WO2021123387A1 - Prédiction de manifestations cliniques d'une dysbiose de microbiote intestinal - Google Patents

Prédiction de manifestations cliniques d'une dysbiose de microbiote intestinal Download PDF

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WO2021123387A1
WO2021123387A1 PCT/EP2020/087329 EP2020087329W WO2021123387A1 WO 2021123387 A1 WO2021123387 A1 WO 2021123387A1 EP 2020087329 W EP2020087329 W EP 2020087329W WO 2021123387 A1 WO2021123387 A1 WO 2021123387A1
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bacteria
ratio
relative abundances
relative
bifidobacterium
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PCT/EP2020/087329
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English (en)
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Matilda BERKELL
Mohamed Mysara Abdelwahab AHMED
Surbhi MALHOTRA
Basil Britto XAVIER
Cornelis Hendrinus VAN WERKHOVEN
Marcus Johannes Marie Bonten
Jean De Gunzburg
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Da Volterra
Universiteit Antwerpen
University Medical Center Utrecht Holding B.V.
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Publication of WO2021123387A1 publication Critical patent/WO2021123387A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to a method for predicting whether a subject is potentially at risk of clinical manifestations of gut microbiota dysbiosis, also called intestinal microbiota dysbiosis, such as a Clostridioides difficile infection (CD I).
  • gut microbiota dysbiosis also called intestinal microbiota dysbiosis
  • CD I Clostridioides difficile infection
  • the gastrointestinal tract is the residence of trillions of microorganisms that include bacteria, archaea, fungi and viruses. All these microorganisms form a microbial community that is most often called gut microbiota or intestinal microbiota but also sometimes called gut flora, intestinal flora, gut microflora or intestinal microflora. The collective genomes of the whole microbial community form what is called the gut microbiome, also called intestinal microbiome. Diet, age, stress, diseases and medical treatments, for example antibiotic treatments, cause increases or decreases in relative abundance and diversity of bacterial species of the microbiota.
  • dysbiosis a persistent imbalance of the gut's microbial community, named dysbiosis, relates to many clinical manifestations such as, non-exhaustively, inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS), diabetes, obesity, cancer, cardiovascular and central nervous system disorders, C. difficile infection and disruption of the immune system.
  • IBD inflammatory bowel diseases
  • IBS irritable bowel syndrome
  • Clostridioides difficile (formerly named Clostridium difficile) is an opportunistic, anaerobic, Gram positive, spore-forming, toxin-producing bacterium of the gut microbiota. It can provoke C. difficile infection (CDI), with symptoms ranging from mild diarrhoea to potentially fatal pseudomembranous colitis. C. difficile is acquired by ingesting spores transmitted from other patients or healthcare personnel, either through the hands or the environment. CDI is responsible for 15-25% of nosocomial cases of antibiotic-associated diarrhoea.
  • Clostridioides difficile infections are considered the leading cause of infectious healthcare-related diarrhoea worldwide and substantial costs have been attributed to its management (Lessa et ah, 2015; Zhang et ah, 2018).
  • C. difficile The incidence of C. difficile is heterogeneous in the population: it is generally very low in the general population but can be much higher in specific patient groups. This makes preventive interventions often burdensome and not cost-efficient in the medical practice although the medical need is high considering the limited preventative and curative treatment options and debilitating aspects of the disease as well as its tendency to spread with spores and a few reported episodes of epidemics. Identification of patients potentially at risk of developing CDI is of paramount importance for diagnostic and therapeutic purposes and would help offer better personalized care to patients without submerging the available hospital resources. For example, this would be extremely interesting when patients are to receive antibiotics, widely recognized to increase the risk of occurrence CDI, to evaluate the risk-benefit balance and adapt the care accordingly.
  • the present invention relates to a method for predicting whether a subject is potentially at risk of clinical manifestations of gut microbiota dysbiosis, the method comprising detecting the presence, absence, quantity or relative abundance of a marker gene with a specific sequence belonging to each of the following taxa: Enterococcus, Finegoldia, Blautia, Ruminococcus , Clostridium, Akkermansia, Porphyromonas, Roseburia, Alistipes, Ezakiella, Bacteroides, Bifidobacterium, Escherichia, Shigella, Pseudescherichia, Pectobacterium, Erwinia, Pantoea, Cronobacter or Anaerocuccus in the gut microbiota of said subject or in a fecal sample of said subject.
  • the method comprises detecting the relative abundance of said bacteria.
  • the present invention also relates to a method for predicting whether a subject is potentially at risk of developing Clostridioides difficile infection (CDI), the method comprising detecting the presence, absence, quantity or relative abundance of a marker gene with a specific sequence belonging to each of the following taxa: Enterococcus, Finegoldia, Blautia, Ruminococcus, Clostridium, Akkermansia, Porphyromonas, Roseburia, Alistipes, Ezakiella, Bacteroides, Bifidobacterium, Escherichia, Shigella, Pseudescherichia, Pectobacterium, Erwinia, Pantoea, Cronobacter or Anaerocuccus in the gut microbiota of said subject or in a fecal sample of said subject.
  • the method comprises detecting the relative abundance of said bacteria.
  • the invention also relates to a method for predicting whether a subject is potentially at risk of developing CDI, the method comprising measuring the ratio of the relative abundance of a first bacteria and the relative abundance of a second bacteria in a gut microbiota sample or fecal sample of said subject.
