both the hospital and the ambulatory setting. Carbapenem-resistant Enterobacteriaceae are regarded as an urgent threat by the CDC in the US. Preferred is therefore the bacterium for use according to the present invention, wherein said Enterobacteriaceae is / are multi-resistant Enterobacteriaceae, such as P. mirabilis, E. cloacae, or E. coli and / or Klebsiella pneumoniae. Further preferred is therefore the bacterium for use according to the present invention, wherein said pathogenic and / or antibiotic resistant Acinetobacter is a CRAB-strain. Preferred is an E. coli strain or a Klebsiella oxytoca for use according to the present invention, wherein said microbiome is located in the gut of said animal, in particular said avian or mammalian subject. Preferred is an E. coli strain or a Klebsiella oxytoca for use according to the present invention, wherein said microbiome is located in the gut of said animal, in particular an avian or mammalian subject, and wherein said subject is selected from poultry, such as chicken or geese, a mouse, rat, cat, dog, rabbit, goat, sheep, horse, camel, lama, cow, monkey, a farm animal, a sport animal, and a pet, and a human. - 12 - The intestinal microbiota plays beneficial roles in many physiological processes of the host. Dysbiosis, a disruption of microbial composition by various stresses, has been implicated in inflammatory bowel disease (IBD), colon cancer, obesity, asthma, and other diseases. In particular, excessive dosing of antibiotics elicits the loss of naturally occurring intestinal microbiota. Such loss increases the numbers of yeasts, such as Candida albicans, and bacteria, such as Proteus, Staphylococcus, and Clostridium difficile (C. difficile), that normally exist at low numbers, leading to depression of digestive functions or the occurrence of intestine-related diseases. The idea that probiotics could improve or prevent diarrhea began with the notion that these gut-associated diseases were caused by the collapse of “colonization resistance” due to the absence of normal bacterial flora. Probiotics are known to be very effective for the treatment of antibiotic- associated diarrhea, with Saccharomyces boulardii (S. boulardii), E. coli Nissle 1917, Lactobacillus, and Bifidobacterium as the main focus of research. Application of FMT and probiotics for eradication of gastrointestinal diseases and enteropathogens exhibits the potential to restore the degraded ecosystem and protection against colonization and proliferation of enteropathogens. Therefore, in another aspect of the present invention, the bacterium is used and / or is for use according to the present invention in reestablishing colonization resistance after antibiotic dysbiosis in the microbiome of an avian or mammalian subject. Another aspect of the present invention then relates to the bacterium for use according to the present invention, wherein said prevention and / or treatment of said pathogenic and / or antibiotic resistant Enterobacteriaceae, Enterococci and / or Acinetobacter and / or said reestablishing colonization resistance after treatment or antibiotic dysbiosis is in the context of bacterial infection, bloodstream infection, allogeneic hematopoietic stem cell transplantation, and bacterial invasion into the liver, spleen and / or mesenteric lymph nodes (MLN) in said animal, in particular an avian or mammalian subject. As further described below, luminal and tissue invasion of K. pneumoniae MDR1 was assessed in the gastrointestinal organs including small intestine, cecum and colon as well as in the liver, and lymphatic organs including spleen and mesenteric lymph nodes (MLN) on day 6 p.c. A strong reduction in the gastrointestinal organ content as well as in the tissues in both pre-colonized groups compared to the control groups was observed. In addition, Klebsiella oxytoca colonized groups did not have any bacteria in the liver, spleen or MLN - 13 - in contrast to the control animals, suggesting a systemic spread of the pathogen in control mice, elevating the risk for blood stream infections. This experiment demonstrated a broad-spectrum activity of Klebsiella oxytoca in all gastrointestinal organs including reduction of pathogenic bacteria in the lumen and tissue and preventing the systemic spread into the liver and lymphatic tissues such as spleen and MLN. The bacterium for use according to the present invention can be administered to the avian or mammalian subject in any suitable way that allows for colonization, in particular a substantial colonization, with said bacterium, for example as a pharmaceutical composition comprising an effective amount of an E. coli strain and / or a Klebsiella michiganensis and / or Klebsiella oxytoca for use according to the present invention, and a pharmaceutically or therapeutically acceptable excipient or carrier. Compositions are for fecal microbiota transplant (FMT), comprise a suitable medium, such as sterile saline, are used as a suitable solid dosage form, such as a suppository or capsule, in particular stomach-acid resistant, and / or as a probiotic. A combination of the strains (see also above) is explicitly included. The term “pharmaceutically or therapeutically acceptable excipient or carrier” refers to a solid or liquid filler, diluent or encapsulating substance which does not interfere with the effectiveness or the biological activity of the active ingredients (here, bacteria) and which is not toxic to the host, which may be either humans or animals, to which it is administered. Depending upon the particular route of administration, a variety of pharmaceutically-acceptable carriers such as those well known in the art may be used. Non-limiting examples include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water. Pharmaceutically acceptable carriers or excipients also include diluents (fillers, bulking agents, e.g. lactose, microcrystalline cellulose), disintegrants (e.g. sodium starch glycolate, croscarmellose sodium), binders (e.g. PVP, HPMC), lubricants (e.g. magnesium stearate), glidants (e.g. colloidal SiO2), solvents / co-solvents (e.g. aqueous vehicle, Propylene glycol, glycerol), buffering agents (e.g. citrate, gluconates, lactates), preservatives (e.g. Na benzoate, parabens (Me, Pr and Bu), BKC), anti -oxidants (e.g. BHT, BHA, Ascorbic acid), wetting agents (e.g. polysorbates, sorbitan esters), thickening - 14 - agents (e.g. methylcellulose or hydroxyethylcellulose), sweetening agents (e.g. sorbitol, saccharin, aspartame, acesulfame), flavoring agents (e.g. peppermint, lemon oils, butterscotch, etc.), humectants (e.g. propylene, glycol, glycerol, sorbitol). Other suitable pharmaceutically acceptable excipients are inter alia described in Remington's Pharmaceutical Sciences, 15thEd., Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologic, 5thEd., Govi-Verlag Frankfurt (1997). The person skilled in the art knows suitable formulations for use according to the present invention and will readily be able to choose suitable pharmaceutically acceptable carriers or excipients, depending, e.g., on the formulation and administration route of the pharmaceutical composition. In a preferred example, the E. coli strain and / or the Klebsiella michiganensis and / or Klebsiella oxytoca for use according to the present invention is provided as a probiotic, i.e. a live microbial food or feed supplement (preparation) which beneficially affects the host animal by improving its intestinal microbial balance. In addition to the aforementioned bacteria for use according to the invention, the pharmaceutical composition as administered can contain other therapeutically active substances – for example with medicaments already known for the treatment of the aforementioned conditions and / or diseases, in particular suitable nutrients and supplements for bacterial growth as desired, e.g. butyrate, but also suitable antibiotics, whereby in the latter case a favorable additive, amplifying or preferably synergistically effect is noticed. Also preferred is the E. coli strain or the Klebsiella michiganensis and / or Klebsiella oxytoca for use according to the present invention, wherein said use is in combination with at least one bacterial species selected from the group consisting of Bacteroidetes, Actinobacteria, and Firmicutes spec., and preferably from the families Lachnospiraceae (in particular the genus Schaedlerella, Chodladocola), Acutalibacteraceae (in particular the genus Eubacterium_R) Lactobacillaceae (in particular the genus Ligilactobacillus), Eggerthellaceae (in particular the genus Adlercreutzia), and Rikenelleaceae (in particular genus the Alistipes), and in particular human isolates thereof, and optionally with at least one additional therapeutically active substance as above. - 15 - The dosage of the pharmaceutical composition to be administered according to the present invention can be appropriately selected according to the route of administration, the subject to be administered, the target disease and its severity, age, sex weight, individual differences and disease state. Dosage may be repeated several times a day. Preferred is a bacterium for use according to the present invention, wherein said use is in a (single) dosage of between 1012and 107CFU, preferably between 1010and 108CFU of said bacterium, e.g. an E. coli strain or a strain of Klebsiella michiganensis and / or Klebsiella oxytoca. Doses can be applied several times, as needed. Yet another aspect of the present invention then relates to a method for preventing and / or treating pathogenic and / or antibiotic resistant Enterobacteriaceae, such as E. coli and / or Klebsiella pneumoniae, in particular multi-resistant (MDR) Enterobacteriaceae, Enterococci and / or Acinetobacter in the microbiome of an animal, in particular an avian or mammalian subject, such as a human, comprising administering to said animal, in particular said avian or mammalian subject an effective amount of a bacterium selected from the group consisting of at least one of E. coli strains MR010, MK031, MR043, MR045, MR102, MR134, MR158, MK241, MK249, MK267, MK270, MK289, MR107, MR050, MK254, MR174, MR084, MK251, MK062, MR097, MK270, MR138, MK262, and MK179. Preferred is the method according to the present invention, wherein said preventing and / or treating is in combination with at least one bacterium selected from the group of Klebsiella michiganensis and / or Klebsiella oxytoca, in particular from the strain MK01, MR08, MR050, and species related to Klebsiella oxytoca MK01. By “treatment” or “treating” is meant any treatment of a disease or disorder, in a mammal, including: preventing or protecting against the disease or disorder, that is, causing, the clinical symptoms of the disease not to develop; inhibiting the