TECHNICAL FIELD

The present disclosure relates to methods of diagnosing a dysbiosis in a subject, methods of determining a suitable treatment, and methods of treating a dysbiosis.

BACKGROUND

The human microbiota consists of several trillion microorganisms, most of which are of bacterial origin and are non-pathogenic. The microbiota plays a crucial role in human health and functions jointly with the host's immune system to protect against the invasion and colonisation of pathogens. It also has an essential metabolic function by providing a source of essential vitamins and nutrients as well as assisting in the extraction of energy and nutrients from food, such as amino acids and short-chain fatty acids. In this regard, the host is highly dependent on its microbiota for a number of critical biological functions, which significantly contribute to health.

There is a growing body of evidence to suggest that dysbiosis of the human microbiota is associated with a number of diseases. However, it is difficult to determine or predict the exact impact that the microbiota has on human health and its involvement in human disease, due to the highly complex interplay between bacterial species in the microbiota. Thus, there is a need for methods that can effectively diagnose a dysbiosis in order to identify subjects at risk of developing associated diseases.

SUMMARY

The present inventors have developed a method of diagnosing a dysbiosis in a subject which involves analysing the microbiome of the subject and comparing it to a reference sample. Additionally, the inventors have identified that there are several bacterial species and genera whose abundance in the microbiome is informative of dysbioses, and subtypes thereof, that are associated with a particular disease, including IBS, autism, and intestinal senescence.

Accordingly, the present disclosure provides a method of determining a subtype of IBS in a subject, comprising determining the abundance of at least 10 genera of bacteria in a sample from the subject or the subject's environment, wherein the at least 10 genera are selected fromAlkaliphilus, Sphingomonas, Pelotomaculum, Eggerthella, Eubacterium, Paracoccus, Lachnoclostridium, Bacillus, Anaerorhabdus, Actinomyces, Methylobacterium, Pseudomonas, Streptococcus, Staphylococcus, Peptoclostridium, Erysipelatoclostridium, Anaerostipes, Sutterella, Brevundimonas, Clostridium, Peptostreptococcaceae, Slackia, Blastomonas, Lactobacillus, Klebsiella, Agrobacterium, andPhyllobacterium.

In one embodiment, the at least 10 genera of bacteria include at least 5 genera selected fromAlkaliphilus, Sphingomonas, Pelotomaculum, Eggerthella, Eubacterium, Paracoccus, Lachnoclostridium, Bacillus, Anaerorhabdus, andActinomyces.

In one embodiment, the at least 10 genera of bacteria includeAlkaliphilus, Sphingomonas, Pelotomaculum, Eggerthella, Eubacterium, Paracoccus, Lachnoclostridium, Bacillus, Anaerorhabdus, andActinomyces.

In one embodiment, the abundance of at least 20 genera of bacteria is determined.

The present disclosure also provides a method of determining a subtype of IBS in a subject, comprising determining an abundance of at least 5 species of bacteria in a sample from the subject or the subject's environment, wherein the at least 5 species of bacteria are selected fromChristensenella minuta, Papillibacter cinnamivorans, Bilophila wadsworthia, Ruminococcus bromii, Soleaferrea massiliensis, Akkermansia muciniphila, Oscillibacter valericigenes, Desulfitobacterium frappieri, Anaerofilum pentosovorans, Lactobacillus japonicas, Catabacter hongkongensis, Clostridium sporosphaeroides, andFaecalibacterium prausnitzii.

In one embodiment, the at least 5 species of bacteria include at least 2 species selected fromChristensenella minuta, Papillibacter cinnamivorans, Bilophila wadsworthia, Ruminococcus bromii, andSoleaferrea massiliensis.

In one embodiment, the at least 5 species of bacteria includeChristensenella minuta, Papillibacter cinnamivorans, Bilophila wadsworthia, Ruminococcus bromii, andSoleaferrea massiliensis.

In one embodiment, the abundance of at least 10 species of bacteria is determined.