  • the first bacteria is an Enterococcus bacteria or a Finegoldia bacteria.
  • Another aspect of the invention relates to a method for the treatment of an infection in a subject, the method comprising administering to said subject a therapeutically effective amount of an antibiotic suitable for the treatment of said infection, wherein decision to treat or not to treat said subject is based on the prediction of said subject to potentially be at risk of developing CDI according to the methods described herein.
  • Another aspect of the invention relates to a method for the prevention of CDI in a subject, the method comprising administering a vaccine or another product for prevention of Clostridioides difficile infection or isolating the patient in the hospital ward or even deploying advanced cleaning techniques to limit the exposure of the patient to spores of C. difficile, wherein decision to proceed with these procedures is based on the prediction of said subject to potentially be at risk of developing CDI according to the methods described herein.
  • Another aspect of the invention relates to a method for the prevention of CDI in a subject, the method comprising administering a live biotherapeutic product, such as a probiotic or a fecal microbiota transplant, wherein decision to proceed with these procedures is based on the prediction of said subject to potentially be at risk of developing CDI according to the methods described herein.
  • a live biotherapeutic product such as a probiotic or a fecal microbiota transplant
  • Another aspect of the invention relates to a method for the prevention of CDI in a subject, the method comprising stool biobanking from the patient prior to therapy/at the time of admission foreseeing a need for a fecal microbiota transplant following unavoidable antibiotic therapy thus giving the possibility to use the patient’s own stool and avoid the known risks associated with a foreign donor fecal transplantation.
  • the present inventors have shown that the risk of developing clinical manifestations of dysbiosis, such as CDI, can be correlated to the presence, absence, quantity or relative abundance of certain bacteria in the gut microbiota (also known as the gut flora or gut microflora). These bacteria can thus serve as biomarkers to detect subjects potentially at risk of developing CDI, from a gut microbiota sample or fecal sample of said subjects.
  • the potential of a subject to be at risk of developing CDI is determined based on a detection of the presence or absence, or on the measure of the quantity or relative abundance of bacteria from at least one bacterial species from a bacterial genus selected in the group consisting of Enterococcus, Finegoldia, Blautia, Ruminococcus , Clostridium, Akkermansia, Porphyromonas, Roseburia, Alistipes, Ezakiella, Bacteroides or Bifidobacterium in the gut microbiota sample or a fecal sample of the subject.
  • the measured quantity or relative abundance of said bacteria can be compared to a reference value, wherein a decreased or increased measured value as compared to the reference value can be indicative of a subject potentially at risk of developing CDI.
  • the potential of a subject to be at risk of developing CDI is determined based on the measure of the ratio of the relative abundance of a first bacteria species from a first bacteria genus to the relative abundance of a second bacteria species from a second bacteria genus.
  • the ratio measured in the method can be compared to a reference value, wherein a decreased or increased ratio compared to the reference value can be indicative of a subject potentially at risk of developing CDI.
  • the potential of a subject to be at risk of developing CDI is determined based on the measure of the abundance of a first bacteria and a second bacteria, and the abundance of the first bacteria compared with a first threshold (or first cutoff value) and the abundance of the second bacteria compared with a second threshold (or second cutoff value).
  • the approach presented above can be extended to the abundance for three, or four, or more bacteria compared with three thresholds values.
  • the subject can be a subject that has been diagnosed with an infection and who receives, or will receive, an antibiotic for the treatment of said infection. Thanks to the method of the present invention, prediction can be made whether the subject who will receive the antibiotic treatment will be potentially at risk of developing clinical manifestations of gut microbiota dysbiosis such as CDI as a consequence of said treatment. A prediction of a potential risk of developing CDI can allow managing the subject with knowledge of this information.
  • Such management can include, for example, further explorations of the most suitable antibiotic to prescribe to the subject to avoid the development of CDI, the co administration together with the antibiotic of a treatment designed to protect/preserve the gut microbiota during such treatment (this could consist in the administration of an enzyme to hydrolyze antibiotic residues in situ in the gut, or of an adsorbent to sequester antibiotic residues in the gut), administration of a microbiota complementation or replacement therapy (such as a fecal microbiota transplant, or one or several bacterial strains extracted from natural sources, or laboratory cultured and formulated, inch non toxigenic C.
  • a treatment designed to protect/preserve the gut microbiota during such treatment this could consist in the administration of an enzyme to hydrolyze antibiotic residues in situ in the gut, or of an adsorbent to sequester antibiotic residues in the gut
  • administration of a microbiota complementation or replacement therapy such as a fecal microbiota transplant, or one or several bacterial strains extracted
  • the subject can also be a patient receiving a prophylaxis with antibiotics to avoid a bacterial infection for example when the patient is immuno-compromised.
  • a prophylaxis with antibiotics to avoid a bacterial infection for example when the patient is immuno-compromised.
  • the detection of a high risk of CDI could help healthcare providers adapt the care offered to the patient.
  • the subject can also be a patient with a history of CDI, i.e. previous infections by C. difficile in his/her medical history.
  • a patient is known to be at risk and it could be interesting to detect patients at even higher risk of CDI.
  • the detection of a high risk of CDI could help healthcare providers adapt the care offered to the patient.
  • the subject can also be a patient that is screened to be enrolled in a clinical study to assess the efficacy of a drug or medical device to prevent CDI.