disease, that is, arresting or suppressing the development of clinical symptoms; and / or relieving the disease, that is, causing the regression of clinical symptoms. By “amelioration” is meant the prevention, reduction or palliation of a state, or improvement of the state of a subject; the amelioration of a stress is the counteracting of the negative aspects of a stress. Amelioration includes but does not require complete recovery or complete prevention of - 16 - a stress. As mentioned above, in addition to the bacteria for use according to the invention, the pharmaceutical composition as administered can contain other therapeutically active substances – for example with medicaments already known for the treatment of the aforementioned conditions and / or diseases, in particular suitable nutrients, such as butyrate, and supplements for bacterial growth as desired, but also suitable antibiotics, whereby in the latter case a favorable additive, amplifying or preferably synergistically effect is noticed. Also preferred is method as above, wherein said prevention and / or treatment is in combination with at least one bacterial species selected from the group consisting of Bacteroidetes, Actinobacteria, and Firmicutes spec., and preferably from the families Lachnospiraceae (in particular the genus Schaedlerella, Chodladocola), Acutalibacteraceae (in particular the genus Eubacterium_R), Lactobacillaceae (in particular the genus Ligilactobacillus), Eggerthellaceae (in particular the genus Adlercreutzia), and Rikenelleaceae (in particular genus the Alistipes), and in particular human isolates thereof, and optionally with at least one additional therapeutically active substance as above. It is to be understood that the present bacteria and / or a pharmaceutical composition comprising the present bacteria is for use to be administered to an animal, such as an avian or mammalian, e.g. a human patient. The term "administering" means administration of a sole therapeutic bacterial agent or in combination with another therapeutic agent. It is thus envisaged that the pharmaceutical composition of the present invention are employed in co-therapy approaches, i.e. in co-administration with other medicaments or drugs and / or any other therapeutic agent which might be beneficial in the context of the methods of the present invention. Nevertheless, the other medicaments or drugs and / or any other therapeutic agent can be administered separately from the compound for use, if required, as long as they act in combination (i.e. directly and / or indirectly, preferably synergistically) with the present compound(s) (for use). The dosage of the pharmaceutical composition to be administered in the method according to the present invention can be appropriately selected according to the route of administration, the subject to be administered, the target disease and its severity, age, sex - 17 - weight, individual differences and disease state. Dosage may be repeated several times a day. Preferred is a bacterium for use, such as a strain of E. coli and / or Klebsiella oxytoca for use according to the present invention, wherein said use is in a (single) dosage of between 1012and 107CFU, preferably between 1010and 108CFU of said strain of E. coli and / or Klebsiella oxytoca. Doses can be applied several times, as needed. The prevention and / or treatment of said pathogenic and / or antibiotic resistant Enterobacteriaceae, Enterococci and / or Acinetobacter and / or said reestablishing colonization resistance after antibiotic dysbiosis can be in the context of bacterial infection, bloodstream infection, allogeneic hematopoietic stem cell transplantation, and bacterial invasion into the liver, spleen and / or mesenteric lymph nodes (MLN) in said animal, in particular an avian or mammalian subject. According to the present invention, the animal, in particular the avian or mammalian subject to be treated can be preferably selected from poultry, such as chicken or geese, a mouse, rat, cat, dog, rabbit, goat, sheep, horse, camel, lama, cow, monkey, a farm animal, a sport animal, and a pet, and a human. After establishing different animal models, the inventors isolated promising candidates for a competition with MDR E. coli strains. As direct competition for environmental niches in the gut is a fundamental requirement for invading species to successfully establish in the host, which is facilitated after antibiotic perturbation of the resident flora, the inventors aimed at identifying commensal bacterial species as candidates to compete with pathogenic and / or MDR E. coli and / or K. pneumoniae, Enterococci and / or Acinetobacter strains. These strains would also exploit similar nutrient sources and grow under an oxidative environment. The present invention relates to the following items. Item 1. A bacterium selected from the group of consisting of at least one of E. coli strains MR010, MK031, MR043, MR045, MR102, MR134, MR158, MK241, MK249, MK267, MK270, MK289, MR107, MR050, MK254, MR174, MR084, MK251, MK062, MR097, MK270, MR138, MK262, and MK179 for use in the prevention and / or treatment of antibiotic resistant Enterobacteriaceae, such as E. coli and / or Klebsiella pneumoniae, in - 18 - particular multi-resistant (MDR) Enterobacteriaceae, Enterococci and / or Acinetobacter in the microbiome of an animal, in particular an avian or mammalian subject. Item 2. Bacterium for use according to Item 1, wherein said use is in combination with at least one bacterium selected from the group of Klebsiella michiganensis and / or Klebsiella oxytoca, in particular from the strain MK01, MR08, MR050, and species related to Klebsiella oxytoca MK01, and wherein preferably the combination shows a synergistic effect in the prevention and / or treatment. Item 3. Bacterium for use according to Item 2, wherein the combination is selected from the group of E. coli strains MR102, and MK289 with Klebsiella oxytoca MK01, preferably MK289 with Klebsiella oxytoca MK01 and / or MR08 or MR102 with Klebsiella oxytoca MK01 and / or MR08. Item 4. Bacterium for use according to any one of Items 1 to 3, wherein said antibiotic resistant Enterobacteriaceae is selected from at least one of E. coli strains MHH, 56922, 56236, 56589, 56231, 55652, 55838, 54519, 55476, 56035, 21236, 26260, 53221, 54875, 44583, 51583, PBIO729, PBIO730, and EPEC, preferably MHH, 56922, 56231, 21236, 53221, PBIO729, and EPEC. Item 5. Bacterium for use according to any one of Items 1 to 4, wherein said use further comprises the addition of N-acetyl-D-galactosamine. Item 6. Bacterium for use according to any one of Items 1 to 4, wherein said pathogenic and / or antibiotic resistant Acinetobacter is selected from at least one of Acinetobacter baumannii, preferably strains A. baumannii FR4326 and FR3462, and CRAB strains, preferably CRAB-strains. Item 7. Bacterium for use according to any one of claims 1 to 6, wherein said use is in combination with supplemented lactose and / or maltose, for example with about 10g / L of each sugar. Item 8. Bacterium for use according to any one of Items 2 to 7, wherein said species - 19 - related to Klebsiella oxytoca is selected from the group consisting of K. michiganensis, K. grimontii, K. aerogenes, and Klebsiella that have a carbohydrate utilization pattern substantially similar to K. oxytoca strain MK01, or MR08. Item 9. Bacterium for use according to any one of Items 1 to 8 in reestablishing colonization resistance after treatment and / or antibiotic dysbiosis in the microbiome of an animal, in particular an avian or mammalian subject. Item 10. Bacterium for use according to any one of Items 1 to 9, wherein said microbiome is located in the gut of said animal, in particular said avian or mammalian subject. Item 11. Bacterium for use according to any one of Items 1 to 10, wherein said prevention and / or treatment of said pathogenic and / or antibiotic resistant Enterobacteriaceae and / or said reestablishing colonization resistance after treatment and / or antibiotic dysbiosis is in the context of bacterial infection, irritated bowl disease (IBD), bloodstream infection, sepsis, allogeneic hematopoietic stem cell transplantation, and bacterial invasion into the liver, spleen and / or mesenteric lymph nodes (MLN) in said animal, in particular said avian or mammalian subject. Item 12. Bacterium for use according to any one of Items 1 to 11, wherein said use comprises fecal microbiota transplant (FMT), in a suitable medium, such as sterile saline, or in a suitable solid dosage form, such as a suppository or capsule, in particular stomach- acid resistant, or as a probiotic. Item 13. Bacterium for use according to any one of Items 1 to 12, wherein said use is in a dosage of between 1012and 107CFU, preferably between 1010and 108CFU of said bacterium. Item 14. Bacterium for use according to any one of Items 1 to 13, wherein said use is in combination with at least one bacterial species selected from the group consisting of Bacteroidetes, Actinobacteria, and Firmicutes spec., and preferably from the families Lachnospiraceae, such as, for example, Schaedlerella, Chodladocola, Acutalibacteraceae, such as for example Eubacterium_R, Lactobacillaceae, such as, for example, - 20 - Ligilactobacillus, Eggerthellaceae, such as, for example, Adlercreutzia, and Rikenelleaceae, such as, for example, Alistipes, and in particular human isolates thereof. Item 15. A method for preventing and / or treating pathogenic and / or antibiotic resistant Enterobacteriaceae, such as E. coli and / or Klebsiella pneumoniae, in particular multi- resistant (MDR) Enterobacteriaceae, Enterococci and / or Acinetobacter in the microbiome of an animal, in particular an avian or mammalian subject, such as a human, comprising administering to said animal, in particular said avian or mammalian subject an effective amount of a bacterium selected from the group consisting of at least one of E. coli strains MR010, MK031, MR043, MR045, MR102, MR134, MR158, MK241, MK249, MK267, MK270, MK289, MR107, MR050, MK254, MR174, MR084, MK251, MK062, MR097, MK270, MR138, MK262, and MK179. Item 16. The method according to Item 15, wherein said preventing and / or treating is in combination with at least one bacterium selected from the group of Klebsiella michiganensis and / or Klebsiella oxytoca, in particular from the strain MK01, MR08, MR050 and species related to Klebsiella oxytoca MK01 or MR08. Item 17. The method according to Item 16, wherein the combination is selected from the group of E. coli strains MR102, and MK289 with Klebsiella oxytoca MK01, preferably MK289 with Klebsiella oxytoca MK01 or MR102 with Klebsiella oxytoca MK01 and / or MR08. Item 18. The method according to any one of Items 15 to 17, wherein said antibiotic resistant