The present disclosure also provides a method of diagnosing autism, comprising determining an abundance of at least 10 genera of bacteria in a sample from a subject or the subject's environment, wherein the at least 10 genera are selected fromRuminiclostridium, Sarcina, Lachnoclostridium, Asaccharospora, Lachnobacterium, Anaerostipes, Faecalibacterium, Bacteroides, Mogibacterium, Haemophilus, Intestinibacter, Mobiluncus, Lactobacillus, Alistipes, Dorea, Ferrimonas, Romboutsia, Actinobacillus, Anaerofilum, Erwinia, Phascolarctobacterium, Selenomonas, Microbacterium, Ureibacillus, Proteus, Megamonas, Christensenella, Butyricimonas, Arcobacter, Yersinia, Lachnoanaerobaculum, Variovorax, Citrobacter, Paenibacillus, Anaeroplasma, Fictibacillus, Eisenbergiella, Lautropia, andHowardella.

In one embodiment, the at least 10 genera of bacteria include at least 5 genera selected fromRuminiclostridium, Sarcina, Lachnoclostridium, Asaccharospora, Lachnobacterium, Anaerostipes, Faecalibacterium, Bacteroides, Mogibacterium, andHaemophilus.

In one embodiment, the at least 10 genera of bacteria includeRuminiclostridium, Sarcina, Lachnoclostridium, Asaccharospora, Lachnobacterium, Anaerostipes, Faecalibacterium, Bacteroides, Mogibacterium, andHaemophilus.

In one embodiment, the abundance of at least 20 genera of bacteria is determined. In one embodiment, the abundance of at least 30 genera of bacteria is determined.

The present disclosure also provides a method of diagnosing a dysbiosis in a subject, comprising determining an abundance of at least 5 species of bacteria in a sample from the subject, wherein the at least 5 species of bacteria are selected fromCorynebacterium minutissimum, Prevotella oulora, Fusobacterium naviforme, Prevotella ruminicola, Bifidobacterium thermacidophilum, Dysgonomonas wimpennyi, Propionibacterium acnes, Corynebacterium tuberculostearicum, Brevibacterium casei, Lachnobacterium bovis, Prevotella dentasini, Prevotella albensis, Veillonella atypica, Kytococcus schroeteri, Prevotella copri, Bacteroides barnesiae, Prevotella conceptionensis, Anaerofustis stercorihominis, Bifidobacterium thermophilum, Prevotella brevis, Roseburia intestinalis, Clostridium symbiosum, Barnesiella intestinihominis, Bacteroides fragilis, Anaerostipes rhamnosus, Collinsella aerofaciens, Clostridium bolteae, Arthrobacter creatinolyticus, Atopobium fossor, Prevotella paludivivens, andPelotomaculum isophthalicicum.

In one embodiment, the at least 5 species include at least 2 species selected fromCorynebacterium minutissimum, Prevotella oulora, Fusobacterium naviforme, Prevotella ruminicola, andBifidobacterium thermacidophilum.

In one embodiment, the at least 5 species includeCorynebacterium minutissimum, Prevotella oulora, Fusobacterium naviforme, Prevotella ruminicola, andBifidobacterium thermacidophilum.

In one embodiment, the abundance of at least 10 species of bacteria is determined. In one embodiment, the abundance of at least 20 species of bacteria is determined.

The present disclosure also provides a method of diagnosing a dysbiosis in a subject, comprising determining an abundance of at least 5 genera of bacteria in a sample from the subject, wherein the at least 5 genera of bacteria are selected fromCorynebacterium, Lachnobacterium, Propionibacterium, Kytococcus, Fusobacterium, Veillonella, Prevotella, Anaerofustis, Arthrobacter, Dysgonomonas, Calothrix, Atopobium, Brevibacterium, Micrococcus, Burkholderia, Veillonella, Pelotomaculum, Acidaminococcus, Mitsuokella, Allisonella, Odoribacter, Bacteroides, Coprobacter,Alistipes, Ruminococcus, Ferrimonas, Alkaliphilus, andLautropia.