  • the prediction of a potential risk of developing CDI will help decide if the patient is to be enrolled or not in the study given the objectives of clinical demonstration in the study.
  • the gut microbiota sample can be obtained through an ileostomy or obtained with a smart pill ingested by the patient and collecting gut microbiota content directly in the gut while travelling within the gastro-intestinal tract.
  • the microbiota sample can be stored, for example to minimize exposure to air, in an adequate preparation media and/or frozen until further use in the methods of the present invention.
  • the fecal sample can be a stool sample or a rectal swab. These fecal samples can be obtained from a subject according to methods known in the art. Collection of a stool sample or rectal swab can be carried out in a clinical setting, or by the subject at home.
  • the stool sample or rectal swab can be stored in an adequate preparation media or frozen until further use in the method of the present invention.
  • the fecal sample or rectal swab can also be mailed by the patient to a laboratory that performs the methods of the present invention.
  • Detection or measure of a bacterial genus or species presence, absence, quantity or relative abundance can be carried out by any method known to those skilled in the art, for example and without limitation all the methods mentioned in Song et ak, 2018 or in Fraher et ah, 2012.
  • the detection can be performed by culture i.e. isolation of bacteria in selective media.
  • it can comprise detecting or measuring the level of a DNA, RNA or protein unique to the bacterial genus or species of interest and can rely on techniques such as PCR, qPCR, DGGE, T-RFLP, FISH or DNA microarrays.
  • the detection or measure can be performed by sequencing of 16S rRNA gene of the bacteria, such as the V3-V4 regions, or it can be performed by shotgun sequencing.
  • NextGen Sequencing (NGS) techniques can also be used as those described in Chiu & Miller, 2019 or any new technique emerging in the future that allows detection of bacterial genera, species or a chromosomal gene specific to the bacterial or species including but not restricted to antibiotic resistance determinants.
  • nucleic acids e.g. DNA, RNA
  • proteins or other molecules from bacteria or produced by bacteria present in gut microbiota samples or fecal samples can be performed using established techniques that are known in the art and routinely used.
  • sample preparations in particular DNA extraction can be operated with the methods presented in Lim et ak, 2018 and in Costea et ak, 2017.
  • the DNA or RNA extraction can be performed with kits commercially available, compatible with the techniques contemplated for the next steps of analysis.
  • DNA or mRNA is extracted from a sample, the amount of a bacterial DNA from a gene or a portion of gene whose sequence is unique to a bacterial species or strain, or the amount of RNA transcribed from a bacterial gene whose sequence or portions thereof are unique to a bacterial species or strain may be quantified.
  • the preferred method for determining the DNA or RNA level is an amplification-based method, such as by polymerase chain reaction (PCR), including reverse transcription-polymerase chain reaction (RT-PCR) for RNA quantitative analysis, and detection by an appropriate method known in the art.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription-polymerase chain reaction
  • the nucleic acids may also be obtained through in vitro amplification methods such as those described herein and in Berger, Sambrook, and Ausubel, as well as Mullis et ak, (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et ak, eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Amheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et ak (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et ak (1990) Proc.
  • nucleic acids will not be amplified before they are quantified.
  • nucleic acid hybridization and/or amplification methods are used to detect and quantify nucleic acid sequences corresponding to specific bacterial groups that are to be detected or quantified in the methods of the invention.
  • an immunoassay or other assay to detect or quantify one or more specific proteins determinative of one or more of the bacteria can be used.
  • solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to specifically detect a protein. See Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988) for a description of suitable immunoassay formats and conditions.
  • DNA sequencing can be performed using known sequencing methodologies. Typically, a sample is sequenced using a large-scale sequencing method that provides the ability to obtain sequence information from many reads.
  • sequencing platforms include for example those commercialized by Roche 454 Life Sciences (GS systems), Illumina (e.g., HiSeq, MiSeq), Life Technologies (e.g., SOLiD systems), Thermo Fisher Scientific (Ion Torrent), Oxford Nanopore Technologies (e.g. MinlON), and Pacific Biosciences (e.g. RSII, Sequel).
  • Short-read sequencing technologies such as Roche 454 Life Sciences, Illumina, and Thermo Fisher Scientific technologies relies on the sequencing by synthesis principle. This involves either an emulsion PCR where DNA fragments are immobilized onto beads or an on-chip bridge amplification where DNA fragments hybridizes to a planar surface. Subsequent incorporation of bases is detected using fluorescence or ionic discharge. Methods that employ sequencing by hybridization may also be used. Such methods, e.g., used in the Life Technologies SOLiD4+ technology uses a pool of all possible oligonucleotides of a fixed length, labelled according to the sequence. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position.
  • the sequence can be determined using any other DNA sequencing method including, e.g., methods that use semiconductor technology to detect nucleotides that are incorporated into an extended primer by measuring changes in current that occur when a nucleotide is incorporated (see, e.g., U.S. Patent Application Publication Nos. 20090127589 and 20100035252).
  • Other techniques include direct label- free exonuclease sequencing in which nucleotides cleaved from the nucleic acid are detected by passing through a nanopore (Oxford Nanopore) (Clark et al., Nature Nanotechnology 4: 265-270, 2009); and Single Molecule Real Time (SMRTTM) DNA sequencing technology (Pacific Biosciences),
  • Deep sequencing can also be used to quantify the number of copies of a particular sequence in a sample and then also be used to determine the relative abundance of different sequences in a sample. Deep sequencing refers to sequencing of a nucleic acid sequence, for example such that the original number of copies of a sequence in a sample can be determined or estimated. See, e.g., Mirebrahim, Hamid et al., Bioinformatics 31 (12): i9-i 16 (2015).