Enterobacteriaceae is selected from at least one of E. coli strains MHH, 56922, 56236, 56589, 56231, 55652, 55838, 54519, 55476, 56035, 21236, 26260, 53221, 54875, 44583, 51583, PBIO729, PBIO730, and EPEC, preferably MHH, 56922, 56231, 21236, 53221, PBIO729, and EPEC. Item 19. The method according to any one of Items 15 to 17 wherein said pathogenic and / or antibiotic resistant Acinetobacter is selected from at least one of Acinetobacter baumannii, preferably strains A. baumannii FR4326 and FR3462, and CRAB strains, preferably CRAB-strains. - 21 - Item 20. The method according to any one of Items 15 to 19 wherein the treatment is in combination with supplemented lactose and / or maltose, for example with about 10g / L of each sugar. Item 21. The method according to any one of Items 15 to 20, further comprising administering to said animal, in particular said avian or mammalian subject, an effective amount of a bacterium selected from the group consisting of Bacteroidetes, Actinobacteria, and Firmicutes spec., and preferably from the families Lachnospiraceae, such as, for example, Schaedlerella, Chodladocola, Acutalibacteraceae, such as for example Eubacterium_R, Lactobacillaceae, such as, for example, Ligilactobacillus, Eggerthellaceae, such as, for example, Adlercreutzia, and Rikenelleaceae, such as, for example, Alistipes, and in particular human isolates thereof. The present invention will now be described further in the following examples with reference to the accompanying Figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. Figure 1 shows the fold change of the growth of an MDR E. coli strain (MHH, ST617) following co-cultivation with several commensal E. coli-strains. Individual values of an experiment that was performed in triplicate. Figure 2 shows in (A) the pre-colonization of several commensal E. coli strains, in (B) the colonization kinetics of an MDR E. coli (strain MHH, ST617) in pre-colonized and control-SPF-mice that were treated with ampicillin, and in (C) the clearance-rates of MDR E. coli MHH in pre-colonized und control animals. Geometric mean of two to three experiments with n=9-17 mice per group. Figure 3 shows a heatmap of the quantification of the growth of several MDR-E. coli- strains following co-cultivation with several commensal E. coli-strains or mixtures with K. oxytoca. The data are from three independent experiments. - 22 - Figure 4 shows in (A) the colonization kinetics des MDR E. coli (strain 21236, ST131) in pre-colonized und control-SPF-mice that were treated with ampicillin, und in (B) die the colonization kinetics des MDR E. coli (strain 55652, ST167) in pre-colonized und control-SPF-mice that were treated with ampicillin. The data are from two to three experiments with n=9-17 mice per group. Figure 5 shows the colonization kinetics of the strains E. coli MR102 and K. oxytoca MK01 in a co-colonization in SPF mice that were treated with ampicillin. Data from one experiment with n=5 mice per group. Figure 6 shows in (A) die pre-colonization of E. coli MR102, K. oxtyoca MK01 and of both strains in co-colonization, in (B) the colonization kinetics of MDR E. coli (strain 55652 ST167) in pre-colonized and control-SPF- mice that were treated with ampicillin, and in (C) the clearance-rates of MDR E. coli MHH in pre-colonized and control animals. Geometric mean of two experiments with n=8-10 mice per group. Figure 7 shows in (A-D) colonization kinetics of different MDR-E strains (A: E. cloaceae, B: E. coli ST617, C: K. pneumoniae, D: P. mirabilis) and in (E) of S. Typhimurium. Geometric mean of two experiments with n=9-10 mice per group. Figure 8 shows the area under the curve (AUC) of growth curves of different strains of E. coli in minimal medium, supplemented with different sugars. The results are depicted as a heatmap of the AUC after 72 hours of incubation. Figure 9 shows the interaction of E. coli and A. baumannii under oxygen limitation. (A, B) Strain-dependent A. baumannii suppression by E. coli candidates in maltose- containing media. (C–F) Single-strain probiotic candidates (E. coli, K. oxytoca) or their combination. (G, H) Carbohydrate utilization-based suppression using E. coli MR102 and its lactose- / mannose-deficient mutants. Figure 10 illustrates the colonization kinetics of CFUs and clearance rates of three different MDR-E strains in SPF mice treated with ampicillin. Mice were colonized with the respective MDR-E strains and treated with PBS or a mixture of E. coli MR102 and K. - 23 - oxytoca MK01 four days later. Experiments were conducted with MDR E. coli MDR1 (A, B), MDR2 (C, D), and MDR K. pneumoniae (E, F). Data represents one experiment with n=3-5 mice per group. Figure 11 illustrates the colonization kinetics of CFUs (A) and clearance rates (B) of E. coli MDR2 (ST131) in SPF mice treated with ampicillin. The mice were initially colonized with E. coli MDR2 (ST131) and then treated with either PBS or E. coli MR102, four days later. This represents the mean of one experiment with n=4 mice per group. EXAMPLES Material and Methods Human cohorts Human sample and data collections have been performed in agreement with the guidelines of the Helmholtz Center for Infection Research, Braunschweig, Germany, the Ethics Committee Lower Saxony (permit No. 8629_BO_K_2019; No. 8750_BO_K_2019) and the European Data Protection Laws (Europäische Datenschutz- Grundverordnung DSGVO). All human donors have signed a letter of informed consent by the World Medical Association Declaration of Helsinki (Version 2013). Mice All animals which were used for in vivo experiments were gender and age-matched, female and male mice with an age of 8-16 weeks and a weight of 16-30 g at the beginning of the experiment. Mice were kept under a 12 h light cycle (lights on from 7 am to 7 pm). C57BL / 6N mice were bred at the animal facility of the Helmholtz Centre of Infection Research. SPF mice were bred under specific pathogen-free (SPF) conditions (Stehr et al. 2009) and germ-free C57BL / N6Tac mice were bred in isolators (Getinge). Mice were killed by euthanization with CO2and cervical dislocation. Bacterial strains Multidrug-resistant (OXA-48 carbapenemase) E. coli MDR1 (2365332, ST617), obtained from the Hannover medical school, and isolated from a rectal swab of a patient, was used for most in vivo colonization experiments and in vitro assays. Multidrug-resistant E. coli - 24 - NRZ-21236 and NRZ-55652, obtained from the National Reference Center for Multidrug-resistant Gram-negative Bacteria (Bochum) was used for two in vivo colonization experiments. Further commensal E. coli isolates used for in vivo and in vitro competition experiments were isolated from the human cohort studies at the HZI (MikroResist=MR, MikroKids=MK, LöwenKids=LK, RheumaVor=RV). Strain isolation Fresh stool samples were homogenized by bead-beating in a Mini-Beadbeater (BioSpec) with 1 mm zirconia / silica beads in 1 ml PBS for 50 s. Stored fecal samples were directly taken from glycerol stock. All samples were serially diluted and plated on CHROMagar Orientation plates and MacConkey agar plates. Colonies were isolated and identified by 16S-PCR and whole genome sequencing. Quantification of E. coli colony-forming units To determine the CFU of bacteria from culture or feces samples under different conditions, samples were spread on plates. Fresh fecal samples were collected and weighted for quantification of CFUs from fecal samples. Afterward, samples were prepared by adding 1 mm Zirconia beads and 1 ml PBS. Samples were homogenized by bead-beating for 50 s (Mini-Beadbeater, BioSpec). To determine CFUs, 25µl of serial dilutions of samples were plated on LB plates with respective antibiotics or MacConkey agar supplemented with Maltose. Plates were cultivated overnight at 37 °C under aerobic conditions before counting. Colony counts were normalized to plated volume and / or weight of feces. Ex vivo competition in germ-free cecum content Germ-free mice were sacrificed, and cecal contents were isolated and diluted 1:1 in PBS. Bacteria were grown in 5 ml LB for 16 h at 37 °C under aerobic conditions before adjusting the OD600. MDR E. coli strains were adjusted to OD600of 1, and the commensal strains were adjusted to OD600 of 0.2. The assay was performed in 96-well plates with 250 µl diluted cecum content with co-cultures of 20 µl of commensal and 10 µl of MDR E. coli strain. Single culturing of the MDR E. coli strain served as a control. In vitro competition for carbohydrates - 25 - To investigate direct competition or specific carbohydrates, bacteria were co-cultivated in MM9 media supplemented with 5 g / L of the respective carbon source. To do so, bacterial strains were cultivated on R2A agar plates (Difco) overnight, and bacteria were adjusted to an OD600 of 0.2 in PBS.99 µl of MM9 media with the respective carbon source was filled into a 96-well plate, and 1 µl of each strain was added. Co-cultures were cultivated under aerobic conditions at 37 °C for 24 h. After incubation, CFUs were determined as previously described. Phenotypic microarrays For carbohydrate utilization screenings, the phenotypic microarrays PM1 and PM2a from BiologTMwere used. Bacterial strains were cultivated on R2A agar plates (Difco) overnight. Colonies were scraped and adjusted to an OD600of 0.2 in PBS. Each well of the PM1 and PM2a plates was filled with 99 µl of MM9 without carbon sources and inoculated with 1 µl of bacterial suspension. For competition experiments, each well was inoculated with 1 µl of bacterial suspension of each strain. The plates were incubated aerobically in a microplate spectrophotometer with continuous shaking, OD600 was measured hourly for 24 h. Mixtures were plated on selective agar plates as previously described. Growth analysis Bacterial strains were cultivated on R2A agar plates (Difco) overnight. Colonies were scraped and adjusted to OD600of 0.2 in PBS. Subsequently, a flat-bottomed 96-well plate (Corning®) was filled with 99 µL of MM9 medium supplemented with the respective carbon source and inoculated with 1 µL of the bacterial suspension. Sterile media served as a negative control. Plates were incubated in a microplate spectrophotometer with continuous shaking (BioTek LogPhase 600 Microbiology Reader). OD600 was measured hourly over a time course of 72 hours. In vivo E. coli colonization SPF mice were treated with ampicillin (0.5 g / l) 4 d before starting the experiment. OMM12and OMM19mice did not receive ampicillin. On the day of colonization, respective bacteria were cultivated in 25 ml LB media (1:25) at 37 °C for 3-4 h. Afterwards, the culture was centrifuged at 500 g for 15 min. The pellet was suspended in 10 ml of PBS - 26 - and the required amounts were calculated. Mice were inoculated by oral gavage of 108CFUs of E. coli diluted in 200 µl PBS. The body weight of the mice was monitored, and feces were collected at different time points (1, 3, 6, 9, 14, 21, 28, and 42 days after gavage) to measure fecal E. coli burden by plating and 16S rRNA sequencing. In vivo decolonization experiments SPF mice were treated with ampicillin (0.5 g / l) 4 d before starting the experiment. OMM12and OMM19mice did not receive ampicillin. On the day of colonization, bacterial cultures were prepared as described previously. Mice were precolonized by oral gavage with 108CFUs / 200 µl in PBS.4 days after precolonization fecal CFUs of respective E. coli strains were checked by plating on selective agar plates. After successful precolonization (CFU / g of at least 109CFUs), the competing E. coli / MDR-E strain was administered by oral gavage (108CFUs in 200 µl PBS). The body weight of the mice was monitored, and feces were collected at different time points (1, 3, 6, 9, 14, 21, 28, and 42 days after gavage) to measure fecal E. coli burden by plating and 16S rRNA sequencing. DNA isolation The microbial community 16S rRNA gene DNA was extracted using precipitation with phenol-chloroform. 