In one embodiment, the at least 5 genera include at least 2 genera selected fromCorynebacterium, Lachnobacterium, Propionibacterium, Kytococcus, andFusobacterium.

In one embodiment, the at least 5 genera includeCorynebacterium, Lachnobacterium, Propionibacterium, Kytococcus, andFusobacterium.

In one embodiment, the abundance of at least 10 genera of bacteria is determined. In one embodiment, the abundance of at least 20 genera of bacteria is determined.

In one embodiment, the dysbiosis is IBS.

The present disclosure also provides a method of determining a subtype of IBS in a subject with IBS, comprising determining an abundance of at least 5 species of bacteria in a sample from the subject, wherein the at least 5 species of bacteria are selected fromChristensenella minuta, Soleaferrea massiliensis, Papillibacter cinnamivorans, Oscillibacter valericigenes, Ruminococcus bromii, Gemmiger formicilis, Desulfitobacterium frappieri, Alistipes obesi, Anaerofilum pentosovorans, Akkermansia muciniphila, Alkaliphilus crotonatoxidans, Eubacterium sulci, Bdellovibrio exovorus, Curtobacterium pusillum, Flavonifractor plautii, Ruminococcus lactaris, Mogibacterium neglectum, Roseburia inulinivorans, Butyricimonas virosa, Intestinimonas butyriciproducens, Butyrivibrio crossotus, Barnesiella intestinihominis, Flavobacterium resistens, Flavobacterium cauense, Clostridium glycyrrhizinilyticum, Anaerostipes hadrus, Prevotella ruminicola, Blautia wexlerae, andAnaerostipes coli.

In one embodiment, the at least 5 species of bacteria include at least 2 species selected fromChristensenella minuta, Soleaferrea massiliensis, Papillibacter cinnamivorans, Oscillibacter valericigenes, andRuminococcus bromii.

In one embodiment, the at least 5 species of bacteria includeChristensenella minuta, Soleaferrea massiliensis, Papillibacter cinnamivorans, Oscillibacter valericigenes, andRuminococcus bromii.

In one embodiment, the abundance of at least 10 species of bacteria is determined. In one embodiment, the abundance of at least 20 species of bacteria is determined.

The present disclosure also provides a method comprising diagnosing IBS in a subject according to the methods described herein and, if the subject is diagnosed with IBS, subsequently determining the subtype of IBS according to the methods described herein.

The present disclosure also provides a method of determining a subtype of IBS in a subject who has been diagnosed with IBS-M, comprising determining an abundance of at least 5 species of bacteria in a sample from the subject and assigning the subject as having constipation-dominant IBS-M (IBS-MC) or diarrhoea-dominant IBS-M (IBS-MD) based on the abundance of the at least 5 species of bacteria.

In one embodiment, the at least 5 species of bacteria are selected fromPeptoniphilus coxii, Clostridium clariflavum, Bacteroides thetaiotaomicron, Bacteroides coprocola, Oscillibacter valericigenes, Clostridium hveragerdense, Ruminiclostridium clariflavum, Bacteroides xylanisolvens, Clostridium chauvoei, Clostridium tepidiprofundi, Papillibacter cinnamivorans, Lactococcus fujiensis, Bacteroides uniformis, Bacillus thuringiensis, Johnsonella ignava, Pseudoflavonifractor capillosus, Christensenella minuta, Enterococcus azikeevi, Intestinimonas butyriciproducens, Eubacterium sulci, Flavobacterium resistens, Anaerotruncus colihominis, Pseudoflavonifractor capillosus, Bacteroides fluxus, Ruminococcus lactaris, Butyrivibrio crossotus, Eubacterium rectale, Prevotella ruminicola, Gemmiger formicilis, Ruminococcus flavefaciens, andFaecalibacterium prausnitzii.

In one embodiment, the at least 5 species of bacteria include at least 2 species selected fromPeptoniphilus coxii, Clostridium clariflavum, Bacteroides thetaiotaomicron, Bacteroides coprocola, andOscillibacter valericigenes.