  • specific sequences in the sample can be targeted for amplification and/or sequencing.
  • specific primers can be used to detect and sequence bacterial sequences of interest and corresponding to the bacterial species to detect in the method of the invention.
  • whole genome sequencing methods that sequence random DNA fragments in a sample can be used.
  • Resulting sequence reads can be further classified by the use of single-nucleotide resolution methods by comparing the resulting sequence reads to known sequences in a genomic database.
  • Illustrative algorithms that are suitable for determining percent sequence identity and sequence similarity and thus aligning and identifying sequence reads are for example the BLAST and BLAST 2.0 algorithms. Accordingly, for the sequence reads generated, a subset of these reads can be aligned to one or more bacterial genomes of the bacterial species of interest for the invention. For example, one can align a read with a database of bacterial sequences and the read can be designated as from a particular bacteria if that read has the best alignment to a DNA sequence from that bacteria in the database.
  • the genes of interest to identify the bacterial species of the invention may be placed on DNA microarrays or DNA chips to permit a fast detection and measure of the bacterial species of interest for the invention.
  • Microarray technology is a high throughput platform used to study numerous samples and to detect thousands of nucleic acid sequences simultaneously making it fast and user friendly.
  • Phylogenetic DNA microarrays consist of several thousand probes, usually designed from rRNA gene sequence database targeting either specific organisms (e.g. pathogenic bacteria) or the whole microbiota at various taxonomic ICVCIST
  • Several microarrays addressing the gut microbiota have been developed over the last decade, showing differences in their design and the aims of study.
  • taxonomic identification can be carried out using the SILVA high quality ribosomal RNA databases (https://www.arb-silva.de/). Greengenes or any other suitable ribosomal RNA database.
  • the invention relates to a method for predicting whether a subject is potentially at risk of developing CDI, the method comprising the step of detecting the presence, absence, quantity or relative abundance of at least one specific bacteria species (also referred to as a "test bacteria") in a gut microbiota sample or fecal sample of the subject.
  • the test bacteria is a bacteria species whose presence, absence, quantity or relative abundance differs in a sample of a subject at risk as compared to samples from subjects who are not at risk.
  • the method comprises the step of measuring the quantity or the relative abundance of the bacteria species.
  • the method comprises the step of measuring the relative abundance of the bacteria species.
  • Illustrative genus to which such bacteria pertain may include those of the Enterococcus, Finegoldia, Blautia, Ruminococcus , Clostridium, Akkermansia, Porphyromonas, Roseburia, Alistipes, Ezakiella, Bacteroides, Bifidobacterium, Escherichia, Shigella, Pseudescherichia, Pectobacterium, Erwinia, Pantoea, Cronobacter or Anaerocuccus genus.
  • Illustrative Enterococcus bacteria useful in the practice of the invention include, without limitation, bacteria selected from the group consisting of Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus villorum, Enterococcus faecalis and Enterococcus ratti.
  • the Enterococcus bacteria is selected from Enterococcus faecium, Enterococcus durans and Enterococcus hirae.
  • Illustrative Finegoldia bacteria include, without limitation, Finegoldia magna.
  • Illustrative Blautia bacteria include, without limitation, Blautia wexlerae, Blautia obeum, Blautia faecis, and Blautia luti.
  • Illustrative Ruminococcus bacteria include, without limitation, Ruminococcus torques, Ruminococcus faecis, Ruminococcus bromii and Ruminococcus lactaris .
  • Clostridium bacteria include, without limitation, Clostridium glycyrrhizinilyticum
  • Illustrative Akkermansia bacteria include, without limitation, Akkermansia muciniphila.
  • Porphyromonas bacteria include, without limitation, Porphyromonas bennonis, Porphyromonas asaccharolytica, Porphyromonas coagulans and Porphyromonas uenonis.
  • Illustrative Roseburia bacteria include, without limitation, Roseburia faecis.
  • Illustrative Alistipes bacteria include, without limitation, Alistipes onderdonkii, Alistipes fmegoldii and Alistipes timonensis.
  • Illustrative Ezakiella bacteria include, without limitation, Ezakiella massiliensis
  • Illustrative Bacteroides bacteria include, without limitation, Bacteroides coagulans, Bacteroides xylanisolvens, Bacteroides acidifaciens, Bacteroides ovatus, Bacteroides kribbi, Bacteroides koreensis and Bacteroides finegoldi.
  • Illustrative Bifidobacterium bacteria include, without limitation, Bifidobacterium dentium, Bifidobacterium adolescentis, Bifidobacterium faecale, Bifidobacterium stercoris, Bifidobacterium pseudocatenulatum, Bifidobacterium kashiwanohense, Bifidobacterium catenulatum.
  • Illustrative Escherichia bacteria include, without limitation, Escherichia fergusonii, Escherichia coli, Escherichia albertii, and Escherichia marmotae.
  • Shigella bacteria include, without limitation, Shigella sonnei, Shigella boydii, Shigella dysenteriae and Shigella flexneri.