0.1 mm Zirconia beads were added to the feces sample until the bottom part of the tube was covered. Afterwards, 500 µl of 2x Buffer A and 200 µl of 20 % SDS were added to each sample. Then 500 µl of the bottom phase of Phenol:Chloroform: IAA was added. This mixture was shaken by a bead-beater for 2 min, then cooled down at 4 °C for 2 min, and then again shaken for 2 min. For phase separation, samples were centrifuged at 8000 rpm for 5 min at 4 °C. The aqueous phase was transferred to a fresh 1.5 ml tube and 600 µl of Phenol:Chloroform: IAA was added and mixed by inverting samples several times. Then, samples were centrifuged at 12700 rpm for 5 min at 4 °C for the second phase separation. Again, the aqueous phase was transferred to a fresh 1.5 ml tube.600 µl of 100 % Isopropanol (stored at -20 °C) and 60 µl (1 / 10 volume) of 3 M NaOAc (pH = 5.5) were added. The mixture was vortexed and then stored at -20 °C for at least 1 h or overnight. After incubation at -20 °C, samples were centrifuged at 12700 rpm for 20 min at 4 °C. Then, all liquid was sucked off. The remaining pellet was washed with 1 ml of 70 % EtOH (RT) and centrifuged at 12700 rpm for 5 min at 4 °C. Afterwards all liquid was sucked off carefully and the pellet was dried - 27 - in a speedvac machine for 15 min using no heat. The pellet was resuspended in 100 µl TE-buffer and incubated at 50 °C for 30 min on a shaker at 1000 rpm. When the pellet was completely dissolved, 1 µl of 10 µg / ml RNAse was added. Crude DNA was column purified (BioBasic Inc.) to remove PCR inhibitors. 16S rRNA gene amplification and sequencing 16S rRNA gene amplification of the V4 region (F515 / R806) was performed according to an established protocol previously described60. Briefly, DNA was normalized to 25 ng / µl and used for sequencing PCR with unique 12-base Golary barcodes incorporated via specific primers (obtained from Sigma). PCR was performed using Q5 polymerase (NewEnglandBiolabs) in triplicates for each sample, using PCR conditions of initial denaturation for 30 s at 98°C, followed by 25 cycles (10 s at 98°C, 20 s at 55°C, and 20 s at 72°C). After pooling and normalization to 10 nM, PCR amplicons were sequenced on an Illumina MiSeq platform via 300 bp paired-end sequencing (PE300). Using the Usearch8.1 software package (http: / / www.drive5.com / usearch / ) the resulting reads were assembled, filtered, and clustered. Sequences were filtered for low-quality reads and binned based on sample-specific barcodes using QIIME v1.8.0. Merging was performed using -fastq_mergepairs – with fastq_maxdiffs 30. Quality filtering was conducted with fastq_filter (-fastq_maxee 1), using a minimum read length of 250 bp and a minimum number of reads per sample = 1000. Reads were clustered into 97% ID OTUs by open- reference OTU picking and representative sequences were determined by use of the UPARSE algorithm (Edgar 2010). Abundance filtering (OTUs cluster >0.5%) and taxonomic classification were performed using the RDP Classifier executed at 80% bootstrap confidence cut-off. Sequences without matching reference datasets, were assembled as de novo using UCLUST. Phylogenetic relationships between OTUs were determined using FastTree to the PyNAST alignment (Price, Dehal, and Arkin 2010). The resulting OTU absolute abundance table and mapping file were used for statistical analyses and data visualization in the R statistical programming environment package phyloseq. Whole genome sequencing To assess the taxonomy of all E. coli isolates, bacteria were sent for whole genome sequencing. First, genomic DNA was extracted using the ZymoBIOMICS 96 MagBead - 28 - DNA Kit according to the manufacturer's instructions. Afterwards, libraries of each isolate were prepared using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina®. DNA was fragmented, ligated to adaptors, enriched, and assessed on a Bioanalyzer. Afterwards, libraries of each isolate were prepared using the Illumina DNA PCR-Free Prep and quantified with KAPA Library Quantification Kit Illumina Platforms. Samples were pooled and sent for whole genome sequencing performed by the group of Genome analysis at Helmholtz-HZI Center for Infection Research using NovaSeq 6000 S4 Reagent Kit v1.5 (300 cycles) and targeting depth of 1 million reads per sample. The resulting E. coli genomes have been deposited in GenBank (see Data availability). Identification of virulence-associated genes (VAGs), AMR, and colibactin genome islands The inventors identified genes related to virulence and antimicrobial resistance by running the abritamr tool (v1.0.14) on the nucleotide sequences of the isolates. In order to compare the commensal isolates from this invention to pathogenic ones, the inventors performed the same analysis on a collection of 912 clinical E. coli isolates from patients with bloodstream infections. The inventors further identified isolates carrying the colibactin genome island (pks+) following a similar approach developed previously. The inventors aligned the proteomes of all isolates against the protein sequences of the clb genes encoded in E. coli strain IHE3034 (GenBank accession AM229678.1), using Blast+ v2.12.0 with an e-value threshold of 1·10-4, a query and subject coverage > 80% and protein sequence identity > 70%. The inventors flagged an isolate to be pks+ if the inventors found a homolog of 16 of the 18 clb genes and if the average nucleotide distance between genes encoded in the same contig was below 5kbp. Association analysis To facilitate the annotation of association hits the inventors included 8 E. coli closed reference genomes from RefSeq, downloaded using the ncbi-genome-download package v0.3.3. The inventors included the following genomes: 536 (GCF_000013305.1), CFT073 (GCF_000007445.1), ED1a (GCF_000026305.1), IAI1 (GCF_000026265.1), IAI39 (GCF_000026345.1), MG1655 (GCF_000005845.2), UMN026 (GCF_000026325.1), and UTI89 (GCF_000013265.1). For each genome the inventors assigned their sequence type (ST) and Escherichia phylogroup using mlst v2.16 and - 29 - ClermontTyping (commit 740c59c), respectively. The inventors generated a de Bruijn graph from all input genomes and extracted unitigs and their presence / absence patterns across all samples using unitig-counter v1.1.0.71,72Each unitig with allele frequency < 1 encodes for a genetic variant, which can be a large one such as a gene transferred horizontally, as well as short variants such as single nucleotide polymorphisms (SNPs). In order to run associations between variants and the protective phenotype, a kinship matrix is required; this can be derived from a core genome alignment, which the inventors generated by first computing the pangeome with panaroo v1.3.0, using the “--clean-mode strict” argument, followed by a concatenation of individual alignments for each nucleotide coding sequence belonging to core genes (i.e. with frequency >= 95%). The inventors then used snp-sites v2.5.1 and bcftools v1.13 to convert the full core genome alignment to a VCF file containing the variant sites with minimum allele frequency (MAF) > 1%, and used this file to derive the kinship matrix using a python script. The inventors estimated narrow-sense heritability for the protective phenotype, using two different covariance matrices; one built from the STs of each strain (“lineage”) and another using the kinship matrix described above. The inventors used Limix v3.04, assuming normal errors for the point estimate and the inventors computed the 95% confidence intervals using the ALBI package (commit 90d819e). The inventors then tested the association between each unitig with MAF > 1% and the protective phenotype using a linear-mixed model as implemented in pyseer v1.3.6. The inventors determined an appropriate significance threshold by counting the number of unique unitigs presence / absence patterns tested, which reduces the risk of excessively deflating association p-values. The inventors mapped the unitigs passing the significance threshold back to all input genomes using bwa v0.7.17 and bedtools v2.31.1, using the output of panaroo to assign each unitig to a gene cluster. The unitigs were further filtered to reduce the number of spurious associations: unitigs were excluded if they were shorter than 30bp, if they were mapped to multiple locations in each individual genome, if they mapped to less than 9 samples (~2% of the sample size) and if they were mapped to more than 10 different genes across all samples. The inventors further computed the odds ratio - 30 - for each gene cluster using the average frequency and effect size as input for a previously developed method for binary phenotypes. The inventors further annotated the gene families with mapped unitigs by taking a representative protein sequence from the genomes encoding each gene family, giving priority to the 8 closed reference genomes, and using them as an input for eggnog-mapper v2.1.3. The derived annotations include COG categories, GO terms and mapping to KEGG entries; the inventors tested an enrichment for each of these annotation systems using the associated gene families as foreground and the annotation for E. coli IAI39 as the background, running a Fisher’s exact test for each annotation item, and the inventors used an FDR-corrected p-value threshold of 0.05 to indicate annotation items enriched in the associated gene families. The inventors generated a phylogenetic tree of all samples using the concatenated core genome alignment as an input for FastTree v2.1.11, using the GTR+CAT model. The tree and associated metadata was visualized using the microreact web interface.