In one embodiment, the at least 5 species of bacteria includePeptoniphilus coxii, Clostridium clariflavum, Bacteroides thetaiotaomicron, Bacteroides coprocola, andOscillibacter valericigenes.

In one embodiment, the abundance of at least 10 species of bacteria is determined. In one embodiment, the abundance of at least 20 species of bacteria is determined.

The present disclosure also provides a method of diagnosing autism in a subject with IBS, comprising determining an abundance of at least 5 species of bacteria in a sample from the subject, wherein the at least 5 species of bacteria are selected fromEubacterium hallii, Eubacterium rectale, Lachnobacterium bovis, Lachnoclostridium glycyrrhizinilyticum, Blautia glucerasea, Eubacterium desmolans, Anoxystipes fissicatena, Blautia coccoides, Faecalibacterium prausnitzii, Clostridium symbiosum, Roseburia inulinivorans, Anaerostipes coli, Coprococcus comes, Lachnospira pectinoschiza, Arthrobacter creatinolyticus, Clostridium nexile, Bifidobacterium thermacidophilum, Anaerostipes rhamnosus, Clostridium clariflavum, Blautia wexlerae, Fusicatenibacter saccharivorans, Tolumonas auensis, Ruminococcus gnavus, Peptococcus niger, Dorea formicigenerans, Roseburia intestinalis, Blautia wexlerae, Clostridium populeti, Dorea massiliensis, andEubacterium eligens.

In one embodiment, the at least 5 species of bacteria include at least 2 species selected fromEubacterium hallii, Eubacterium rectale, Lachnobacterium bovis, Lachnoclostridium glycyrrhizinilyticum, andBlautia glucerasea.

In one embodiment, the at least 5 species of bacteria includeEubacterium hallii, Eubacterium rectale, Lachnobacterium bovis, Lachnoclostridium glycyrrhizinilyticum, andBlautia glucerasea.

In one embodiment, the abundance of at least 10 species of bacteria is determined. In one embodiment, the abundance of at least 20 species of bacteria is determined.

The present disclosure also provides a method of diagnosing a dysbiosis associated with intestinal senescence, the method comprising determining an abundance of at least 5 species of bacteria in a sample from the subject, wherein the at least 5 species of bacteria are selected fromPseudobutyrivibrio xylanivorans, Dorea massiliensis, Blautia glucerasea, Lachnoclostridium herbivorans, Faecalibacterium prausnitzii, Romboutsia lituseburense, Peptoniphilus methioninivorax, Blautia coccoides, Megamonas funiformis, Eubacterium rectale, Clostridium bifermentans, Roseburia intestinalis, Clostridium populeti, Clostridium hiranonis, Peptoclostridium difficile, Lactonifactor longoviformis, Clostridium caliptrosporum, Asaccharospora irregulare, Clostridium malenominatum, Clostridium symbiosum, Clostridium thermoalcaliphilum, Lachnoclostridium glycyrrhizinilyticum, Ruminococcus faecis, Blautia schinkii, Pseudoflavonifractor capillosus, Clostridium chromoreductans, Clostridium ghonii, Clostridium innocuum, Christensenella minuta, Dorea formicigenerans, andClostridium tertium.

In one embodiment, the at least 5 species of bacteria include at least 2 species selected fromPseudobutyrivibrio xylanivorans, Dorea massiliensis, Blautia glucerasea, Lachnoclostridium herbivorans, andFaecalibacterium prausnitzii.

In one embodiment, the at least 5 species of bacteria includePseudobutyrivibrio xylanivorans, Dorea massiliensis, Blautia glucerasea, Lachnoclostridium herbivorans, andFaecalibacterium prausnitzii.

In one embodiment, the abundance of at least 10 species of bacteria is determined. In one embodiment, the abundance of at least 20 species of bacteria is determined.