  • Illustrative Brenneria bacteria include, without limitation, Brenneria alni.
  • Illustrative Pseudescherichia bacteria include, without limitation, Pseudescherichia vulenris.
  • Illustrative Pectobacterium bacteria include, without limitation, Pectobacterium carotovorum.
  • Illustrative Erwinia bacteria include, without limitation, Erwinia iniecta.
  • Pantoea bacteria include, without limitation, Pantoea Bertjingensis and Pantoea wallissii.
  • Cronobacter bacteria include, without limitation, Cronobacter condimenti and Cronobacter turicensis.
  • Anaerococcus bacteria include, without limitation, Anaerocuccus mediterranneensis, Anaerocuccus murdochii, Aanerocccus degeneri, Anaerococcus prevotii, Anaerococcus lactolyticus, and Anaerococus tetradius.
  • a specific embodiment of the method of the invention comprises the detection of the absence, presence, quantity or relative abundance of bacteria from at least one bacterial species selected in the group consisting of Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus villorum, Enterococcus ratti, Enterococcus faecalis, Finegoldia magna, Blautia wexlerae, Blautia luti, Blautia obeum, Blautia fiaecis, Ruminococcus torques, Ruminococcus fiaecis, Ruminococcus bromii, Ruminococcus lactaris, Clostridium glycyrrhizinilyticum, Akkermansia muciniphila, Porphyromonas bennonis, Porphyromonas asaccharolytica, Porphyromonas coagulans, Porphyromonas u
  • bacteria species quantity or relative abundance may be increased in subjects potentially at risk of developing CDI.
  • bacteria species one can cite, without limitation, bacteria selected from the group consisting of Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus villorum, Enterococcus ratti, Enterococcus faecalis, Finegoldia magna, Bacteroides xylanisolvens, Bacteroides acidifaciens , Bacteroides ovatus, Bacteroides kribbi, Bacteroides koreensis and Bacteroides fimegoldi, in particular from the group consisting of Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus villorum, Enterococcus ratti and Finegoldia magna, such as from the group consisting of Entero
  • bacteria species have a quantity or relative abundance which is increased in subjects who are not potentially at risk of developing CDI.
  • Such bacteria include, without limitation, Blautia wexlerae, Blautia luti, Blautia obeum, Blautia faecis, Ruminococcus torques, Ruminococcus faecis, Ruminococcus bromii, Ruminococcus lactaris, Clostridium glycyrrhizinilyticum, Akkermansia muciniphila, Porphyromonas bennonis, Porphyromonas asaccharolytica, Porphyromonas coagulans, Porphyromonas uenonis, Roseburia faecis, Alistipes onderdonkii, Alistipes fmegoldii, Alistipes timonensis, Bacteroides coagulans, Bifidobacterium dentium, Bifidobacter
  • the method of the invention may include the determination of the presence or absence, or the measure of the quantity or relative abundance of one or more than one bacteria species, in particular of more than one bacteria species.
  • different combinations can be implemented to increase the predictive value, the sensitivity and/or the specificity of the method of the invention.
  • the combinations of measures can be implemented according to different methods.
  • the measure for each bacteria species can be compared to a threshold value corresponding to a predetermined value under which or over which the measured value will be considered predictive or not predictive of a potential risk of developing CDI. For example, for bacteria species whose quantity or relative abundance was determined to be increased in subjects potentially at risk of developing CDI, a measure higher than such predetermined threshold value is indicative of a potential risk of developing CDI.
  • a measure lower than a predetermined threshold value is also indicative of a potential risk of developing CDI.
  • combination of both information i.e. a measure higher than a threshold value for a bacteria associated to subjects at risk of CDI and a measure lower than a threshold value for a bacteria associated to subjects not at risk of CDI, can achieve a better prediction than each measure considered separately.
  • the measures obtained for more than one bacteria species can be used to determine a ratio.
  • the measure of the relative abundance of two bacterial species can be used to determine a ratio of relative abundances.
  • the ratio is calculated of the relative abundance of a bacteria species associated to subjects potentially at risk of developing CDI to the relative abundance of a bacteria species associated to subjects not potentially at risk of developing CDI.
  • the ratio calculated from the measures carried out from the gut microbiota sample or the fecal sample of the subject can be compared to a predetermined control ratio.
  • Such control ratio can be set so that a calculated ratio higher than this control ratio is indicative of a potential risk of developing CDI, while a calculated ratio lower than this control ratio is not indicative of a potential risk of developing CDI.
  • the method of the invention may include the measure of the quantity or the relative abundance, in particular the relative abundance, of at least two bacteria species.
  • the bacteria species include at least one bacteria selected from Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus villorum, Enterococcus ratti, Enterococcus faecalis, Finegoldia magna, Bacteroides xylanisolvens, Bacteroides acidifaciens , Bacteroides ovatus, Bacteroides kribbi, Bacteroides koreensis and Bacteroides fmegoldi, in particular from the group consisting of Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus villorum, Enterococcus ratti and Finegoldia magna, such as at
  • the combinations are used in the variant described above wherein each measure is compared to a threshold value.