[0002] - 31 - Table 1: Overview over strains as used in the context of the present invention. Several of the strains were deposited at the Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstraße 7B, 38124 Braunschweig, Germany on May 24, 2024 under the numbers as indicated. The Biosample numbers refer to the Bioproject PRJEB76066 (for E. coli), and PRJEB42167 for MK01, respectively (website https: / / www.ncbi.nlm.nih.gov / bioproject ). Indicated is furthermore the competitive “strength“ of the strains, as “fold change to control“, i.e. compared to a single culture of a respective MDR strain. The genomes of the Klebsiella strains are also disclosed in Osbelt, L., Almási, É.d.H., Wende, M. et al. Klebsiella oxytoca inhibits Salmonella infection through multiple microbiota-context-dependent mechanisms. Nat Microbiol (2024). https: / / doi.org / 10.1038 / s41564-024-01710-0. The phylogroup refers to the groups as described in Jolley, K. A. & Maiden, M. C. J. (in: BIGSdb: Scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics 11, (2010)), and Beghain, J., et al. (in: Typing: An easy-to-use and accurate in silico method for Escherichia genus strain phylotyping. Microb. Genomics 4, 1–8 (2018)). The determination of virulence genes and antimicrobial resistance (AMR) genes was made according to Sherry, N. L. et al. (in: An ISO-certified genomics workflow for identification and surveillance of antimicrobial resistance. Nat. Commun.14, (2023)), and Denamur, E. et al. (in: Genome wide association study of Escherichia coli bloodstream infection isolates identifies genetic determinants for the portal of entry but not fatal outcome. PLoS Genet. 18, 1–20 (2022)).