The present disclosure also provides a method of treating a subject with a dysbiosis, comprising determining the subtype of IBS according to the methods described herein, or diagnosing the subject according to the methods described herein, and administering a composition to the subject which increases and/or decreases the abundance of one or more species of bacteria in the subject, thereby treating the subject.

In one embodiment, the composition is a nutraceutical. In one embodiment, the composition is a probiotic. In one embodiment, the composition is a faecal microbiota transplant.

The present disclosure also provides a method of monitoring effectiveness of the treatments described herein, comprising measuring the abundance of one or more species of bacteria in a sample from the subject, wherein an increase and/or decrease in the abundance of one or more species of bacteria is indicative of the effectiveness of the treatment.

The present disclosure also provides a method of determining a suitable treatment for a subject with a dysbiosis, comprising determining the subtype of IBS according to the methods described herein, or diagnosing the subject according to the methods described herein, and determining a suitable treatment based on the results of the determination of subtype of IBS or diagnosis.

In one embodiment, the subject is a human.

In one embodiment, the subject has previously been administered a chemotherapy or an antibiotic.

In one embodiment, the sample is a faecal sample.

The present disclosure also provides a method of diagnosing a dysbiosis ina subject, comprisingi) aligning sequences of nucleic acids in a sample from the subject to a database of bacterial 16S rRNA sequences;ii) using the alignment in step i) to determine the identity and relative abundance of one or more species of bacteria present in the sample; andiii) comparing the abundance of the one or more species of bacteria in the sample to the abundance of the one or more species of bacteria in a reference population of healthy individuals,wherein an increase or decrease in abundance of a species of bacteria in the sample relative to the reference population is indicative of a dysbiosis.

In one embodiment, the database essentially consists of 16S rRNA sequences from human gastrointestinal microbiota.

In one embodiment, the database consists of DNA sequences. In one embodiment, the database consists of RNA sequences.

In one embodiment, the reference population is age-matched to the subject.

In one embodiment, the sequences of nucleic acids in the sample are obtained by high-throughput nucleic acid sequencing.

In one embodiment, the identity and relative abundance of at least 50 species of bacteria in the sample is determined. In one embodiment, the identity and relative abundance of at least 100 species of bacteria in the sample is determined. In one embodiment, the identity and relative abundance of at least 200 species of bacteria in the sample is determined.

In one embodiment, the subject is a human. In one embodiment, the subject is a horse.

In one embodiment, the sample is a faecal sample. In one embodiment, the sample is obtained from the subject's mouth. In one embodiment, the sample is obtained from the subject's vagina. In one embodiment, the sample is obtained from the subject's surrounding environment.

In one embodiment, the dysbiosis is a dysbiosis of the gastrointestinal tract.

In one embodiment, the dysbiosis is associated with irritable bowel syndrome (IBS). In one embodiment, the dysbiosis is associated with an inflammatory disease. In one embodiment, the dysbiosis is associated with an immune disease. In one embodiment, the dysbiosis is associated with a metabolic disorder. In one embodiment, the dysbiosis is associated with autism. In one embodiment, the dysbiosis is associated with intestinal senescence. In one embodiment, the dysbiosis is associated with chronic fatigue.

In one embodiment, the subject has previously been administered a chemotherapy.

The present disclosure also provides a method of diagnosing a dysbiosis in a subject, comprisingi) aligning sequences of nucleic acids in a sample from the subject to a database of bacterial 16S rRNA sequences;ii) using the alignment in step i) to determine the identity and relative abundance of one or more species of bacteria present in the sample, thereby obtaining a sample dataset; andiii) applying a machine learning algorithm to the sample dataset to diagnose the dysbiosis in the subject,wherein the machine learning algorithm has been trained with a training dataset comprising identities and abundances of bacterial species from a reference population comprising individuals having the dysbiosis.

In one embodiment, the machine learning algorithm is a Bayesian algorithm. In one embodiment, the machine learning algorithm is a Random Forest algorithm.

In one embodiment, the reference population further comprises healthy individuals.