  • Illustrative variants of implementation of this embodiment include, for example, a prediction made on the following basis:
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Blautia wexlerae lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Blautia luti lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Blautia obeum lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Blautia faecis lower than a threshold value is indicative of a potential risk of developing CDI
  • - a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Ruminococcus torques lower than a threshold value is indicative of a potential risk of developing CDI
  • - a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Ruminococcus faecis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Ruminococcus bromii lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Ruminococcus lactaris lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Clostridium glycyrrhizinilyticum lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Akkermansia muciniphila lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Roseburia faecis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Porphyromonas bennonis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Porphyromonas asaccharolytica lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Porphyromonas coagulans lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Porphyromonas uenonis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Alistipes onderdonkii lower than a threshold value is indicative of a potential risk of developing CDI
  • - a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Alistipes finegoldii lower than a threshold value is indicative of a potential risk of developing CDI
  • - a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Alistipes timonensis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Bacteroides coagulans lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Bifidobacterium adolescentis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Bifidobacterium faecale lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Bifidobacterium stercoris lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Bifidobacterium pseudocatenulatum lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Bifidobacterium kashiwanohense lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Enterococcus faecium higher than a threshold value and a measure of the relative abundance of Bifidobacterium catenulatum lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Blautia wexlerae lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Blautia luti lower than a threshold value is indicative of a potential risk of developing CDI
  • - a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Blautia obeum lower than a threshold value is indicative of a potential risk of developing CDI
  • - a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Blautia faecis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Ruminococcus torques lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Ruminococcus faecis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Ruminococcus bromii lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Ruminococcus lactaris lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Clostridium glycyrrhizinilyticum lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Akkermansia muciniphila lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Roseburia faecis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Porphyromonas bennonis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Porphyromonas coagulans lower than a threshold value is indicative of a potential risk of developing CDI
  • - a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Porphyromonas uenonis lower than a threshold value is indicative of a potential risk of developing CDI
  • - a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of A lisiipes onderdonkii lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Alistipes finegoldii lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Alistipes timonensis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Bacteroides coagulans lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Bifidobacterium dentium lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Bifidobacterium adolescentis lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Bifidobacterium faecale lower than a threshold value is indicative of a potential risk of developing CDI
  • a measure of the relative abundance of Finegoldia magna higher than a threshold value and a measure of the relative abundance of Bifidobacterium stercoris lower than a threshold value is indicative of a potential risk of developing CDI
  • the combinations are used in the variant described above wherein each measure is combined to determine a ratio of relative abundances.
  • Illustrative variants of implementation of this embodiment include, for example, a prediction made on the following basis: - ratio of Enterococcus faecium to Blautia wexlerae relative abundances;
  • the ratio Enterococcus faecium / Blautia luti is compared to a predetermined control ratio (or threshold ratio).
  • the control ratio is of 26, and when the determined Enterococcus faecium / Blautia luti ratio is equal to or higher than 26, the subject is deemed to be potentially at risk of CDI.
  • the relative abundance of Blautia wexlerae and of Enteroccus faecium are compared to predetermined control relative abundances (or threshold relative abundances).
  • the predetermined control relative abundance are respectively of 0.093% and 0.086%, and when the relative abundance of Blautia wexlerae in the sample is lower than 0.093% and the relative abundance of Enteroccus faecium in the sample is equal to or higher than 0.086%, the subject is deemed to be potentially at risk of CDI.
  • the method comprises the following steps:
  • the method comprises the following steps:
  • predictive models can be established for the development of CDI, based on the quantity or relative abundance of the bacteria species disclosed above. For example, as mentioned above, such predictive models can result in the comparison of the relative abundance of a first bacteria species to a predetermined threshold value and the comparison of the relative abundance of a second bacteria to another predetermined threshold value. The predictive models can also result in the comparison of a ratio of relative abundance between a first bacteria species and a second bacteria species to a predetermined threshold ratio. The predictive models can usefully be used to determine whether a subject is potentially at risk of CDI, in particular potentially at risk of CDI within a specific time window.
  • the predictive models and methods of the invention can usefully be designed to determine the potential risk for a subject to develop CDI within 1 day, within 7 days, within 10 days, within 20 days, within 30 days, within 40 days, within 50 days, within 60 days, within 70 days, within 80 days, within 90 days, or within more than 90 days.
  • the predictive models and methods of the invention can usefully be designed to determine the potential risk for a subject to develop CDI within 30 days or within 90 days.
  • the invention can advantageously be used to determine how an infection in a subject can be handled. For example, thanks to the invention, decision can be made to determine whether a particular antibiotic can be administered to the subject.
  • the invention can also allow a physician or healthcare providers to determine whether a therapeutics prescription must be made, whether a therapeutics prescription should be altered, or whether other measures should be used to decrease the chances of nosocomial disease in the subject. Thanks to the invention, the routine care given to the subject can also be adapted, or other measures can be used to predict and/or detect any outbreak of CDI in the facility.
  • Another aspect of the invention relates to a method for the treatment of an infection in a subject, the method comprising administering to said subject a therapeutically effective amount of an antibiotic suitable for the treatment of said infection, wherein decision to treat or not to treat said subject is based on the prediction of said subject to potentially be at risk of developing CDI according to the methods described above.
  • Another aspect of the invention relates to a method for the prevention of CDI in a subject, the method comprising administering a vaccine or another product for prevention of Clostridioides difficile infection or isolating the patient in the hospital ward or even deploying advanced cleaning techniques to limit the exposure of the patient to spores of C. difficile, wherein decision to proceed with these procedures is based on the prediction of said subject to potentially be at risk of developing CDI according to the methods described herein.