[0003]

[0004]

[0005] - 34 - Commensal E. coli strains show different competitive effects against MDR E. coli in an ex vivo screening assay Since the species E. coli is characterized by a large genomic diversity, as many strains as possible were screened. To enable a screening of 430 strains, the inventors found that the ex vivo setup is the most feasible. Thus, to characterize the commensal E. coli strains from the generated strain collection regarding their ability to compete with an MDR E. coli strain, the inventors utilized an ex vivo screening assay (1). Co-cultivation in cecum content of germ-free (GF) animals under microaerophilic conditions enables to mimic the gut environment and nutritional landscape of an in vivo setting without the influence of a resident microbiota. The inventors co-cultivated the human-derived commensal E. coli strains from the strain collection together with one clinical isolate of a multi-drug resistant (MDR) E. coli strain (strain MHH, patient isolate, ST617, origin: Hanover) in a 10:1 ratio (commensal: MDR) in the isolated cecal contents diluted in PBS, for 24 h at 37°C. The inventors used a single culture of the E. coli MHH in GF cecum content as a growth control and quantified the CFU / ml of the E. coli MHH in single and co-cultures by plating on selective agar plates. To present the results of this assay, the fold change of the co- cultures to the growth control was calculated. Here, a fold change of 1 means no influence on the growth of the E. coli MHH, a fold change of <1 means a reduction of growth, and a fold change of >1 means increased growth compared to the growth control. The inventors could observe a broad range of fold changes after co-cultivation with different commensal isolates. The fold change ranged from 16.8 (least competitive) to 0.004 (most competitive). The majority of strains showed a fold change of 1 to 0.1. (Figure 1). Comparing the four cohorts to each other, a similar pattern could be observed for each cohort, indicating that competitive or non-competitive effects are generally distributed and not linked to a specific cohort or age group. Specific commensal E. coli strains enable the decolonization of an MDR E. coli strain in a preventive manner in vivo To elucidate the potential protective effects shown in the ex vivo assay in vivo, the inventors selected a panel of commensal E. coli strains exhibiting different competitive effects. An SPF mouse model with ampicillin treatment was chosen to validate the results in vivo. This model was used to investigate the competitive properties of - 35 - Enterobacteriaceae before (1). Due to ampicillin treatment, strains with intrinsic ampicillin resistance were selected. The inventors selected the strains E. coli MR102, MR158, and RV228 as they belonged to the strains with a fold change below 0.02, thus showing a strong competition, as competitive strains and E. coli strains MR103, LK91, MK192, and LK192 as non-competitive strains (Fold-change >0.1) (Figure 1, strains marked in light gray). Mice were treated with 0.5 g / L ampicillin in their drinking water for four days before pre-colonization with 108CFUs of the respective commensal strain by oral gavage. Another group was treated with PBS as a control. After four days, pre- colonization levels were monitored by plating of fecal samples on selective agar plates and all mice were challenged by oral gavage with 108CFUs of the MDR E. coli MHH. Afterward, fecal colonization levels of the MDR E. coli MHH were monitored on days 1, 3, 6, 9, 14, 21, 28, and 42 by plating of fecal samples on selective agar plates. Ampicillin treatment was stopped on day three after administration of the MDR E. coli MHH, and mice were switched back to regular drinking water. A comparison of the pre-colonization levels of all strains shows that all strains can colonize mice to mean values of 109to 1010CFU / g (Figure 2A). Monitoring the fecal colonization kinetics, the inventors observed that pre-colonization with different commensal E. coli strains results in different colonization kinetics of the MDR E. coli MHH strain, compared to the control group (Figure 2B). It was observed that groups pre- colonized with E. coli strains MR102, MR158, RV228, MR193, LK91, and MK192 showed a 100-fold reduction of CFUs of MDR E. coli MHH by day three and a 10-fold reduction in the group pre-colonized with E. coli LK192, compared to the PBS treated group. On day nine, clearance in the groups pre-colonized with competitive isolates could be observed. Five out of 15 mice pre-colonized with MR102 and five out of ten mice pre- colonized with RV228 could clear out the MDR E. coli MHH from feces. This effect is increased at day 28 with clearance rates of 70-80 % in groups pre-colonized with competitive isolates. In the groups pre-colonized with E. coli MR103, LK91, and MK192 clearance rates of 40- 60 % were observed, whereas mice pre-colonized with E. coli LK192 showed a clearance rate of 10 %, and only 1 / 10 mice could clear the MDR E. coli MHH below the detection limit (Figure 2C). - 36 - These experiments support the inventor’s hypothesis that specific commensal E. coli isolates may harbor competitive effects, which are not exhibited by all strains. Furthermore, it was demonstrated that the conducted ex vivo assay is a valid tool for pre- screening of potential probiotic strains. The inventors selected the E. coli MR102 as a representative strain with probiotic potential and used it for further experiments. Analysis of carbohydrate utilization patterns of MDR and commensal E. coli strains To elucidate the ability of different E. coli strains to utilize various carbon sources, the inventors performed growth analysis of representative E. coli strains classified as MDR (MHH, 21236, 55652) since they were included in the following in vivo competition experiments and commensal strains, which were previously screened for their competitive phenotypes in vivo (MR102, MR158, RV228, MR103, LK91, MK192, LK192). Growth curves were performed in basal minimal media (MM9) over 72 h in a microplate reader under aerobic conditions. Bacteria were grown overnight on R2A agar plates to ensure the loss of storage of nutrients. Afterwards, cultures were diluted and inoculated in MM9 supplemented with 5 g / L of the respective carbon source. The area under the curve (AUC) was calculated and different carbohydrate utilization capabilities by different E. coli strains were observed. The strains investigated here displayed different carbohydrate utilization patterns (Figure 8). Among the MDR strains, MDR E. coli MHH (ST617) was able to grow in 20 out of the 39 carbon sources tested, while MDR E. coli 55652 (ST167) exhibited growth in 24 carbon sources and MDR E. coli 21236 (ST131) showed growth in 17 carbon sources. Competitive strains E. coli MR102, E. coli MR158, and E. coli RV228 utilized 22, 21, and 22 carbon sources, respectively. Intermediate and non-competitive strains displayed highly variable utilization patterns. E. coli MR103 was able to utilize up to 24 carbon sources, while E. coli LK91 could only utilize 13 carbon sources in total and E. coli MK192 was able to utilize 21 out of the 39 tested carbon sources and E. coli LK192 was able to utilize 20 carbon sources. Visualizing the number of carbon sources, which resulted in strong growth (AUC >20) for each strain, E. coli MR102 could utilize the most carbon sources with AUC >20 (n=15). To focus on the utilization patterns of E. coli MHH, MR102, and LK192, the inventors observed a significant degree of overlap (15 / 39 tested carbon sources). Furthermore, E. coli MR102 shows an overlap in utilized carbon sources with MDR E. coli MHH (D- - 37 - malic acid, lactulose) and E. coli LK192 (maltotriose, maltodextrin, maltose). Moreover, distinct utilization patterns become evident, as certain sugars were exclusively metabolized by the single strains. D-cellobiose, D-salicin, and D-fructose could solely be utilized by MDR E. coli MHH, N-acetyl-D-galactosamin by E. coli MR102, and L- rhamnose by E. coli LK192. In vivo supplementation with cellobiose diminishes the protective effect To investigate whether carbohydrate competition is involved in the protective effect of E. coli MR102 against MDR E. coli MHH in vivo, mice were supplemented with cellobiose, a carbon source that can only be utilized by MDR E. coli MHH, but not E. coli MR102. Sugar supplementation was started with the start of ampicillin treatment at day -7 and continued until day 14. The fecal burden of MDR E. coli MHH in cellobiose-treated and E. coli MR102 pre-colonized mice was compared to PBS and E. coli MR102-treated mice without sugar supplementation. CFU / g of MDR E. coli MHH were significantly higher in cellobiose-treated groups compared to E. coli MR102 control on day 14, reaching a 10,000-fold difference on day 42. On top of that, clearance of E. coli MR102 was significantly delayed and decreased in cellobiose-treated mice. Mice pre-colonized with E. coli MR102 and supplemented with cellobiose reached a final clearance rate of 50 %, compared to 80 % in the non-cellobiose treated group. To investigate whether cellobiose supplementation affects E. coli MR102 colonization and thereby interferes with the protective effect, the inventors monitored the fecal colonization levels of E. coli MR102 with or without cellobiose treatment at different time points of the experiments. No differences could be observed between both groups for the pre-colonization levels and the day six levels. Notably, at day 28 colonization of E. coli MR102 was rather slightly increased in cellobiose-treated mice. This leads to the hypothesis that the diminished protective effect is not caused by lower colonization levels of protective E. coli MR102. - 38 - Evaluation of the strain specificity of commensal isolates and the potential to broaden the target spectrum by a combination of different strains The competitive effect of different commensals is strain-specific Since strains belonging to the species E. coli are characterized by a large genomic variability and diversity, including between MDR E. coli strains, the inventors wanted to assess whether the protective effect is strain-specific. To investigate the competitive effects of commensal isolates against a variety of strains, the inventors conducted an ex vivo assay, screening different commensal isolates against a panel of 18 MDR E. coli strains and one EPEC strain.16 of the MDR strains represent different STs associated with infections and gut colonization in a hospital setting and were isolated from rectal swabs or human fecal samples. Two additional strains isolated from a dog and a crow, respectively, and one EPEC strain were included. The inventors co-cultivated these strains together with either one of four commensal E. coli strains from the strain collection (MR102, MR103, MR134, and MK289) or E. coli Nissle. Furthermore, the inventors included K. oxytoca MK01, which was previously described to have the ability to promote gut decolonization of MDR K. pneumoniae (1), as well as mixtures of the commensal E. coli strains and K. oxytoca MK01. The respective commensal strains were classified by the ability to reduce the CFUs <10- fold (non-competitive), <10-fold (intermediate), or <100-fold (competitive) (Figure 3). The inventors found that the commensal strains show different competitive effects. K. oxytoca MK01 could reduce the CFUs of most of the strains (12 / 19), followed by the E. coli strains MR102 and MK289 (7 / 19), then MR103 and MR134 (5 / 19). The probiotic E. coli Nissle could only reduce the CFUs of two of the MDR strains. Interestingly, the inventors could identify one MDR strain (21236, ST131) that was reduced by all of the strains and one MDR strain (55652, ST167), which could not be reduced by any of the tested isolates. Moreover, the spectrum of competitive effects of each commensal could be broadened by combining commensal E. coli strains with K. oxytoca MK01. A mixture of K. oxytoca MK01 and E. coli MK289 could reduce the CFUs of all 19 strains and the combination of K. oxytoca MK01 and E. coli MR102 could reduce the CFUs of 84% (16 / 19) of the tested strains. - 39 - Surprisingly, the combination of K. oxytoca MK01 and E. coli Nissle showed the least competitive effects and could reduce the CFUs of only 13 of the 19 tested strains. Together, these experiments showed that combinations of specific commensal strains could broaden the protective effect. E. coli MR102 is able to compete with MDR E. coli 55652 (ST167) and 21236 (ST131) To validate the results of the ex vivo assay, two MDR E. coli strains were selected for in vivo experiments. The strain E. coli 21236 (ST131) was selected since ST131 is the most prevalent and urgent sequence type of MDR E. coli strains regarding colonization and infections in hospital settings5,6. In addition, the strain E. coli 55652 (ST167) was selected, because it could not be reduced by any of the commensal E. coli strains or K. oxytoca MK01 alone. Both strains were screened in the SPF ampicillin mouse model. SPF mice were treated with 0.5 g / L ampicillin in their drinking water and pre-colonized with 108CFUs of the E. coli MR102 or PBS, after four days of pre-colonization all mice were challenged with 108CFUs of the respective MDR E . coli strain. Afterwards, fecal colonization levels of MDR E. coli were monitored over six weeks, by plating on selective agar plates. CFUs of MDR E. coli 21236 (ST131) in the group pre-colonized with E. coli MR102 were reduced more than 1000-fold already at day one, from 1010CFU / g in PBS treated group to 106CFU / g. This effect further increased by day six. At day nine, all mice pre-colonized with E. coli MR102, reduced the CFU / g of the MDR E. coli strain 21236 below the detection limit, while PBS-treated mice were still colonized to 109CFU / g. Until the end of the experiment, the pre-colonized mice remained without detectable MDR E. coli 21236, while PBS-treated mice maintained colonization levels up to 105CFU / g until day 42 (Figure 4A). In contrast to MDR E. coli strain 21236 (ST131), which was well cleared, strain 55652 (ST167) showed different colonization kinetics throughout the experiment. During ampicillin treatment, the inventors could