  • Another aspect of the invention relates to a method for the prevention of CDI in a subject, the method comprising administering a live biotherapeutic product, such as a probiotic or a fecal microbiota transplant, wherein decision to proceed with these procedures is based on the prediction of said subject to potentially be at risk of developing CDI according to the methods described herein.
  • a live biotherapeutic product such as a probiotic or a fecal microbiota transplant
  • the invention also provides a kit for detecting a subject potentially at risk of developing CDI.
  • the kit of the invention includes: i) an agent suitable for the measure of the relative abundance of a first bacteria species; and ii) an agent suitable for the measure of the relative abundance of a second bacteria species.
  • the kit of the invention may further include: iii) a standard control that provides a reference amount of the first bacteria species and a standard control of the second bacteria species, allowing the measure from said standard controls of a control threshold value or of a relative abundance ratio of the first and second bacteria species.
  • agents i) and ii) are polynucleotide probes that bind specifically to a DNA or RNA species unique to the first and second bacteria species, respectively.
  • agents i) and ii) can comprise a set of two oligonucleotide primers suitable for specifically amplifying a portion of a DNA molecule in a DNA amplification reaction, such as in a PCR, quantitative PCR, RT-PCR or quantitative RT-PCR.
  • a DNA amplification reaction such as in a PCR, quantitative PCR, RT-PCR or quantitative RT-PCR.
  • other components can be included such as a polynucleotide probe or fluorescent dyes.
  • Agents i) and ii) can also correspond to agents that specifically bind to a protein unique to the first bacteria species, and to a protein unique to the second bacteria species, respectively.
  • the agents can be selected from antibodies, antibody fragments and aptamers.
  • the invention also provides kits for sampling gut microbiota or fecal samples containing suitable preservation medium and probes to detect the first and second bacteria species mentioned above.
  • the present invention also provides compositions and methods for preventing CDI.
  • the invention also relates to a composition comprising at least one bacteria, identified as more abundant in subjects that will not develop CDI.
  • the bacteria is/are selected in the group consisting of a Blautia bacteria, Ruminococcus bacteria, Clostridium bacteria, Akkermansia bacteria, Porphyromonas bacteria, Roseburia bacteria, Alistipes bacteria, Ezakiella bacteria, Bacteroides bacteria, Bifidobacterium bacteria, Escherichia bacteria, Shigella bacteria, Pseudescherichia bacteria, Pectobacterium bacteria, Erwinia bacteria, Pantoea bacteria, Cronobacter bacteria or Anaerocuccus bacteria.
  • the bacteria is/are selected in the group consisting of Blautia wexlerae, Blautia luti, Blautia obeum, Blautia fiaecis, Ruminococcus torques, Ruminococcus fiaecis, Ruminococcus bromii, Ruminococcus lactaris, Clostridium glycyrrhizinilyticum, Akkermansia muciniphila, Roseburia fiaecis, Porphyromonas bennonis, Porphyromonas asaccharolytica, Porphyromonas coagulans, Porphyromonas uenonis, Alistipes onderdonkii, Alistipes fimegoldii, Alistipes timonensis, Bacteroides coagulans, Bifidobacterium dentium, Bifidobacterium adolescentis, Bifidobacterium fae
  • the administered bacteria is/are selected in the group consisting of a Blautia bacteria, Ruminococcus bacteria, Clostridium bacteria, Akkermansia bacteria, Porphyromonas bacteria, Roseburia bacteria, Alistipes bacteria, Ezakiella bacteria, Bacteroides bacteria, Bifidobacterium bacteria, Escherichia bacteria, Shigella bacteria, Pseudescherichia bacteria, Pectobacterium bacteria, Erwinia bacteria, Pantoea bacteria, Cronobacter bacteria or Anaerocuccus bacteria.
  • the bacteria is/are selected in the group consisting of Blautia wexlerae, Blautia luti, Blautia obeum, Blautia fiaecis, Ruminococcus torques, Ruminococcus fiaecis, Ruminococcus bromii, Ruminococcus lactaris, Clostridium glycyrrhizinilyticum, Akkermansia muciniphila, Roseburia fiaecis, Porphyromonas bennonis, Porphyromonas asaccharolytica, Porphyromonas coagulans, Porphyromonas uenonis, Alistipes onderdonkii, Alistipes fimegoldii, Alistipes timonensis, Bacteroides coagulans, Bifidobacterium dentium, Bifidobacterium adolescentis, Bifidobacterium fae
  • the invention also relates to a composition comprising a substance able to slow the growth or kill at least one bacteria, identified as less abundant in subjects that will not develop CDI.
  • a composition could comprise, without limitation, specific antibiotics able to selectively kill a bacteria, combination of antibiotics whose combined use selectively kills one bacteria, bacteriophages selected to be targeting the specific bacteria, CRISPR-CAS systems able to selectively alter the DNA of the specific bacteria.
  • the bacteria to be killed or whose growth is to be slowed is selected in the group consisting of Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus villorum, Enterococcus ratti, Finegoldia magna, Bacteroides xylanisolvens , Bacteroides acidifaciens , Bacteroides ovatus, Bacteroides kribbi, Bacteroides koreensis and Bacteroides fmegoldi, in particular from the group consisting of E. faecium, E. durans, E. hirae, E. villorum, E. ratti and Finegoldia magna, such as from the group consisting of Enterococcus faecium, Enterococcus durans, Enterococcus hirae and Finegoldia magna.