not observe a reduction of CFUs between the PBS- and E. coli MR102-treated groups. Beginning on day six, a 10 to 100-fold reduction of CFU / g was observed between the two groups. Notably, four out of nine mice could even clear out the MDR E. coli 55652, whereas the other mice remained colonized to 107- 108CFU / g at the end of the experiment. The PBS-treated group maintained colonization levels of 109-1010CFU / g until day 42 (Figure 4B). - 40 - Thus, the inventors could demonstrate that the E. coli strain MR102 harbors the potential to decolonize different STs of MDR E. coli strains. In the case of the competition between E. coli MR102 and E. coli 55652, the inventors observed no competition ex vivo, but clearance in 50 % of mice screened in vivo (Figure 4B). Combination of E. coli MR102 and K. oxytoca MK01 shows the ability to decolonize different strains of MDR Enterobacteriaceae Since the inventors could demonstrate ex vivo that a combination of E. coli MR102 and K. oxytoca MK01 enables competition against a broader spectrum of E. coli isolates, the inventors wanted to investigate the decolonization potential of strain combinations in vivo. Since combinations of candidate probiotic strains could also interfere with each other, the inventors first tested whether they could co-colonize SPF mice. Therefore, SPF mice treated with 0.5 g / L ampicillin in their drinking water, were colonized with a 1:1 mixture of E. coli MR102 and K. oxytoca MK01 (108CFUs in total). Ampicillin treatment was stopped at day three and fecal colonization of both strains was monitored at different time points for a time course of six weeks. Notably, both strains showed similar colonization levels in the feces at all time points demonstrating that they can co-exist in the gut. During ampicillin treatment on days one and three, colonization levels were at 1010CFU / g. After withdrawal of the antibiotic, the colonization levels decreased to 109CFU / g on day 21 and remained stable at 106CFU / g until the end of the experiment on day 42 (Figure 5). Since the inventors could demonstrate that the commensal strains E. coli MR102 and K. oxytoca MK01 can co-colonize the gut of SPF mice, the inventors selected the MDR E. coli strain 55652 (ST167) as a challenge strain, since the inventors observed that this strain cannot be fully cleared by E. coli MR102 alone. To investigate whether cooperation improves clearance, the inventors compared mice colonized with either E. coli, K. oxytoca, or the mixture to control mice. Of note, pre- colonization levels of the two commensal strains were comparable at 1010CFU / g, but slightly lower in co-colonized mice (Figure 6A). After a challenge with 108CFUs of the MDR E. coli 55652, CFUs in groups pre-colonized with either E. coli MR102 or K. - 41 - oxytoca MK01 were reduced 5 to 10-fold at day one and three compared to the PBS- treated group, whereas the group pre-colonized with the mixture of both strains reduced the CFU / g 1000-fold. This effect further increased throughout the experiment. While the groups pre-colonized with K. oxytoca MK01 or E. coli MR102 could only reduce the CFUs of E. coli 55652, groups pre-colonized with the combination of both strains could clear out the MDR E. coli 55652 to the detection limit by day nine. The CFUs remained stable throughout the rest of the experiment, the PBS-treated group showed 105CFU / g, and the groups pre-colonized with one of the strains showed 104-105CFU / g (Figure 6B). Comparing the clearance kinetics, the inventors could observe 100 % clearance in the groups pre-colonized with the mix and 60% clearance for groups pre- colonized with either one of the strains (Figure 6C), demonstrating that the combination of probiotic candidates is more effective in displacing E. coli strain 55652 (ST167). Based on the results that a mixture of E. coli MR102 and K. oxytoca MK01 can decolonize MDR E. coli 55652 specifically, the inventors wanted to analyze whether the combination is also able to compete against different MDR-E strains or gastrointestinal pathogens in vivo. The inventors selected a panel of four MDR-E strains comprising a strain of E. coli (strain MHH), E. cloaceae (strain MHH), K. pneumoniae (strain MD), P. mirabilis (strain MHH), and the enteric pathogen S. Typhimurium (strain EM12442). Of note, it was shown previously that E. coli MR102 alone could decolonize the E. coli MHH and that K. pneumoniae MD can be decolonized by K. oxytoca MK01. Thus, for these two instances, it was of great interest to observe if the protective effect will be increased or diminished due to a co-colonization with E. coli MR102 and K. oxytoca MK01. To investigate the competitive effects in vivo, mice were pre-colonized with either E. coli MR102 or K. oxytoca MK01 alone or in combination, and another group was treated with PBS. After a successful pre-colonization, 108CFUs of the respective MDR-E strain or 105CFUs of S. Typhimurium were administered. CFU / g of the MDR E. cloaceae strain could be reduced to the detection limit by day six in mice pre-colonized with the combination. Pre-colonization with one of the two strains alone led to a reduction of CFUs to the detection limit one day later on day nine. Colonization levels in the PBS-treated group remained at 107CFU / g at day 42 (Figure 7A). E. coli MHH and K. pneumoniae - 42 - MD could also be reduced to the detection limit by day 42 (E. coli MHH) or day 14 (K. pneumoniae MD) (Figure 7B, C). The inventors could not observe a reduction of colonization levels more than 10-fold of the MDR P. mirabilis strain in groups pre- colonized with the single strains or the combination (Figure 7D). S. Typhimurium colonization levels were strongly reduced after pre-colonization with either E. coli MR102 or K. oxytoca MK01 and the combination. Five days after the challenge with S. Typhimurium, CFU / g in groups pre-colonized with the single strains were reduced 1000- fold, whereas a pre-colonization with the combination led to a 10.000-fold reduction (Figure 7E). Clearance levels of 100 % could be observed for MDR E. cloaceae, E. coli, and K. pneumoniae. Application of probiotic E. coli strains alone and in probiotic combinations against A. baumannii To simulate an established intestinal commensal community, in vitro coculture assays were conducted with a 10:1 ratio of commensal bacteria to A. baumannii under oxygen- limited conditions using the AnaeroGen system. Two CRAB clinical isolates were included: FR4326, which originated from a rectal swab, and FR3462, isolated from the urine of a systemic infection case. First, to assess the interaction between E. coli and A. baumannii, bacterial cocultures of E. coli strains EC103, MK289, and MR102, as well as K. oxytoca MK01 and A. baumannii FR4326 or FR3462, were established under oxygen-limiting conditions in a tryptone-based medium without and with 10 g / L maltose supplementation. With maltose supplementation, all tested bacteria were able to suppress A. baumannii by at least 102-fold (Figure 9A, B). Depending on the E. coli strain, maltose-dependent reductions were observed of up to 104-fold for MR102 against both Acinetobacter strains, while K. oxytoca MK01 consistently reduced A. baumannii to the assay's detection limit. To investigate whether similar maltose-dependent reductions occur in the coculture of two A. baumannii effectively reducing bacterial strains (MK01, MR102) with FR3462 or FR4326, assays were repeated for single and combined probiotic candidates while monitoring A. baumannii (Figure 9C, D) and probiotic bacterial counts (Figure 9E, F). MK01 and MR102 were consistently detected at high colony-forming units (CFU / mL) - 43 - exceeding 10^8, while both tested A. baumannii strains showed significantly reduced CFU / mL. The combination of probiotic candidates K. oxytoca MK01 and E. coli MR102 (“mix”) exhibited a similar Acinetobacter effective reduction compared to the single strains. In vivo presence of maltose promotes the competitive effect All observed effects depended on the presence of maltose, a common disaccharide that is part of human nutrition and can be utilized by humans as well as many intestinal microbes. The inventors hypothesized that, similar to intraspecies E. coli competitive behavior, carbohydrate presence is a key factor here as well. Therefore, the inventor tested a coculture assay in tryptone medium supplemented with either 10 g / L maltose, lactose, or mannose to determine the influence of different simple sugars on the interaction of E. coli MR102 and non-carbohydrate-utilizing mutants for lactose (∆lacZ) and mannose (∆manA) with the intestinal isolate A. baumannii FR4326. Lactose and mannose supplementation similarly enabled MR102 wild-type to suppress FR4326 (Figure 9G). Mutants of MR102 that lack the ability to metabolize lactose or mannose were, respectively, unable to suppress A. baumannii, confirming that specific carbohydrate metabolism is necessary for probiotic effectiveness. All MR102 strains were detected at similar CFU / mL under all supplementation conditions (Figure 9H). Decolonization of MDR-E in mice The use of probiotic strains was tested further in mice that were first colonized with the respective MDR-E strain and then treated with either a combination of E. coli MR102 and K. oxytoca MK01 (Fig.10) or E. coli MR102 alone (Fig.11). Mice were treated with 0.5g / L ampicillin from day -7 to day 3. Three days after starting the ampicillin treatment (day -4), mice were orally gavaged with 10^8 CFUs with the MDR-E strains. After successful colonization of the MDR-E, four days later, mice were treated with PBS or 10^8 CFUs of the probiotic strains (d0). Colonization of the MDR-E strains was then monitored on days 1, 3, 6, 9, 14, 21, 28, and 42, by selective plating. The analysis demonstrated a significant reduction in colonization levels. Specifically, mice treated with the probiotic mixture showed a 10- to 100-fold decrease in bacterial load compared to the PBS-treated control group. This effect was observed for E. coli - 44 - MDR1 (Fig. 10A), E. coli MDR2 (Fig. 10C), and MDR K. pneumoniae (Fig. 10E). Furthermore, the inventors observed high clearance rates: after 42 days, E. coli MDR1 was eliminated in 75% of treated mice (Fig.10B), while E. coli MDR2 (Fig.10D) and MDR K. pneumoniae (Fig.10F) exhibited clearance rates of 80% in probiotic-treated groups. Notably, treatment of E. coli MDR2-colonized mice with E. coli MR102 alone resulted in complete clearance in 100% of cases (Figure 11). The findings of the inventors show that administering the probiotic strain E. coli MR102—whether alone or in combination with K. oxytoca MK01—represents an effective strategy for reducing the colonization of already established MDR-E strains. These promising results highlight the potential of probiotic-based approaches to combat multidrug-resistant pathogens within the gastrointestinal tract. To summarize, the inventors could demonstrate that a combination of E. coli MR102 and K. oxytoca MK01 can decolonize a variety of clinically relevant MDR-E strains and gastrointestinal pathogens. By combining the two probiotic strains, the target spectrum was broadened in comparison to the use of one strain by itself, and this indicates a synergistic effect. Furthermore, the use of probiotic E. coli MR102 was identified as a novel strategy to combat multidrug-resistant A. baumannii, representing a non-antibiotic alternative for intestinal decolonization. The carbohydrate-dependent suppression mechanism opens new possibilities for dietary interventions to enhance probiotic efficacy. Furthermore, the potential for multispecies probiotic formulations strengthens the case for developing next- generation biotherapeutics against MDR pathogens. The present invention relates to the use of probiotic E. coli MR102 alone or in combination with targeted carbohydrate supplementation and multispecies formulations to prevent and reduce A. baumannii colonization in high-risk individuals. Further screening is required to validate and expand the target spectrum of the combinations as tested. Furthermore, it needs to be investigated how the bacteria enable competition against such a broad spectrum of relevant pathogens. - 45 - References as cited 1. Osbelt, L. et al. Klebsiella oxytoca causes colonization resistance against multidrug- resistant K. pneumoniae in the gut via cooperative carbohydrate competition. Cell Host Microbe 29, 1663-1679.e7 (2021). 2. Eisenhard, L. Investigating the ecological niche of commensal and multidrug-resistant Escherichia coli strains in the gut environment. Tech. Univ. Braunschweig Masterthesis, (2023). 3. Eberl, C. et al. E. coli enhance colonization resistance against Salmonella Typhimurium by competing for galactitol, a context-dependent limiting carbon source. Cell Host Microbe 29, 1680-1692.e7 (2021). 4. Oliveira, R. A. et al. Klebsiella michiganensis transmission enhances resistance to Enterobacteriaceae gut invasion by nutrition competition. Nat. Microbiol.5, 630–641 (2020). 5. Banerjee, R. & Johnson, J. R. A new clone sweeps clean: The enigmatic emergence of Escherichia coli sequence type 131. Antimicrob. Agents Chemother. 58, 4997–5004 (2014). 6. Johnson, J. R., Johnston, B., Clabots, C., Kuskowski, M. A. & Castanheira, M. Escherichia coli sequence type ST131 as the major cause of serious multidrug-resistant E. coli infections in the United States. Clin. Infect. Dis.51, 286–294 (2010). 7. McConnell, M. J., Actis, L. & Pachón, J. Acinetobacter baumannii: human infections, factors contributing to pathogenesis and animal models. FEMS microbiology reviews 37, 130–155; 10.1111 / j.1574-6976.2012.00344.x (2013). 8. Guidelines for the prevention and control of carbapenem-resistant Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa in health care facilities (World Health Organization, Geneva, Switzerland, 2017). 9. Teng, J. et al. Combatting resistance: Understanding multi-drug resistant pathogens in intensive care units. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 167, 115564; 10.1016 / j.biopha.2023.115564 (2023). 10. Antimicrobial Resistance Division (AMR). WHO bacterial priority pathogens list, 2024: Bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance. World Health Organization (2024).

Claims

- 46 - H34511WO Claims 1. A bacterium selected from the group of consisting of at least one of E. coli strains MR010, MK031, MR043, MR045, MR102, MR134, MR158, MK241, MK249, MK267, MK270, MK289, MR107, MR050, MK254, MR174, MR084, MK251, MK062, MR097, MK270, MR138, MK262, and MK179 for use in the prevention and / or treatment of pathogenic and / or antibiotic resistant Enterobacteriaceae, such as E. coli and / or Klebsiella pneumoniae, in particular multi-resistant (MDR) Enterobacteriaceae, Enterococci and / or Acinetobacter in the microbiome of an animal, in particular an avian or mammalian subject.

2. Bacterium for use according to claim 1, wherein said use is in combination with at least one bacterium selected from the group of Klebsiella michiganensis and / or Klebsiella oxytoca, in particular from the strain MK01, MR08, MR050, and species related to Klebsiella oxytoca MK01, and wherein preferably the combination shows a synergistic effect in the prevention and / or treatment.

3. Bacterium for use according to claim 2, wherein the combination is selected from the group of E. coli strains MR102, and MK289 with Klebsiella oxytoca MK01, MR08, or Klebsiella michiganensis MR050, preferably MK289 with Klebsiella oxytoca MK01 or MR102 with Klebsiella oxytoca MK01 and / or MR08.

4. Bacterium for use according to any one of claims 1 to 3, wherein said pathogenic and / or antibiotic resistant Enterobacteriaceae is selected from at least one of E. coli strains MHH, 56922, 56236, 56589, 56231, 55652, 55838, 54519, 55476, 56035, 21236, 26260, 53221, 54875, 44583, 51583, PBIO729, PBIO730, and EPEC, preferably MHH, 56922, 56231, 21236, 53221, PBIO729, and EPEC.

5. Bacterium for use according to any one of claims 1 to 3, wherein said pathogenic and / or antibiotic resistant Acinetobacter is selected from at least one of Acinetobacter baumannii, preferably strains A. baumannii FR4326 and FR3462, and CRAB strains, preferably CRAB-strains.- 47 - 6. Bacterium for use according to any one of claims 1 to 5, wherein said use is in combination with supplemented lactose and / or maltose, for example with about 10g / L of each sugar.

7. Bacterium for use according to any one of claims 2 to 6, wherein said species related to Klebsiella oxytoca is selected from the group consisting of K. michiganensis, and Klebsiella that are related to or have a carbohydrate utilization pattern substantially identical to K. oxytoca strain MK01, MR08, or Klebsiella michiganensis MR050.

8. Bacterium for use according to any one of claims 1 to 7 in reestablishing colonization resistance after treatment and / or antibiotic dysbiosis in the microbiome of an animal, in particular an avian or mammalian subject.

9. Bacterium for use according to any one of claims 1 to 8, wherein said microbiome is located in the gut of said animal, in particular said an avian or mammalian subject.

10. Bacterium for use according to any one of claims 1 to 9, wherein said prevention and / or treatment of said pathogenic and / or antibiotic-resistant Enterobacteriaceae and / or Acinetobacter and / or said reestablishing colonization resistance after treatment and / or antibiotic dysbiosis is in the context of bacterial infection, inflammatory bowel disease (IBD), bloodstream infection, sepsis, allogeneic hematopoietic stem cell transplantation, and bacterial invasion into the liver, spleen and / or mesenteric lymph nodes (MLN) in said animal, in particular said avian or mammalian subject.

11. Bacterium for use according to any one of claims 1 to 10, wherein said use comprises fecal microbiota transplant (FMT), in a suitable medium, such as sterile saline, or in a suitable solid dosage form, such as a suppository or capsule, in particular stomach-acid resistant, or as a probiotic.

12. Bacterium for use according to any one of claims 1 to 11, wherein said use is in a dosage of between 1012and 107CFU, preferably between 1010and 108CFU of said bacterium.- 48 - 13. Bacterium for use according to any one of claims 1 to 12, wherein said use is in combination with at least one bacterial species selected from the group consisting of Bacteroidetes, Actinobacteria, and Firmicutes spec., and preferably from the families Lachnospiraceae, such as, for example, Schaedlerella, Chodladocola, Acutalibacteraceae, such as for example Eubacterium_R, Lactobacillaceae, such as, for example, Ligilactobacillus, Eggerthellaceae, such as, for example, Adlercreutzia, and Rikenelleaceae, such as, for example, Alistipes, and in particular human isolates thereof.

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