  • Reads were processed through a bioinformatic pipeline, and reads were given taxonomic identifications using the SILVA database. The relative abundance of the various identified taxa was calculated from the OTU table.
  • a LEfSe analysis (Segata et ah, 2011) was performed to identify taxa enriched in patients that developed a CDI within 90 days, and those enriched in patients that did not develop a CDI. Table 1 lists bacteria taxa identified thanks to this analysis.
  • Table 1 Taxa whose relative abundance is significantly increased in patients that will develop CDI, or that will not develop CDI following antibiotic treatment
  • Group 1 Taxa whose relative abundance is significantly increased in patients that will develop CDI Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus villorum, Enterococcus ratti, Enterococcus faecalis Finegoldia magna
  • Bacteroides xylanisolvens Bacteroides acidifaciens, Bacteroides ovatus, Bacteroides kribbi, Bacteroides koreensis, Bacteroides finegoldi
  • Group 2 Taxa whose relative abundance is significantly increased in patients that will not develop CDI Blautia wexlerae, Blautia luti, Blautia obeum, Blautia faecis
  • Shigella sonnei Shigella boydii, Shigella dysenteriae, Shigella flexneri
  • Anaerocuccus mediterranneensis Anaerocuccus murdochii, Aanerocccus degeneri, Anaerococcus prevotii, Anaerococcus lactolyticus, Anaerococus tetradius
  • Ratio OTUl/OTU648> 26 207 12.8 5.37% (1.79-12.70) 5.83 (2.10-21.44)
  • OTU 1 encompasses Enterococcus bacteria belonging from one or several of Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus villorum, Enterococcus ratti, Enterococcus fiaecalis and OTU 648 encompasses Blautia bacteria, corresponding essentially to Blautia luti
  • OTU30 encompasses Blautia bacteria, from one or several of Blautia wexlerae, Blautia obeum, Blautia faecis, and OTU 1 is defined as above
  • Example 2 application of the invention - management of a patient by a physician
  • a fecal swab is taken at hospital admission.
  • DNA is extracted from the fecal swab using the FAST DNA SPIN kit (MP Biomedicals), and the V3- V4 regions of the 16S rRNA genes is sequenced using Illumina MiSeq technology.
  • Reads are processed through a bioinformatic pipeline, and reads are given taxonomic identifications using the SILVA database. The relative abundance of the various identified bacteria are compared to thresholds. In particular:
  • Example 3 application of the invention - management of a patient by a physician
  • a fecal swab is taken at hospital admission.
  • the swab is discharged in a sterile tube on 0.5 ml water, and one drop is inserted in the analysis cassette of a diagnostic multiplex PCR machine, containing DNA primers specific for several bacterial taxa, among which Enterococcus faecium, Blautia luti and Blautia wexlerae. Quantitative detection of the amplified DNA is performed within the machine either using a fluorescent taqman probe for each of these taxa, or by analysis on a microarray slide.
  • the relative abundance of the various identified bacteria are compared to thresholds.
  • thresholds In particular:
  • Example 4 application of the invention - management of a patient by a physician
  • a fecal sample is collected from the patient with a rectal swab and sent to the laboratory where the DNA content of the swab is extracted, amplified by PCR in a high multiplex PCR reaction using primer pairs adapted to amplify DNA from the various bacteria of interest, and placed on a phylogenetic microarray that permits the evaluation of the relative abundance of different species.
  • the laboratory sends back to the physician a report with an evaluation of the predictive risk of occurrence of CDI in the patient.
  • the physician can then alter the antibiotic prescription or recommend additional measures to decrease the chances of nosocomial disease in the patient. Or, if the antibiotic prescription is unavoidable, the physician can request stool biobanking from the patient for an autologous fecal transplant following antibiotic therapy.
  • Example 5 application of the invention - management of a patient in a long-term care facility
  • a fecal sample is taken from each patient at the time of the next stool and the sample sent to a central laboratory.
  • the stool sample is processed and nucleic acids are extracted and analysed in a next generation sequencing device that performs a quantification of the diverse bacteria present initially in the stool sample.
  • the risk of occurrence of CDI is estimated and communicated to the long-term care facility healthcare providers to adapt the routine care given to the patient and detect any outbreak of CDI at the earliest possible timepoint.
  • Example 6 application of the invention - recruitment in a clinical study
  • a clinical trial for a CDI-preventing drug or device is held.
  • patients with a high level of risk for CDI should be enrolled in the trial.
  • a fecal sample is taken from each patient and sent to the central laboratory where it is tested on a specific DNA array chip able to detect bacteria whose abundance is high or low among bacteria predictive of a high risk of occurrence of CDI.
  • the risk of occurrence of CDI is estimated and communicated to the physician who can then decide to recruit the patient in the clinical trial or not.

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Abstract

La présente invention concerne un procédé pour prédire si un sujet est potentiellement susceptible de développer des manifestations cliniques d'une dysbiose de microbiote d'intestin, également appelée dysbiose de microbiote intestinal, telle que le développement d'une infection par Clostridioides difficile (CDI).
PCT/EP2020/087329 2019-12-20 2020-12-18 Prédiction de manifestations cliniques d'une dysbiose de microbiote intestinal WO2021123387A1 (fr)

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